Bob Lowenberg

Value of different modalities of granulocyte-macrophage colony-stimulating factor applied during or after induction therapy of acute myeloid leukemia.

PURPOSEnThe hematopoietic growth factors (HGFs) introduced into induction chemotherapy (CT) of acute myeloid leukemia (AML) might be of benefit to treatment outcome by at least two mechanisms. HGFs given on days simultaneously with CT might sensitize the leukemic cells and enhance their susceptibility to CT. HGFs applied after CT might hasten hematopoietic recovery and reduce morbidity or mortality.nnnMATERIALS AND METHODSnWe set out to evaluate the use of granulocyte-macrophage colony-stimulating factor (GM-CSF; 5 microg/kg) in a prospective randomized study of factorial design (yes or no GM-CSF during CT, and yes or no GM-CSF after CT) in patients aged 15 to 60 years (mean, 42) with newly diagnosed AML. GM-CSF was applied as follows: during CT only (+/-, n = 64 assessable patients), GM-CSF during and following CT (+/+, n = 66), no GM-CSF (-/-, n = 63), or GM-CSF after CT only (-/+, n = 60).nnnRESULTSnThe complete response (CR) rate was 77%. At a median follow-up time of 42 months, probabilities of overall survival (OS) and disease-free survival (DFS) at 3 years were 38% and 37% in all patients. CR rates, OS, and DFS did not differ between the treatment groups (intention-to-treat analysis). Neutrophil recovery (1.0 x 10(9)/L) and monocyte recovery were significantly faster in patients who received GM-CSF after CT (26 days v 30 days; neutrophils, P < .001; monocytes, P < .005). Platelet regeneration, transfusion requirements, use of antibiotics, frequency of infections, and duration of hospitalization did not vary as a function of any of the therapeutic GM-CSF modalities. More frequent side effects (eg, fever and fluid retention) were noted in GM-CSF-treated patients predominantly related to the use of GM-CSF during CT.nnnCONCLUSIONnPriming of AML cells to the cytotoxic effects of CT by the use of GM-CSF during CT or accelerating myeloid recovery by the use of GM-CSF after CT does not significantly improve treatment outcome of young and middle-aged adults with newly diagnosed AML.

Intensive postremission chemotherapy without maintenance therapy in adults with acute lymphoblastic leukemia. Dutch Hemato-Oncology Research Group.

PURPOSEnTo investigate the value of intensive consolidation chemotherapy not followed by maintenance therapy in adult acute lymphoblastic leukemia (ALL).nnnMATERIALS AND METHODSnA multicenter phase II trial was conducted in 130 adult patients with ALL between 16 and 60 years of age. After standard induction therapy, postinduction chemotherapy was given: three courses of high-dose cytarabine (2,000 mg/m2 every 12 hours for four doses) in combination with amsacrine (course one), mitoxantrone (course two), and etoposide (course three). CNS prophylaxis consisted of 10 injections of intrathecal methotrexate (IT MTX). Patients younger than 50 years with an HLA-identical sibling were eligible to receive allogeneic bone marrow transplantation (BMT).nnnRESULTSnNinety-five patients (73%) achieved complete remission (CR); 82% were younger than 50 years and 41% were older than 50 years. Seventeen patients (13%) were resistant to chemotherapy, and 18 (14%) died during induction treatment. Only age and performance status were significantly associated with response (P<.001 and .03, respectively). Death during consolidation occurred in four patients. The estimated 5-year overall survival (OS) was 22% for the entire group and 26% for patients younger than 35 years. Disease-free survival (DFS) at 5 years was 28% +/- 6 for patients younger than 35 years, 25% +/- 9 for patients between 35 and 50 years, and 0% for patients older than 50 years. Increasing age (P<.01) and expression of CD34 (P<.01) were adverse factors. Only three patients (3%) developed an isolated CNS relapse.nnnCONCLUSIONnIntensive consolidation including high-dose cytarabine not followed by maintenance therapy provides an outcome for adult patients with ALL that may be worse or even inferior compared with studies using long-term maintenance therapy. High-dose cytarabine in combination with IT MTX was effective for CNS prophylaxis.

