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The spontaneous formation of colony-forming units granulocyte/macrophage (CFU-GM) in semisolid cultures has been shown to be due to the endogenous release of cytokines and/or to the hypersensitivity of cells against growth factors. We have reported that increased autonomous CFU-GM growth is an in vitro characteristic of myelofibrosis (MF) which may reflect aberrant hematopoiesis in vivo. Because of its cytokine synthesis-inhibiting action, we speculated that interleukin-10 (IL-10) may inhibit pathological overproduction of myeloid cells in MF by suppression of autonomous myelopoiesis. In this study, IL-10 significantly inhibited autonomous CFU-GM formation in vitro from peripheral blood mononuclear cells (PB MNC) in 10 of 11 patients with MF tested. In all patients, there was a mean inhibition of 69% ranging from 35% to 100%. Suppression of autonomous CFU-GM formation by IL-10 was dose dependent and reversible by the addition of anti-IL-10 antibodies. Our results indicate that IL-10 is a potentially useful molecule to affect aberrant myelopoiesis in patients with MF.

A total of 20 patients with cold antibody hemolytic anemia were evaluated in a retrospective study of them, 15 had a monoclonal gammopathy of unknown significance (MGUS): 14 with MGUS of immunoglobulin M (IgM) subtype and 1 with immunoglobulin G subtype. One patient had smoldering Waldenström's macroglobulinemia, but four patients had no monoclonal protein and no evidence of lymphoma. However, in three of these patients, we were able to demonstrate a (mono-)clonal rearrangement of their immunoglobulin heavy and/or light chains. Of the 20 patients, 5 had IgHV34 nucleotide sequence indicating that the antibody was directed against the "I" antigen. Two patients exhibited a progressive increase of IgM over time, however without increasing hemolytic activity. Moreover, in two patients with long-term follow-up, we were able to correlate recurrent hemolytic activity with low environmental temperatures. Among four patients treated with rituximab, all four responded to treatment. However, treatment effect was only transient in all of them.

Autoimmune hemolytic anemia is a heterogeneous disease with respect to the type of the antibody involved and the absence or presence of an underlying condition. Treatment decisions should be based on careful diagnostic evaluation. Primary warm antibody autoimmune hemolytic anemias respond well to steroids, but most patients remain steroid-dependent, and many require second-line treatment. Currently, splenectomy can be regarded as the most effective and best-evaluated second-line therapy, but there are still only limited data on long-term efficacy and adverse effects. The monoclonal anti-CD20 antibody rituximab is another second-line therapy with documented short-term efficacy, but there is limited information on long-term efficacy and side effects. The efficacy of immunosuppressants is poorly evaluated. Primary cold antibody autoimmune hemolytic anemias respond well to rituximab but are resistant to steroids and splenectomy. The most common causes of secondary autoimmune hemolytic anemias are malignancies, immune diseases, or drugs. They may be treated in a way similar to primary autoimmune hemolytic anemias, by immunosuppressants or by treatment of the underlying disease.

Autoimmune cytopenias are typical paraneoplastic phenomena in malignant lymphomas. We assessed the occurrence, clinical, and laboratory features as well as the response to treatment of autoimmune hemolytic anemia, autoimmune thrombocytopenia, and neutropenia in published cases of Hodgkin lymphomas (HL). We identified 34 cases of an association between HL and autoimmune hemolytic anemia and 48 cases of an association of autoimmune thrombocytopenia (AITP) and HL. The autoimmune cytopenias (AIC) may occur prior to, concurrent with, at the time of recurrence of lymphoma or in complete remission after treatment. Almost all autoimmune hemolytic anemias were caused by warm antibodies. Patients with HL with AIC are more commonly males and are more likely to have mixed cellularity (MC) HL. HL is the only lymphoma which may be associated with autoimmune neutropenia. In contrast to AIC in diffuse large B-cell lymphoma (DLBCL), early stage is less common in patients with HL with AIC. AIC in HL respond better to antilymphoma treatment than to steroids, with the exception of post-treatment AITP in CR of HL which responds easily and durably to steroids.

