The CD34+ Cell Dose Matters in Hematopoietic Stem Cell Transplantation with Peripheral Blood Stem Cells from Sibling Donors.

The effect of CD34+ cell dose in allogeneic hematopoietic stem cell transplantation (HSCT) on overall survival (OS) and incidence of acute and chronic graft-versus-host disease (GvHD) has not been established and few studies have been performed. Our single center analysis included 189 patients with hematological malignancies who received peripheral blood stem cell (PBSC) grafts from sibling donors. Myeloablative conditioning was used in 88 cases and 101 received reduced intensity conditioning. The median CD34+ cell dose was 5.6 × 106/kg (0.6-17.0). In the multivariate analysis, a CD34 cell dose of 6-7 × 106/kg was associated with better OS and lower transplant-related mortality (TRM), while a dose of <5 × 106/kg led to increased relapse and reduced chronic GVHD (cGVHD). A high CD34 cell-dose (>6.5 × 106/kg) correlated with less acute GVHD (aGVHD) II-IV. We conclude that the CD34 cell dose has an impact on the outcome of HSCT from sibling donor PBSCs.

Impact of malignant stem cell burden on therapy outcome in newly diagnosed chronic myeloid leukemia patients.

Chronic myeloid leukemia (CML) stem cells appear resistant to tyrosine kinase inhibitors (TKIs) in vitro, but their impact and drug sensitivity in vivo has not been systematically assessed. We prospectively analyzed the proportion of Philadelphia chromosome-positive leukemic stem cells (LSCs, Ph+CD34+CD38-) and progenitor cells (LPCs, Ph+CD34+CD38+) from 46 newly diagnosed CML patients both at the diagnosis and during imatinib or dasatinib therapy (ClinicalTrials.gov NCT00852566). At diagnosis, the proportion of LSCs varied markedly (1-100%) between individual patients with a significantly lower median value as compared with LPCs (79% vs 96%, respectively, P=0.0001). The LSC burden correlated with leukocyte count, spleen size, hemoglobin and blast percentage. A low initial LSC percentage was associated with less therapy-related hematological toxicity and superior cytogenetic and molecular responses. After initiation of TKI therapy, the LPCs and LSCs rapidly decreased in both therapy groups, but at 3 months time point the median LPC level was significantly lower in dasatinib group compared with imatinib patients (0.05% vs 0.68%, P=0.032). These data detail for the first time the prognostic significance of the LSC burden at diagnosis and show that in contrast to in vitro data, TKI therapy rapidly eradicates the majority of LSCs in patients.

Ultra-short course sirolimus contributes to effective GVHD prophylaxis after reduced-intensity allogeneic hematopoietic cell transplantation.

Reduced-intensity conditioning (RIC) allo-SCT is a potentially curative treatment approach for patients with relapsed Hodgkin's or non-Hodgkin's lymphoma. In the present study, 37 patients underwent RIC allo-SCT after induction treatment with EPOCH-F(R) using a novel form of dual-agent immunosuppression for GVHD prophylaxis with CsA and sirolimus. With a median follow-up of 28 months among survivors, the probability for OS at 3 and 5 years was 56%. Treatment-related mortality was 16% at day +100 and 30% after 1 year of transplant. Acute GVHD grades II-IV developed in 38% of patients, suggesting that the regimen consisting of CsA and an ultra-short course of sirolimus is effective in the prevention of acute GVHD.

Clonal expansion of T/NK-cells during tyrosine kinase inhibitor dasatinib therapy.