Erythroid defects and increased retrovirally-induced tumor formation in Evi1 transgenic mice

Aberrant expression of the Evi1 (ecotropic virus integration site 1) proto-oncogene has been associated with hematopoietic malignancies in both mice and man. To determine the effect of enforced expression of Evi1 in vivo, we developed a transgenic mouse model utilizing the murine Sca-1 (Ly-6E.1) promoter. Here, we describe the generation and analysis of three independent lines of Evi1 transgenic mice. Transgenic animals of two founder lines developed normally. These mice did not show any obvious hematological abnormalities but showed a significant reduction in the number of bone marrow colony-forming unit erythroid (CFU-E)-derived colonies. This implies a defect of normal erythroid hematopoiesis affecting relatively late erythroid progenitor cells. We also show that when newborn Evi1 transgenic mice of these two lines were infected with Cas-Br-M MuLV, tumor incidence was greatly enhanced in comparison with nontransgenic littermates, indicating an increased susceptibility for leukemia development. Interestingly, analysis of a third founder line revealed that all male progeny consistently displayed severely impaired erythropoiesis with major defects in the bone marrow, spleen and peripheral blood. Taken together, our results present the first evidence of Evi1 disturbing normal erythropoiesis in vivo and provides evidence for cooperative potential of Evi1 in tumor progression.

Induction of granulocytic maturation in acute myeloid leukemia by G-CSF and retinoic acid

AML cells were cultured free of serum with G-CSF in combination with all-trans-retinoic acid (RA), prostaglandin E2 or 8-bromocyclic AMP to see whether the maturation blockade of these cells could be overcome. The combination G-CSF + RA was most effective in inducing morphologic maturation, i.e. in 7/10 cases. Morphological alterations in response to G-CSF + RA indicated progression of the cells along the granulocytic pathway towards metamyelocytes and granulocytes. However, morphologically mature AML cells remained negative for myeloperoxidase and Sudan black stainings, indicators of granulocytic maturation. Chloracetate esterase positivity and CD15 membrane antigens became expressed on cultured AML cells, i.e. on unstimulated and G-CSF/RA exposed blasts. Ingestion of latex beads and reduction of nitroblue tetrazolium salt occurred in cultured AML cells regardless of the presence of inducers. In almost all cases clonogenic cells persisted after exposure to G-CSF + RA suggesting that subpopulations of immature cells escaped the action of these inducers. Thus although G-CSF + RA were capable of inducing maturation of AML cells along the granulocytic lineage, maturation was incomplete and the effect was evident in a subfraction of the cells only.

Primary human acute myeloblastic leukaemia : an analysis of in vitro granulocytic maturation following stimulation with retinoic acid and G-CSF

Acute myeloid leukaemia (AML) is characterized by the inability of myeloid cells to reach terminal maturation. We examined to what degree granulocytic maturation could be achieved by stimulating AML blast cells in an in vitro serum‐free system with a combination of granulocyte‐colony stimulating factor (G‐CSF) and all‐trans‐retinoic acid (RA). Specimens from 41 AML patients were cultured for 7 d and then examined for cytochemistry (myeloperoxidase, Sudan Black, napthyl‐ASD‐chloracetate esterase, periodic acid Schiff) and nitroblue tetrazolium reduction. The expression of CD11a, CD11b. CD11c, CD15, CD18, CD10, CD24 and B13‐3 membrane antigens was also evaluated. Morphological and cytochemical studies were also performed after AML colony culture and culture of normal bone marrow cells (NBM). The comparative analysis of the panel of parameters was indicative of granulocytic maturation although to different degrees.

Mutant Wilms' tumor 1 (WT1) mRNA with premature termination codons in acute myeloid leukemia (AML) is sensitive to nonsense-mediated RNA decay (NMD).