12 patients with severe chronic idiopathic thrombocytopenic purpura (ITP) were treated with i.v. polyvalent intact immunoglobulin (0.14-0.4 g/kg body weight for 5 d) because of various bleeding episodes or prior to surgery. In 9 patients a significant rise in platelet counts was noted, starting on d 2 and reaching its maximum between d 4 and 11. Thereafter, platelet counts decreased slowly, approaching pre-treatment values after 20 d in most cases. Response to immunoglobulin was reproducible, when infusions were repeated. Response to high-dose immunoglobulin was independent of age of patients and duration of disease. Platelet-associated IgG decreased in all patients tested. A pepsin-treated immunoglobulin preparation was ineffective in 3 patients.

Diagnosis and management of immune thrombocytopenic purpura (ITP) remain largely dependent on clinical expertise and observations more than on evidence derived from clinical trials of high scientific quality. One major obstacle to the implementation of such studies and in producing reliable meta-analyses of existing data is a lack of consensus on standardized critical definitions, outcome criteria, and terminology. Moreover, the demand for comparative clinical trials has dramatically increased since the introduction of new classes of therapeutic agents, such as thrombopoietin receptor agonists, and innovative treatment modalities, such as anti-CD 20 antibodies. To overcome the present heterogeneity, an International Working Group of recognized expert clinicians convened a 2-day structured meeting (the Vicenza Consensus Conference) to define standard terminology and definitions for primary ITP and its different phases and criteria for the grading of severity, and clinically meaningful outcomes and response. These consensus criteria and definitions could be used by investigational clinical trials or cohort studies. Adoption of these recommendations would serve to improve communication among investigators, to enhance comparability among clinical trials, to facilitate meta-analyses and development of therapeutic guidelines, and to provide a standardized framework for regulatory agencies.

Autoimmune haemolytic anaemia (AIHA) is an immune disorder caused by antibodies directed against unmodified autologous red cells. The disorder may be a primary (idiopathic) or a secondary disease. The diagnosis is based on the presence of anaemia, signs of haemolysis with reticulocytosis, low haptoglobin, increased lactate dehydrogenase, elevated indirect bilirubin, and a positive direct antiglobulin test (Coombs test). Sometimes, not all of these typical features are present. Most AIHA are caused by warm antibodies, whereas cold antibodies are less commonly detected. While half of the warm antibody-based AIHA are idiopathic anaemias, almost all cold antibody AIHA are secondary anaemias. Underlying diseases are Non Hodgkin's lymphomas and systemic autoimmune disorders, and less frequently organ transplantation, infections, or solid tumors. Moreover, AIHA is an important complication of treatment with nucleoside analogs. Most patients with AIHA require therapy. In warm antibody AIHA, standard first line therapy are glucocorticosteroids with or without high dose immunoglobulins, whereas splenectomy is considered second-line therapy. Response rates of primary AIHA to corticosteroid therapy are high. After initial remission, the dose should be tapered down slowly and with caution, and in some cases, low-dose maintenance therapy is required. The efficacy of standard therapy is low in secondary AIHA that develops in lymphoma patients, posttransplant patients, or tumor patients. Among other immunosuppressive treatments, rituximab (anti-CD20) appears to be highly effective in patients with warm antibody AIHA refractory to standard therapy. Mycophenolate mofetil is quite effective in AIHA patients with an underlying autoimmune or lymphoproliferative disease. Patients with cold agglutinins are refractory to steroids and splenectomy. Half of these patients may respond to rituximab, although responses usually are short-lived. Sometimes, AIHA that is associated with malignant lymphomas or tumors, disappears after successful anti-lymphoma or anti-tumor therapy.

Early allogeneic hematopoietic stem cell transplantation (HSCT) has been proposed as primary treatment modality for patients with chronic myeloid leukemia (CML). This concept has been challenged by transplantation mortality and improved drug therapy. In a randomized study, primary HSCT and best available drug treatment (IFN based) were compared in newly diagnosed chronic phase CML patients. Assignment to treatment strategy was by genetic randomization according to availability of a matched related donor. Evaluation followed the intention-to-treat principle. Six hundred and twenty one patients with chronic phase CML were stratified for eligibility for HSCT. Three hundred and fifty four patients (62% male; median age, 40 years; range, 11-59 years) were eligible and randomized. One hundred and thirty five patients (38%) had a matched related donor, of whom 123 (91%) received a transplant within a median of 10 months (range, 2-106 months) from diagnosis. Two hundred and nineteen patients (62%) had no related donor and received best available drug treatment. With an observation time up to 11.2 years (median, 8.9 years), survival was superior for patients with drug treatment (P = .049), superiority being most pronounced in low-risk patients (P = .032). The general recommendation of HSCT as first-line treatment option in chronic phase CML can no longer be maintained. It should be replaced by a trial with modern drug treatment first.