Dasatinib, a broad-spectrum tyrosine kinase inhibitor (TKI), predominantly targets BCR-ABL and SRC oncoproteins and also inhibits off-target kinases, which may result in unexpected drug responses. We identified 22 patients with marked lymphoproliferation in blood while on dasatinib therapy. Clonality and immunophenotype were analyzed and related clinical information was collected. An abrupt lymphocytosis (peak count range 4-20 x 10(9)/l) with large granular lymphocyte (LGL) morphology was observed after a median of 3 months from the start of therapy and it persisted throughout the therapy. Fifteen patients had a cytotoxic T-cell and seven patients had an NK-cell phenotype. All T-cell expansions were clonal. Adverse effects, such as colitis and pleuritis, were common (18 of 22 patients) and were preceded by LGL lymphocytosis. Accumulation of identical cytotoxic T cells was also detected in pleural effusion and colon biopsy samples. Responses to dasatinib were good and included complete, unexpectedly long-lasting remissions in patients with advanced leukemia. In a phase II clinical study on 46 Philadelphia chromosome-positive acute lymphoblastic leukemia, patients with lymphocytosis had superior survival compared with patients without lymphocytosis. By inhibiting immunoregulatory kinases, dasatinib may induce a reversible state of aberrant immune reactivity associated with good clinical responses and a distinct adverse effect profile.

No benefit from adding GM-CSF to induction chemotherapy in transforming myelodysplastic syndromes: better outcome in patients with less proliferative disease.

In this prospective randomized multicenter trial 93 patients, median age 72 years, with RAEB-t (n=25) and myelodysplastic syndrome (MDS)-AML (n=68) were allocated to a standard induction chemotherapy regimen (TAD 2+7) with or without addition of granulocyte-macrophage-CSF (GM-CSF). The overall complete remission (CR) rate was 43% with no difference between the arms. Median survival times for all patients, CR patients, and non-CR patients were 280, 550, and 100 days, respectively, with no difference between the arms. Response rates were significantly better in patients with serum lactate dehydrogenase (S-LDH) levels

Self-renewal of multipotent long-term repopulating hematopoietic stem cells is negatively regulated by Fas and tumor necrosis factor receptor activation.

Multipotent self-renewing hematopoietic stem cells (HSCs) are responsible for reconstitution of all blood cell lineages. Whereas growth stimulatory cytokines have been demonstrated to promote HSC self-renewal, the potential role of negative regulators remains elusive. Receptors for tumor necrosis factor (TNF) and Fas ligand have been implicated as regulators of steady-state hematopoiesis, and if overexpressed mediate bone marrow failure. However, it has been proposed that hematopoietic progenitors rather than stem cells might be targeted by Fas activation. Here, murine Lin(-)Sca1(+)c-kit(+) stem cells revealed little or no constitutive expression of Fas and failed to respond to an agonistic anti-Fas antibody. However, if induced to undergo self-renewal in the presence of TNF-alpha, the entire short and long-term repopulating HSC pool acquired Fas expression at high levels and concomitant activation of Fas suppressed in vitro growth of Lin(-)Sca1(+)c-kit(+) cells cultured at the single cell level. Moreover, Lin(-)Sca1(+)c-kit(+) stem cells undergoing self-renewal divisions in vitro were severely and irreversibly compromised in their short- and long-term multilineage reconstituting ability if activated by TNF-alpha or through Fas, providing the first evidence for negative regulators of HSC self-renewal.

Tumor necrosis factor (TNF)-mediated activation of the p55 TNF receptor negatively regulates maintenance of cycling reconstituting human hematopoietic stem cells.

Hematopoietic stem cell (HSC) fate decisions between self-renewal and commitment toward differentiation are tightly regulated in vivo. Recent developments in HSC culture and improvements of human HSC assays have facilitated studies of these processes in vitro. Through such studies stimulatory cytokines critically involved in HSC maintenance in vivo have been demonstrated to also promote HSC self-renewing divisions in vitro. Evidence for negative regulators of HSC self-renewal is, however, lacking. Tumor necrosis factor (TNF), if overexpressed, has been implicated to mediate bone marrow suppression. However, whether and how TNF might affect the function of HSC with a combined myeloid and lymphoid reconstitution potential has not been investigated. In the present studies in vitro conditions recently demonstrated to promote HSC self-renewing divisions in vitro were used to study the effect of TNF on human HSCs capable of reconstituting myelopoiesis and lymphopoiesis in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. Although all cord blood and adult bone marrow CD34(+)CD38(-) cells were capable of undergoing cell divisions in the presence of TNF, cycling HSCs exposed to TNF in vitro and in vivo were severely compromised in their ability to reconstitute NOD-SCID mice and long-term cultures. The negative effect of TNF was not dependent on the Fas pathway, and a similar effect could be observed using a mutant TNF exclusively targeting the p55 TNF receptor. TNF did not appear to enhance apoptosis or affect cell-cycle distribution of cultured progenitors, but rather promoted myeloid differentiation. Thus, TNF might regulate HSC fate by promoting their differentiation rather than self-renewal.