Mutant Wilms’ tumor 1 ( WT1 ) mRNA with premature termination codons in acute myeloid leukemia (AML) is sensitive to nonsense-mediated RNA decay (NMD)

Molecular remission of Philadelphia/bcr-abl-positive acute myeloid leukaemia after treatment with anti-CD33 calicheamicin conjugate (gemtuzumab ozogamicin, CMA-676)

The anti‐CD33 monoclonal antibody linked to NAc‐gamma calicheamicin gemtuzumab ozogamicin (CMA‐676) was used to treat a patient with Philadelphia/bcr‐abl‐positive acute myeloid leukaemia. We report a morphological and cytogenetic complete remission after treatment with two doses of gemtuzumab ozogamicin as a single agent. Using real‐time polymerase chain reaction (PCR), gemtuzumab ozogamicin treatment resulted in a 3‐log tumour mass reduction in bone marrow.

Acute lymphoblastic leukaemia in adults: immunological subtypes and clinical features at presentation

SummaryIn 91 of 106 adult patients with acute lymphoblastic leukemia (ALL) enrolled in the treatment protocol ALL HOVON-5 between May 1988 and October 1991, the immunophenotype of the leukemia was determined and correlated with clinical characteristics at presentation. The immunological marker analysis was performed in ten laboratories, all members of the Dutch Study Group on Immunophenotyping of Leukemias and Lymphomas (SIHON). Undifferentiated blasts were found in four patients, 67 had B-lineage ALL, 18 had T-lineage ALL, and two had biphenotypic ALL. The age of T-lineage ALL patients was lower (mean 29.3) than that of B-lineage ALL patients (mean 35.5). Tumor mass, as expressed by leukocyte count, organomegaly, and LDH, was more pronounced in T-lineage ALL. Hemoglobin and platelet count was similar in all (sub)types. CD34 was expressed in 58% of the leukemias, but most frequently in the common B-ALL (70%). Thirteen percent of the leukemias expressed one or more markers not associated with their lineage. In this prospective study immunological data were not evaluable for 15 patients. On four of them data were not available because of dry tap, for six patients the typing was technically insufficient, and for four patients the results were unclassifiable; with one patient the marker analysis was not performed.

Towards Detection of Minimal Disease: Discrimination of AML Precursors from Normal Myeloid Precursors using a Combination of Surface Markers

For monitoring of the effect of chemotherapy in acute myelocytic leukemia (AML), i.e., to assess the quality of a remission by quantitative criteria, and for the diagnosis of early relapse, the detection of small tumor masses is a necessity. Attempts at detecting minimal numbers of AML clonogenic cells (AML-CFU) in the marrow during complete remission can be pursued utilizing phenotypic properties which distinguish AML-CFU from normal marrow stem and progenitor cells. Colony culture assays and a variety of monoclonal antibodies (MCA) reacting with differentiation antigens expressed on the surface of myeloid cells may be used in investigations aimed at disclosing such differences.

Aberrant expression of miR-9/9{*}in myeloid progenitors inhibits neutrophil differentiation by post-transcriptional regulation of ERG

Aberrant post-transcriptional regulation by microRNAs (miRNAs) has been shown to be involved in the pathogenesis of acute myeloid leukemia (AML). In a previous study, we performed a large functional screen using a retroviral barcoded miRNA expression library. Here, we report that overexpression of miR-9/9* in myeloid 32D cell line (32D-miR-9/9*) had profound impact on granulocyte colony-stimulating factor-induced differentiation. Further in vitro studies showed that enforced expression of miR-9/9* blocked normal neutrophil development in 32D and in primary murine lineage-negative bone marrow cells. We examined the expression of miR-9/9* in a cohort of 647 primary human AMLs. In most cases, miR-9 and miR-9* were significantly upregulated and their expression levels varied according to AML subtype, with the highest expression in MLL-related leukemias harboring 11q23 abnormalities and the lowest expression in AML cases with t(8;21) and biallelic mutations in CEBPA. Gene expression profiling of AMLs with high expression of miR-9/9* and 32D-miR-9/9* identified ETS-related gene (Erg) as the only common potential target. Upregulation of ERG in 32D cells rescued miR-9/9*-induced block in neutrophil differentiation. Taken together, this study demonstrates that miR-9/9* are aberrantly expressed in most of AML cases and interfere with normal neutrophil differentiation by downregulation of ERG.