We report on four cases (three women, one man, age at diagnosis 26-61 years) with severe autoimmune thrombocytopenia (AITP) who were refractory to initial steroid therapy (n = 4), to subsequent splenectomy (n = 2), azathioprine (n = 1), and cyclosporine (n = 1). Over years they received low-dose continuous or intermittent steroid therapy. After 6 to 31 years these patients achieved a "spontaneous" complete remission (CR) (n = 3) or partial remission (PR) (n = 1) unrelated to any specific second or third line treatment; CR/PR are sustained for 0.5+ to 9+ years. These data indicate that spontaneous remissions may occur in AITP even after a long duration of the disease.

Background and Objectives Rituximab, a monoclonal chimeric antibody to the CD20 antigen, is an effective therapy for the treatment of non-Hodgkin’s lymphomas. Moreover, rituximab has also shown to be effective in various autoimmune diseases including spontaneous antibodies to factor VIII. The aim of this study was to assess the efficacy of rituximab treatment of spontaneous inhibitors to factor VIII.Design and Methods We studied the efficacy of rituximab by analyzing the data of 42 previously published cases as well as one so far unpublished case. For comparison, we also analyzed 44 patients treated with cyclophosphamide/prednisone reported in the literature.Results Treatment with rituximab resulted in an overall rate of complete remission (CR) of 78.6%. Similar results were found when analyzing patients who had (75%) or had not (77%) received previous treatment with other immunosuppressive drugs. The median time to CR was 8.3 weeks. In follow-up 66% of the patients were still in CR after 2 years and the plateau in the Kaplan-Meier analysis suggests that a substantial number of patients had been cured. Among the 44 patients treated with cyclophosphamide/prednisone reported in the literature, the CR rate was 84.1%, which was slightly higher than that for rituximab. The median time to CR with cyclophosphamide/prednisone treatment was 6.3 weeks, which was similar to that in the rituximab-treated patients; the probability of continuous CR at 2 years was 94%.Interpretation and Conlusions All in all, both treatment schemes are effective therapies in patients with spontaneous antibodies to factor VIII. Our data analysis is only descriptive and no conclusions can be drawn as to the relative efficacy of the two regimens. However, these data may serve as a useful basis for planning randomized studies to definitively resolve these issues.

We analyzed 108 cases of non-CLL non-Hodgkin lymphoma (NHL) associated with autoimmune hemolytic anemia (AIHA) (+/- pure red cell aplasia (PRCA)) or Evans' syndrome. The analysis was based on cases reported in the literature, which were retrieved by means of Pubmed and Medline searches and of an original series of 121 patients with NHL as well as reference lists of papers in the field. The number of cases in various NHL subtypes was small (n = 6-25). Nevertheless, interesting and sometimes unexpected differences in sex prevalence, temporal relationship between onset of lymphoma and AIHA, stage of lymphoma, relative frequency of warm antibody-AIHA (WA-AIHA) and cold antibody (CA-AIHA), association with PRCA and response of AIHA to treatments were noted for various lymphoma entities. WA-AIHA was more frequent in B-cell lymphomas, while CA-AIHA and PRCA predominantly occurred in T-cell lymphomas. Anti-lymphoma treatment seemed to be more effective against AIHA than conventional therapy with steroids or immunoglobulin. Although generated by a literature survey, this compilation of data indicates a complex relation of lymphoma and AIHA and warrants more attention and specific studies.

Primary warm autoimmune hemolytic anemia (WAIHA) is a rare autoimmune disorder frequently responding to corticosteroid first-line treatment and effective second-line treatment options such as splenectomy or anti-CD20 antibody therapy. Disease management is frequently hampered by a lack of evidence. We have investigated the probability of sustained treatment-free remission after steroid induction to facilitate clinical decision making regarding timing and necessity of second-line treatments. Response data from 31 patients with primary WAIHA initially treated with steroids were retrospectively analyzed. All patients responded by achieving a hemoglobin of at least 10 mg/dl. After steroid tapering and final withdrawal, 9 of 30 patients remained in unsustained complete remission (CR). The probability of remaining in CR after steroid treatment only was 38.2 % (2 SD 20.6 %) at 15 months. The median remission duration was 100 + months with a range of 12 + to 163 + months. Of note, none of the remaining patients still on steroids achieved CR beyond 15 + months. These data indicate that a considerable proportion of patients do not need further treatment and that relapses will not occur after 15 months in CR.