Treatment of anemia in myelodysplastic syndromes with granulocyte colony-stimulating factor plus erythropoietin: results from a randomized phase II study and long-term follow-up of 71 patients.

Treatment with erythropoietin (epo) may improve the anemia of myelodysplastic syndromes (MDS) in approximately 20% of patients. Previous studies have suggested that treatment with the combination of granulocyte colony-stimulating factor (G-CSF) and epo may increase this response rate. In the present phase II study, patients with MDS and anemia were randomized to treatment with G-CSF + epo according to one of two alternatives; arm A starting with G-CSF for 4 weeks followed by the combination for 12 weeks, and arm B starting with epo for 8 weeks followed by the combination for 10 weeks. Fifty evaluable patients (10 refractory anemia [RA], 13 refractory anemia with ring sideroblasts [RARS], and 27 refractory anemia with excess blasts [RAEB]) were included in the study, three were evaluable only for epo as monotherapy and 47 for the combined treatment. The overall response rate to G-CSF + epo was 38%, which is identical to that in our previous study. The response rates for patients with RA, RARS, and RAEB were 20%, 46%, and 37%, respectively. Response rates were identical in the two treatment groups indicating that an initial treatment with G-CSF was not neccessary for a response to the combination. Nine patients in arm B showed a response to the combined treatment, but only three of these responded to epo alone. This suggests a synergistic effect in vivo by G-CSF + epo. A long-term follow-up was made on 71 evaluable patients from both the present and the preceding Scandinavian study on G-CSF + epo. Median survival was 26 months, and the overall risk of leukemic transformation during a median follow-up of 43 months was 28%. Twenty patients entered long-term maintenance treatment and showed a median duration of response of 24 months. The international prognostic scoring system (IPSS) was effective to predict survival, leukemic transformation, and to a lesser extent, duration of response, but had no impact on primary response rates.

Transforming growth factor-beta1 abrogates Fas-induced growth suppression and apoptosis of murine bone marrow progenitor cells.

Fas, a member of the tumor necrosis factor (TNF ) receptor superfamily is a critical downregulator of cellular immune responses. Proinflammatory cytokines like interferon-gamma (IFN-gamma) and TNF-alpha can induce Fas expression and render hematopoietic progenitor cells susceptible to Fas-induced growth suppression and apoptosis. Transforming growth factor-beta1 (TGF-beta1 ) is an essential anti-inflammatory cytokine, thought to play a key role in regulating hematopoiesis. In the present studies we investigated whether TGF-beta1 might regulate growth suppression and apoptosis of murine hematopoietic progenitor cells signaled through Fas. In the presence of TNF, activation of Fas almost completely blocked clonogenic growth of lineage-depleted (Lin-) bone marrow (BM) progenitor cells in response to granulocyte-macrophage colony-stimulating factor (GM-CSF ), CSF-1, or a combination of multiple cytokines. Whereas TGF-beta1 alone had no effect or stimulated growth in response to these cytokines, it abrogated Fas-induced growth suppression. Single-cell studies and delayed addition of TGF-beta1 showed that the ability of TGF-beta1 to inhibit Fas-induced growth suppression was directly mediated on the progenitor cells and not indirect through potentially contaminating accessory cells. Furthermore, TGF-beta1 blocked Fas-induced apoptosis of Lin- BM cells, but did not affect Fas-induced apoptosis of thymocytes. TGF-beta1 also downregulated the expression of Fas on Lin- BM cells. Thus, TGF-beta1 potently and directly inhibits activation-dependent and Fas-mediated growth suppression and apoptosis of murine BM progenitor cells, an effect that appears to be distinct from its ability to induce progenitor cell-cycle arrest. Consequently, TGF-beta1 might act to protect hematopoietic progenitor cells from enhanced Fas expression and function associated with proinflammatory responses.

