Limited clinical efficacy of azacitidine in transfusion-dependent, growth factor-resistant, low- and Int-1-risk MDS: Results from the nordic NMDSG08A phase II trial.

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.

Long-term culture of hematopoietic stem cells - validating the stromal component of the CAFC assay.

BACKGROUND: The stroma-based long-term culture is the assay of choice when a functional detection of primitive hematopoietic cells in vitro is sought. However, different stromal cell lines varying in supporting capacity have been raised and applied in different laboratories, resulting in a wide range in published frequencies of LTCIC alternative CAFC. METHODS: In order to identify the most suitable stromal source in terms of supportive capacity, reproducibility, and ease of handling, we have compared some of the most commonly employed murine cell lines to human bone marrow stroma in secondary long-term culture set-ups. RESULTS: Seeking an approximation to the supportive capacity of human BM stroma we found the FBMD-1 cell line supplemented with G-CSF and IL-3 superior to FBMD-1 cells alone, and to M2-10B4 and Sl/Sl cells. Moreover, in co-cultures of CD34(+) cells and the FBMD-1 line, we found week 5 CAFC content highly reproducible (50.5 +/- 6.66 - 54.6 +/- 7.07/10(4) plated cells, p value > 0.95) and the assay was suitable for inter-individual comparison in a clinical setting. In fact, the week 5 CAFC results were even more reproducible than those of the CFU assays (CV 0.03 for the CAFC assay versus 0.13-0.33 for the CFU assays). On the other hand, when extending the culture period to 8 weeks, the cobblestone area formation was best maintained by human BM stroma and the high reproducibility in CAFC enumeration in cultures supported by the FBMD-1 was lost. DISCUSSION: Among the stromal cell sources tested, the FBMD-1 line was found to be superior in terms of ease of handling and week 5 CAFC reproducibility. However, this robustness could not be extended to week 8 CAFC.

Kinetics of BCR-ABL fusion transcript levels in chronic myeloid leukemia patients treated with STI571 measured by quantitative real-time polymerase chain reaction.

The activated tyrosine kinase, which arises as a result of the balanced t(9,22) translocation in chronic myeloid leukemia (CML), is thought to be essential for the development of the leukemic phenotype. Recently, designer drugs have been introduced which specifically inhibit such specific kinases. Among these, STI571 (Glivec) has entered clinical trials and shown promising activities in chronic phase (CP), accelerated phase (AP) and blast crisis (BC) as evidenced by significant hematological and cytogenetic responses in CML patients. To evaluate the effect of STI571 at the molecular level we have employed quantitative real-time PCR (RQ-PCR) to measure the amount of BCR-ABL fusion transcript in a series of 19 patients treated with STI571, either in CP(11) or in (AP)(8) of the disease for 3--9 months (median 6 months). Employing this method, which is able to detect at least one BCR-ABL+ cell in 500,000, in serial blood and bone marrow specimens we found decreases in transcript levels in 10/11 CP patients, but only in 1/8 of the AP patients. When present such decreases were gradual and became evident only after 3 months of STI571 treatment, and their kinetics in blood closely mirrored those seen in parallel marrow samples. Moreover, decreases were between 10- and 100-fold in 11/13 patients, with only two patients reaching residual disease levels below 10(-2) (a 900-fold decrease). Thus, no patient reached PCR negativity. We conclude that the RQ-PCR method is a highly suitable tool for following the effect of STI571 in CML and that further validation of the method, performed in a prospective manner, will contribute significantly to the elucidation of the proper role of STI571 in CML.

[Hematopoietic stem cells I. Basal aspects]. / Haematopoietiske stamceller I. Basale aspekter.