We have originally shown that spontaneous granulocyte/macrophage colony (CFU-GM) formation in semisolid medium is a characteristic in vitro feature of chronic myelomonocytic leukemia (CMML). However, the clinical significance of spontaneous CFU-GM growth in CMML is unknown so far. CFU-GM growth characteristics were studied in semisolid cultures in the absence of exogenous cytokines using peripheral blood mononuclear cells in 30 patients with CMML at first presentation. The median number of CFU-GM/10(5) MNC of all patients was 48.5 (range 0-622) with 18 patients having colony numbers below 100 (low CFU-GM growth) and 12 patients above 100 (high CFU-GM growth). Kaplan-Meier analysis revealed that patients with high CFU-GM growth had a significantly shorter survival than patients with low CFU-GM growth (median 6.5 vs. 44.5 months, p0.00002). The probability of survival after 2 years was 60.5% for patients with low colony growth but 0% in those with high colony formation. Patients with CFU-GM100 had a significantly higher WBC count, a higher LDH, and a higher number of blast cells in blood and bone marrow than patients with low colony growth. Moreover, patients with high colony growth had more often splenomegaly and lower platelet counts. In seven patients, in whom semisolid in vitro cultures were performed after transformation into RAEBT/AML, spontaneous colony growth was significantly increased as compared to CFU-GM growth in patients before transformation (median number/10(5) MNC 533, range 212-4553, p0.005). This study demonstrates that high (100) spontaneous CFU-GM formation in CMML at presentation correlates with increased disease activity and represents a novel and important prognostic factor predicting for short survival of CMML patients.

BACKGROUND: ABO mismatch has not been thought to affect the outcome of patients undergoing myeloablative conditioning and allogeneic HPC transplantation. Data on transplant-related complications after ABO-mismatched transplantation after nonmyeloablative conditioning are limited. STUDY DESIGN AND METHODS: Therefore, 40 patients were analyzed after nonmyeloablative conditioning with regard to ABO compatibility. Eleven received a minor and bidirectional and 8 a major ABO-mismatched graft. RESULTS: Four patients had evidence of hemolysis during engraftment, being lethal in one, and three developed pure RBC aplasia. Six patients in the ABO-mismatched group developed thrombotic microangiopathy, and three of them died. ABO-identical and ABO-mismatched patients had a similar incidence of GVHD. Viral infections occurred in both groups in equal shares. Patients with an ABO-mismatch had to be rehospitalized until Day 100 for a median of 19 days versus 0 days in the identical group (p < 0.05). Overall survival was 60 and 57 percent in the ABO-identical and ABO-mismatch groups, respectively. The probability of transplant-related mortality was 0 versus 28 percent in the identical group compared to patients with an ABO mismatch (p < 0.05). The probability of relapse or progression was 76 versus 25 percent in the ABO-identical group compared to the ABO-mismatched group, respectively. CONCLUSION: Significantly more patients with ABO mismatch showed transplant-associated complications and died as a result of transplant-related causes.

We report on a 35-year-old woman who underwent allogeneic stem cell transplantation (SCT) in second complete remission (CR) of acute myeloid leukemia (AML) after reduced-intensity conditioning with fludarabine and 2 Gy of total body irradiation. For graft-versus-host disease (GVHD) prophylaxis, cyclosporin A (CsA) and mycophenolate mofetil (MMF) were given. On day 27 after SCT complete hematological remission and donor chimerism was documented. However, in CD34(+) bone marrow cells 28% of recipient hematopoiesis persisted. On day +59 leukemic relapse occurred. After discontinuation of CsA and onset of GVHD, complete donor chimerism and hematological CR were achieved which has been maintained for 14 months.