The RAR-RXR as well as the RXR-RXR pathway is involved in signaling growth inhibition of human CD34+ erythroid progenitor cells.

Previous studies have shown that retinoic acid (RA), similar to tumor necrosis factor-alpha (TNF-alpha), can act as a bifunctional regulator of the growth of bone marrow progenitors, in that it can stimulate granulocyte-macrophage colony-stimulating factor (GM-CSF)- or interleukin-3 (IL-3)-induced GM colony formation, but potently inhibit G-CSF-induced growth. The present study, using highly enriched human CD34+ as well as Lin- murine bone marrow progenitor cells, demonstrates a potent inhibitory effect of 9-cis-RA on burst-forming unit-erythroid (BFU-E) colony formation regardless of the cytokine stimulating growth. Specifically, 9-cis-RA potently inhibited the growth of BFU-E response to erythropoietin (Epo) (100%), stem cell factor (SCF) + Epo (92%), IL-3 + Epo (97%), IL-4 + Epo (88%), and IL-9 + Epo (100%). Erythroid colony growth was also inhibited when CD34+ progenitors were seeded at one cell per well, suggesting a direct action of RA. Using synthetic ligands to retinoic acid receptors (RARs) and retinoid X receptors (RXRs) that selectively bind and activate RAR-RXR or RXR-RXR dimers, respectively, we dissected the involvement of the two retinoid response pathways in the regulation of normal myeloid and erythroid progenitor cell growth. Transactivation studies showed that both the RAR (Ro 13-7410) and RXR (Ro 25-6603 and Ro 25-7386) ligands were highly selective at 100 nmol/L. At this concentration, Ro 13-7410 potently inhibited G-CSF-stimulated myeloid as well as SCF + Epo-induced erythroid colony growth. At the same concentration, Ro 25-6603 and Ro 25-7386 had little or no effect on G-CSF-induced colony formation, whereas they inhibited 75% and 53%, respectively, of SCF + Epo-stimulated BFU-E colony growth. Thus, the RAR-RXR response pathway can signal growth inhibition of normal bone marrow myeloid and erythroid progenitor cells. In addition, we demonstrate a unique involvement of the RXR-RXR pathway in mediating growth inhibition of erythroid but not myeloid progenitor cells.

Transforming growth factor beta (TGF-beta), a potent inhibitor of erythropoiesis: neutralizing TGF-beta antibodies show erythropoietin as a potent stimulator of murine burst-forming unit erythroid colony formation in the absence of a burst-promoting activity.

Transforming growth factor beta (TGF-beta) is a bifunctional regulator of the growth of myeloid progenitors and is here demonstrated to directly inhibit the growth of primitive erythroid progenitors by 95% to 100% regardless of the cytokines stimulating growth. Autocrine TGF-beta production of primitive hematopoietic progenitors has previously been reported. In the present study, a neutralizing TGF-beta antibody (anti-TGF-beta) added to serum-containing cultures, resulted in a 3-, 4-, and 25-fold increase in burst-forming unit erythroid (BFU-E) colony formation in response to interleukin-4 (IL-4) plus erythropoietin (Epo), SCF plus Epo, and IL-11 plus Epo, respectively. The growth of BFU-E progenitors has been suggested to require a burst-promoting activity in addition to Epo. Accordingly, we observed no BFU-E colony formation in serum-containing cultures in response to Epo alone. In contrast, 50 BFU-E colonies were formed when anti-TGF-beta was included in the culture. In serum-free cultures, Epo also stimulated BFU-E colony formation in the absence of other cytokines, whereas anti-TGF-beta had no effect on the number of colonies formed. Quantitation of TGF-beta 1 in serum by an enzyme-linked immunosorbent assay method showed predominantly the presence of precursor (latent) TGF-beta 1, but also showed active TGF-beta 1 at a concentration sufficient to potently inhibit erythroid colony formation. Thus, neutralization of active TGF-beta 1 in serum shows that Epo alone is sufficient to stimulate the growth of murine BFU-E progenitors.