The knowledge on basic aspects of hematopoietic stem cells (HSC) has markedly increased during the last decade to the extent that such cells are now routinely employed therapeutically in order to restore hematopoiesis following myeloablative chemotherapy and irradiation in patients with malignant disorders. Different methods can be applied to characterize HSC. Thus, by immunophenotyping and flow cytometry it is possible to delineate subpopulations of cells enriched for HSC. However, since phenotypic characteristics associated with HSC do not necessarily correlate to their developmental potential, measurements of HSC also rely heavily upon functional in vitro and in vivo assays, which demonstrate the presence of HSC through their ability to proliferate and differentiate in a clonogenic fashion. In this review we describe the assays developed to characterize and quantitate immature HSC, which form the basis for their clinical application.

[Hematopoietic stem cells II. Diagnostic and therapeutic aspects]. / Haematopoietiske stamceller II. Diagnostiske og terapeutiske aspekter.

Characterization and isolation of haematopoietic stem cells (HSC) have resulted in their clinical application in patients with malignant disorders and--through gene therapeutic initiatives--also in the treatment of inherited diseases. Autologous stem cell transplantation (ASCT), which was introduced because of the high number of relapses in cancer patients in remission, involves dose-intensification (conditioning), which induces myeloablation. In this setting, reinfusion of HSC is performed to restore haematopoiesis. Flow cytometric determination of CD34+ cells and clonogenic assays for committed myeloid HSC (CFU-GM) are vehicles for quality control of the harvested HSC material and are integrated into the ASCT programs. Moreover, harvest of HSC and purification of CD34+ cells enables new treatment options such as removal of cancer cells from grafts, optimization of gene transduction as well as ex vivo expansion of HSC before reinfusion. In conclusion, the expanding insights into HSC in the 1990's have already been translated into valuable diagnostic and therapeutic modalities.

Natural effector cells in patients with acute myeloid leukemia treated with the immunomodulator Linomide after autologous bone marrow transplantation.

Roquinimex, Linomide, is a quinoline derivative with pleiotropic immunomodulatory activities which has been shown to enhance NK function. As part of a phase III placebo-controlled multicenter study patients were randomized to receive Roquinimex, 0.2 mg/kg body weight, or a placebo twice weekly for a duration of 2 yr following autologous bone marrow transplantation for acute myeloid leukemia in remission. At Arhus University Hospital 7 patients were randomized to receive the active drug and 6 to receive the placebo. Surviving patients were followed for 2 yr with immunological monitoring of their natural immune effector cells (NK- and LAK cell activity). Peripheral heparinized blood samples were obtained twice before the onset of conditioning therapy and at several time points after ABMT, and whole blood samples were analyzed by flow cytometry for the detection of leukocyte differentiation antigens as well as by 4 h 51Cr release assays for cytotoxicity. In contrast to previous experience with Linomide, in the present study we found that at 36 wk or later time points Linomide patients exhibited a significant suppression of circulating natural effector cell number and activity when compared with the control group. These observations underline the need for further exploration into novel and manageable immunostimulators.

Modulation of T lymphocyte function by the angiogenesis inhibitor AGM-1470.

The angiogenesis inhibitor AGM-1470 has recently been reported to inhibit collagen-induced arthritis in rats. To determine if the anti-arthritic effects of AGM-1470 might be due to T cell inhibition, we have studied its effects on T cell responses in vitro. Responses of human cells to tetanus toxoid (TT), and those of murine splenocytes to staphylococcal enterotoxin (SE), mitogens or a mls difference were inhibited by AGM-1470. Responses of human cells to SE, OKT3 and PHA were all partially inhibited on day 2 (d2) but not d3, and in fact were augmented on d6-8. The amount of IL-2 in SEA cultures was augmented on d4 and d5. There were no differences in the expression of CD3, CD4, CD8, CD25, CD45RA, CD45RO, LFA-1, VLA-4 or VLA-6 in inhibited cultures, except for slight decreases in CD25 and CD45RO in TT cultures. These results indicated that the angiogenesis inhibitor AGM-1470 also modulates human and murine lymphocyte function.

1,4-Dihydronaphthoquinones as water-soluble inhibitors of 5-lipoxygenase.