The growth characteristics and the prognostic value of cytokine-stimulated myeloid colony formation from peripheral blood mononuclear cells (PBMC) of patients with myelodysplastic syndromes (MDS) are largely unknown. In this study we have determined the number of myeloid colony-forming units (mCFUs) in semisolid medium from 112 MDS patients and correlated them with French-American-British (FAB) type, the international prognostic scoring system (IPSS), karyotype, peripheral blood (PB) and bone marrow (BM) blast cells, cytopenias, lactate dehydrogenase (LDH), and survival data. Concerning the FAB classification, lower median mCFUs were found in patients with refractory anemia (RA) and refractory anemia with ringed sideroblasts (RARS) compared to refractory anemia with excess of blast cells (RAEB) and refractory anemia with excess of blasts cells in transformation (RAEB-T). In vitro growth in MDS clearly correlated with the cytogenetic risk groups defined by the IPSS (30.5/10(5) PBMCs with favorable karyotypes, 191 in the intermediate prognostic group, 677 with unfavorable cytogenetics, p=0.015 favorable vs unfavorable). BM blast cells >5% (60.5 vs 255 colonies, p=0.032) as well as LDH levels above the normal limit (64.5 vs 425 colonies, p=0.045) were also associated with higher colony formation. Patients were stratified according to the number of circulating mCFUs into a low growth, intermediate growth and high growth group. Median survival was 343 days in the high growth, 1119 days in the low growth, and 2341 days in the intermediate growth group ( p=0.0002). Multivariate analyses revealed colony growth ( p=0.0056), PB blast cells ( p=0.0069), cytogenetic risk group ( p=0.024), and platelet count ( p=0.018) to predict survival in our patients. After inclusion of the IPSS risk categories, mCFU levels remained a highly predictive parameter for survival ( p=0.0056) and acute myeloblastic leukemia (AML) transformation ( p=0.0003).

Background: Mast cells (MCs) are multifunctional effector cells of the immune system. These cells originate from pluripotent hemopoietic progenitors. In contrast to basophils and other leukocytes, MCs exhibit a remarkably long life span (years) in vivo. Although a role for stem cell factor (SCF) and SCF receptor (KIT) in long-term survival of MCs has been proposed, the underlying biochemical mechanisms remain unknown. Materials and Methods:We have examined expression of ‘survival-related’ molecules of the bcl-2 family including bcl-2 and bcl-xL, in primary human MCs and the human MC line HMC-1. Primary MCs were isolated from dispersed lung tissue by cell sorting using an antibody against KIT. mRNA expression was analyzed by RT-PCR and Northern blotting. Results: As assessed by RT-PCR, purified unstimulated lung MCs (>98% pure) exhibited KIT- and bcl-xL mRNA, but did not express bcl-2 mRNA. However, exposure of lung MCS to SCF (100 ng/ml) for 8 h resulted in expression of bcl-2 mRNA. Corresponding results were obtained by immunocytochemistry. In fact, exposure of MC to SCF resulted in expression of the bcl-2 protein whereas unstimulated MCs displayed only the bcl-xL protein without expressing the bcl-2 protein. The human MC leukemia cell line HMC-1, which contains a mutated and intrinsically activated SCF receptor, showed constitutive expression of both bcl-2 and bcl-xL at the mRNA and protein level. Conclusion: Our data show that human MCs can express members of the bcl-2 family. It is hypothesized that bcl-xL plays a role in KIT-independent growth of MCs, whereas bcl-2 may be involved in KIT-dependent functions of MCs.

SummaryVenous thromboembolism represents a significant cause of morbidity worldwide. The factors that underly thrombophilia are manifold. The concept of Virchow defines the well known triad of stasis, humoral factors, and pathologies of the vascular wall. In the current article, an additional factor, the “accumulation of repair cells” is discussed. This novel concept highlights the mast cell that accumulates around thrombosed vessels and provides a number of important repair molecules including heparin, profibrinolytic tPA, and fibrinogenolytic β-tryptase. Thus, mast cell recruitment and activation may result in local thrombolysis and prevention of coagulation. In line with this concept, mast cell-deficient mice are more susceptible to lethal thrombogenic stimuli compared to normal mice. The factors (cytokines) that trigger mast cell accumulation and release of repair molecules have also been identified – the most important one appears to be stem cell factor (SCF). All in all, our novel concept suggests that the patho-physiology of thrombosis may involve a “physiologic” cell that provides the same repair molecules that are used for treatment of thrombotic disorders by the physician. Whether an altered availability of components of this cellular repair system can predispose for thrombophilia remains to be determined.