IL-12 directly enhances in vitro murine erythropoiesis in combination with IL-4 and stem cell factor.

It has been demonstrated recently that in vivo administration of murine IL-12 to mice enhances the activity of cytotoxic NK cells and lymphocyte-activated killer cells, and that it has antitumor and antimetastatic activity. However, one side effect observed in response to systemic IL-12 treatment is anemia. In the present study, we examined for the first time the ability of IL-12 to affect directly the growth of murine erythroid progenitor cells in vitro. Whereas IL-12 alone or in combination with Erythropoietin (Epo) showed no stimulatory effect on erythroid progenitors, IL-12 potently enhanced the number of erythroid burst-forming unit (BFU-E) colonies formed in response to Epo+IL-4 by 63% and Epo+stem cell factor by 80%. The stimulatory effect of IL-12 occurred in a concentration-dependent fashion, with maximum enhancing effect observed at 50 ng/ml. Furthermore, single cell experiments suggested that the stimulatory effect of IL-12 on erythroid colony formation was directly mediated. Thus, IL-12 can directly enhance murine erythropoiesis in vitro, suggesting that IL-12-induced anemia is mediated through an indirect mechanism.

Acute leukemia in middle Norway 1984-88.

One hundred and eight adult patients with acute leukemia were diagnosed in the middle Norwegian health region during the 5-year period 1984-88, giving an incidence rate of 4.6/100,000 per year. Nine patients had acute lymphoblastic leukemia (ALL), 93 acute myeloid leukemia (AML) and 6 patients acute leukemia without definite sub-classification. The median age of AML patients was 66 years. Thirty-five patients (median age 78 years) were found non-suitable for cytotoxic drugs, while 58 AML patients (median age 57 years) were given aplasia-inducing drug combinations according to one of three treatment programs depending on the time of diagnosis and age, in order to induce remission. Six patients were given oral drugs or low dose ara-C. All patients were followed until death or for an observation time of more than 5 years (median 7 years). The overall long term survival was found to be 12/108 for all acute leukemias, 8/93 for AML patients and 4/9 for ALL patients. For the AML patients given intravenous aplasia-inducing drugs the remission rate was 0.65, the median remission duration 12.2 months and the 5-year survival rate 0.19. For 31 AML patients, (median age 41 years), started on an intensive chemotherapy program, the 5-year survival rate was 0.32 and the relapse-free 5-year survival rate for the 22 patients entering complete remission was also 0.32.

A combination of granulocyte colony-stimulating factor and erythropoietin may synergistically improve the anaemia in patients with myelodysplastic syndromes.

In an attempt to obtain a synergistic effect on the hemoglobin levels in anaemic patients with myelodysplastic syndromes (MDS), granulocyte colony-stimulating factor (G-CSF) and erythropoietin (epo) were combined in a clinical phase II trial. Twenty-two patients with MDS were included in the study. G-CSF was given alone for six weeks and then in combination with epo for the following twelve weeks. Eight (38%) of 21 evaluable patients showed a significant increase in hemoglobin. One patient with a previous response and subsequent failure to epo alone improved after the addition of G-CSF. Responses were more frequent in patients with less advanced pancytopenia, lower endogenous levels of serum-epo and in those with ring sideroblasts in the bone marrow. The response frequency of 38% is higher than in any study of epo as monotherapy. Moreover, patients with ring sideroblasts, who respond poorly to epo alone, showed a response rate of 60%. Our findings suggest a synergistic in vivo effect of granulocyte-CSF and erythropoietin in patients with myelodysplastic syndromes.

[Streptococcus viridans and respiratory failure in acute leukemia]. / Streptococcus viridans og respirasjonssvikt ved akutt leukemi.

Through the last 18 months, five of our patients with acute leukaemia have developed septicaemia caused by Streptococcus viridans, followed by acute respiratory failure. Two patients had to be placed in a respirator. In patients with acute leukaemia treated with cytostatic drugs, close clinical observation, including repeated blood gas analysis, is very important if they develop septicaemia caused by Streptococcus viridans. Early administration of high doses of corticosteroids seems to be important in order to prevent serious respiratory failure.