The acetate derivatives of 1,4-dihydronaphthoquinones showed significant inhibition of 5-lipoxygenase. Among them, 1-acetyl-2-n-butyl-4-methoxy-naphthalene and 1-acetyl-2, 3-diethyl- 4- methoxy-naphthalene were found to be the best inhibitors. A series of HCl salts of the amino acid esters and other derivatives of the two parent molecules, 1-hydroxy-2-n-butyl-4-methoxy-naphthalene and 1-hydroxy-2, 3-diethyl-4-methoxynaphthalene, were synthesized as water-soluble potential inhibitors of 5-lipoxygenase to improve the formulation characteristics of this class of compounds. The derivatives were evaluated for leukotriene (LT) C4/D4 and LTB4 inhibitory activity. The HCl salts of the L-valine esters from the two parent molecules exhibited the best potency for inhibition of LTC4/D4 (IC50 0.11-0.90 microM) in ionophore A23187-stimulated rat mononuclear cells and of LTB4 in A23187-stimulated rat blood (55.5-79.2% inhibition) following a single oral dose of 50 mg/kg.

1,4-Dihydronaphthoquinones, hydroindoloquinones, benzofurans, and benzothiophenes as inhibitors of 5-lipoxygenase. Synthesis and structure-activity studies.

A series of substituted 1,4-dihydronaphthoquinones, hydroindoloquinones, benzofuran-4,7-dihydroquinones, and benzothiophene-4,7-dihydroquinones were synthesized and evaluated for inhibitory activity against 5-lipoxygenase. These compounds were found to be active in vitro for LTC4/D4 inhibition with the potencies (IC50's) ranging from 0.2 to 85 microM. Active 1,4-dihydronaphthoquinone acetates (IC50 less than 20 microM) were evaluated in an ex vivo LTB4 inhibition assay. The acetates of 1,4-dihydronaphthoquinones containing the alkyl substituent(s) (2-n-butyl, 11, and 2,3-diethyl, 15) exhibited the best activity in LTC4/D4 inhibition (IC50 = 0.2-0.4 microM, in vitro) as well as in LTB4 inhibition (60-75% inhibition).

Ethylnitrosourea treatment increases lectin binding to mouse germ cells.

Germ cell toxicity was assessed by investigating the binding of FITC-labeled lectins to mouse testis cells before and 18 days after treatment with ethylnitrosourea (ENU). Flow cytometry of testis cells dual-labeled with FITC-lectin plus the DNA stain, propidium iodide, allowed analysis of haploid (1C), diploid (2C), and dividing (4C) cell populations. Soybean agglutinin, wheat germ agglutinin, concanavalin A and Limax flavus agglutinin bound to normal mouse testis cells containing 1C, 2C or 4C DNA. Asparagus pea lectin and Bandeireae simplicifolia I isolectin B4 did not. ENU treatment reduced the number of testis cells and increased lectin binding, particularly of those lectins which bound to untreated cells.

Effect of trypsinization on lectin binding to germ cells from ICR and T/t6 mice.

Flow cytometry was used to quantify the binding of fluorescein isothiocyanate (FITC)-labeled lectins to testis cells from ICR and T/t6 mice before and after trypsin treatment. Soybean agglutinin, wheat germ agglutinin, and concanavalin A bound well to testis cells of both mouse strains. Limax flavus agglutinin (LFA) bound very slightly and Ulex europeas agglutinin (UEA) did not bind at all. Trypsinization increased binding of soybean agglutinin and decreased binding of wheat germ agglutinin in both mouse strains, providing evidence for masked carbohydrate-binding sites on the surface of germ cells. It did not affect binding of the other lectins. Trypsin treatment was an attempt to increase lectin binding, particularly the binding of LFA and UEA to the reported T/t-specific carbohydrates, sialic acid, and L-fucose, respectively. These studies indicate that the T/t6 locus alleles do not alter the surface carbohydrate content of testis cells sufficiently to be detected by lectin-binding differences.