Recent data suggest that mast cells (MCs) in patients with systemic mastocytosis or mast cell leukemia express a CD2-reactive antigen. To explore the biochemical nature and function of this antigen, primary MCs as well as the MC line HMC-1 derived from a patient with mast cell leukemia were examined. Northern blot experiments revealed expression of CD2 messenger RNA in HMC-1, whereas primary nonneoplastic MCs did not express transcripts for CD2. In cell surface staining experiments, bone marrow (BM) MCs in systemic mastocytosis (n = 12) as well as HMC-1 cells (30%-80%) were found to express the T11-1 and T11-2 (but not T11-3) epitopes of CD2. By contrast, BM MCs in myelodysplastic syndromes and nonhematologic disorders (bronchiogenic carcinoma, foreskin phimosis, uterine myeomata ) were consistently CD2−. All MC species analyzed including HMC-1 were found to express LFA-3 (CD58), the natural ligand of CD2. To study the functional role of CD2 on neoplastic MCs, CD2+ and CD2− HMC-1 cells were separated by cell sorting. CD2+ HMC-1 cells were found to form spontaneous aggregates and rosettes with sheep erythrocytes in excess over CD2−cells, and a T11-1 antibody inhibited both the aggregation and rosette formation. Moreover, exposure of CD2+ HMC-1 cells to T11-1 or T11-2 antibody was followed by expression of T11-3. In addition, stimulation of neoplastic MCs through T11-3 and a second CD2 epitope resulted in histamine release. These data show that neoplastic MCs express functionally active CD2. It is hypothesized that expression of CD2 is associated with pathologic accumulation and function of MCs in systemic mastocytosis.

Background The myelodysplastic syndromes (MDS) are a group of clonal haematological disorders characterized by cytopenia(s), reduced differentiation-capacity of myeloid cells, and impaired leukocyte function. However, little is known so far about basophil granulocytes in MDS. Design We have compared the numbers, phenotype and function of basophils in MDS patients with those in healthy subjects. A total numer of 23 patients with MDS (refractory anaemia, n = 8; refractory anaemia with ringsideroblasts, n = 7; refractory anaemia with excess of blasts/refractory anaemia with excess of blasts in transformation, n = 8) and 20 healthy donors were included. Results The numbers of blood basophils in MDS patients (34.6 +/- 62.9 microL-1) was lower compared to healthy controls (58.6 +/- 64.9 microL-1). Correspondingly, whole blood histamine levels were lower in MDS patients (MDS 34.1 +/- 29.1 ng mL-1 vs. normal donors 72.0 +/- 36.9 ng mL-1). Like "normal" basophils, basophils in MDS expressed interleukin-3 receptor alpha (CD123), E-NPP3 (CD203c), CR1 (CD35), CR3 (CD11b), CR4 (CD11c), membrane co-factor protein (CD46), decay-accelerating factor (CD55) and membrane attack complex inhibitory factor (CD59), as well as receptors for C3a, C5a (CD88), and IgE. Recombinant human (rh) C5a and anti-IgE induced significant release of histamine from basophils in both groups of donors without significant differences between MDS and healthy controls. Conclusions The absolute numbers of basophils in MDS patients are lower than in normal donors. However, basophils in MDS do not differ from their "normal counterparts" in terms of complement receptor expression, IgE-receptor expression, or functional responses to respective ligands.

BACKGROUND: Ex vivo expansion strategies with different cytokine combinations are currently used by several groups as a means of increasing the number of HPCs for a variety of special clinical applications. Because there is little information on the potential role of IL-10 in such ex vivo expansion models, the effect of this cytokine on the generation of myeloid progenitor cells in suspension cultures was investigated. STUDY DESIGN AND METHODS: On the basis of data from the literature and from new experiments, the combination of SCF and IL-3 at concentrations of 100 ng per mL and 100 U per mL, respectively, was chosen as the standard cocktail. The addition of IL-10 to such cultures resulted in a marked and dose-dependent potentiation of myeloid progenitor cell production. RESULTS: Using unmanipulated leukapheresis components from 13 individuals (including lymphoma and cancer patients and normal donors), the expansion multiple of CFU–GM after 14 days as compared with pre-expansion values was 9.54 ± 2.31 times by SCF/IL-3 and 46.38 ± 7.37 times by the combination of SCF/IL-3 and 100 ng per mL of IL-10 (p

We have analysed the results of semi-solid bone marrow cultures in 296 patients with de novo acute myeloblastic leukaemia (AML) and correlated them with the leukaemic karyotype. A favourable prognostic karyotype was found in 52 patients (group A, 18·3%), an intermediate karyotype in 163 patients (group B, 57·4%), and unfavourable cytogenetics were observed in 69 patients (group C, 24·3%). Median colony growth according to the three risk groups was 2 (range 0–344) in group A, 14·5 (range 0–5000) in group B and 50·0 (0–3000) in group C (A vs. B, P

Patients with systemic mast cell (MC) disease, but not those with cutaneous mastocytosis, are at a high risk (10-30%) to develop life-threatening myelogenous malignancies. In a significant proportion of cases, myeloid leukemias occur. Using conventional criteria, such leukemias resemble acute myeloid leukemia (AML), chronic myeloid leukemia (CML), or myelomonocytic leukemia (CMML). Mast cell leukemia (MCL) may also occur. Myeloid leukemias (AML, CML, CMML) can develop in indolent or aggressive mastocytosis (skin lesions present or absent) with a variable prephase of MC disease. By contrast, MCL (typically without skin lesions) often develops on a "de novo" basis, and, if at all recognized, a prephase resembling (malignant) mastocytosis, is short. MCL differs from myeloid leukemias (AML, CML, CMML) by morphologic and phenotypic cellular characteristics. In fact, MCL are strongly tryptase-positive, c-kit-positive, myeloperoxidase (MPO) -negative neoplasms with variable metachromasia and chloroacetate esterase expression, whereas an MPO-positive, tryptase-negative phenotype supports the diagnosis of a myeloid non-MC lineage disease. Thus, MCL, but also myeloid non-MC lineage leukemias can develop in patients with (systemic) mastocytosis. Little is known, however, about the pathophysiologic basis of co-evolution. In the present article, the concomitant occurrence of mastocytosis and leukemia is discussed in the light of the literature and of concepts proposed to explain the biologic basis of this phenomenon.

Schnitzler’s syndrome is a rare disease characterized by chronic urticaria, monoclonal IgM, and clinical and laboratory signs of inflammation. In a subset of patients, the urticarial lesions cause pruritus. However, the pathophysiology of the disease and the biochemical basis of urticaria are not known. We describe a female patient with Schnitzler’s syndrome suffering from chronic urticaria associated with pruritus. The patient’s serum was found to contain IgG antibodies recognizing cellular components of the microvasculature. In particular, IgG3 antibodies directed against proteins (14–100 kD) expressed in cultured dermal microvascular endothelial cells and mast cells, were found by immunoblotting. Moreover, IgG2 antibodies specific for the α-chain of the FcεRI were detectable. However, the autoantibodies did not mediate histamine release in mast cells or basophils. In patients with IgM paraproteinemia who did not have Schnitzler`s syndrome, antibodies against endothelial/mast cells or FcεRI were not detectable. In summary, we describe subclass-specific IgG reactivity against microvascular endothelial cells and mast cells indicating Th1 autoimmunity in a patient with Schnitzler’s syndrome. Whether such autoantibodies are recurrently produced in patients with Schnitzler’s syndrome and play a role in the pathophysiology of the disease remains to be determined.

To determine the safety, biologic, and clinical benefits of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF; Amgen, Thousand Oaks, CA) after myelosuppressive chemotherapy in acute myeloid leukemia (AML),108 adult patients with de novo AML were randomized to receive either PEG-rHuMGDF (2.5 mu g/kg/d or 5 mu g/kg/d) for up to 21 doses (group A), a single dose of 2.5 mu g/kg PEG-rHuMGDF, 7 daily doses of 2.5 mu g/kg PEG-rHuMGDF (group B), or placebo. The greatest biologic activity was seen in group A with a median peak platelet count of 1,084 x 10(9)/L, occurring at a median 9 days after the last dose of study drug, compared with 517 x 10(9)/L and 396 x 10(9)/L in group B and placebo group, respectively. Thrombocytosis (platelets >1,000 x 10(9)/L) was seen at rates of 52%, 8%, and 9% in groups A, B, and placebo, respectively, but were not associated with any adverse event. There was no effect on median time to transfusion independent platelet recovery (greater than or equal to 20 x 10(9)/L). The median time to neutrophil recovery (greater than or equal to 500/mu L) and red blood cell transfusion requirements were similar in all groups, and there was no apparent stimulation of leukemia. PEG-rHuMGDF was biologically active and well tolerated. Further investigation of dose and scheduling is required, specifically earlier dosing before and during chemotherapy. (C) 1999 by The American Society of Hematology.

Mast cells (MC) and blood basophils (Ba) are multifunctional effector cells of the immune system and accumulate in areas of ongoing disease. However, despite of similar morphology, MC and Ba differ from each other in terms of cell surface receptor expression, mediator content, and tissue distribution. In order to gain new insights into mechanisms and molecules responsible for the distribution and accumulation of MC and Ba, we have investigated expression of homing receptors on primary human MC (lung, n=28; uterus, n=17), Ba (healthy donors, n=64), the mast cell line HMC-1, and the basophil line KU-812. Expression of cell surface antigens on MC and Ba was analyzed by mAb and indirect immunofluorescence staining techniques. In addition to previous findings, Ba were found to react with mAb against the selectin-ligands sLe(x) (CD15s) and PSGL-1 (CD162), L-selectin (CD62L), beta7-integrin, the 'matrix-receptor' neurothelin (CD147), platelet-endothelial cell tetraspan antigen-3 (PETA-3=CD151), and BST-1 (CD157). Novel antigens detectable on MC (lung and uterus) were CD147, CD151, CD157 and CD49c (VLA-3alpha). By contrast, MC were not recognized by mAb to sLe(x), PSGL-1, L-selectin, or beta7 integrin. No reactivity of Ba or MC with mAb to syndecan-1 (CD138), VE-cadherin (CD144), MUC18/MCAM (CD146), MGC-24 (CD164), or ALCAM (CD166) was found. The cell lines HMC-1 and KU-812 expressed a similar profile of antigens when compared to primary cells. In summary, Ba and MC express a unique profile of homing molecules. Apparently, Ba differ from MC in expression of recognition receptors relevant for binding to endothelium and consecutive transmigration.

A large group of patients relapsing after allogeneic bone marrow transplantation (BMT) have obtained remission after infusion of leukocytes from their original donor, suggesting a graft-versus-myeloma effect. However, side effects such as graft-versus-host disease and myelosuppression are severe, and sometimes fatal, complications of this therapeutic approach. Previously we demonstrated that patients with leukemia who lack donor hematopoiesis in relapse after BMT experience severe and lasting aplasia after infusion of donor leukocytes. In two patients - one with extramedullary and one with marrow relapse after a sex-mismatched transplantation - we analyzed hematopoietic chimerism by cell sorting and bone marrow cultures. CD34-positive cells, CD4-CD8-positive cells, committed progenitors, and LTC-IC were of donor origin, as demonstrated by two-color fluorescence in situ hybridization (FISH). Additionally, in relapse complete donor T-cell chimerism was seen. In contrast, plasma cells were of recipient origin in the patient who had a relapse in the bone marrow. Both patients were treated with infusions of donor leukocytes from their original donor. Neither patient suffered myelosuppression, and one achieved a stable complete remission.

Neutropenic infections are the major cause of morbidity and mortality in the treatment of aplastic anemia (AA) with antilymphocyte globulin (ALG), cyclosporin A (CSA), and methylprednisolone (MP). Recent data suggest a beneficial effect of administering G-CSF as an adjunct to immunosuppression. We have treated 11 consecutive patients with AA using a combined immunosuppressive regimen including ALG, CSA, and MP plus G-CSF at a dose of 5 microg/kg/day until neutropoietic recovery. In addition to measuring routine hematological parameters we have performed serial determinations of reticulocyte counts and in vitro progenitor cell cultures before and after therapy in order to assess their predictive value for treatment response and to determine the impact of therapy on early hematopoiesis. One patient died on day 34 of neutropenic septicemia. At 1 year, 81% of patients showed response to treatment. The median time to ANC values0.5 and1.0 x 10(9)/l were 19 and 35 days, respectively. Reticulocyte counts started to recover after 6 weeks, and transfusion independence was observed on day 52 for red blood cell transfusions and on day 53 for platelet concentrates. All patients with detectable colony formation in peripheral blood achieved a complete hematological remission, as compared with only one of five patients without progenitor cell growth. Although normal ranges were rarely achieved, there was a small but definitive improvement in progenitor cell numbers as compared with baseline values in most patients. Our results confirm the good tolerability and high efficacy of this G-CSF-supported combined immunosuppressive therapy for AA. Detectable colony growth at diagnosis seems to predict a high chance for complete hematological response.

Abstract —Vascular endothelial growth factor (VEGF) is important for the proliferation, differentiation, and survival of microvascular endothelial cells. It is a potent angiogenic factor and a specific endothelial cell mitogen that increases fenestration and extravasation of plasma macromolecules. Recently, large quantities of VEGF were detected in human megakaryocytes. Incubation of human platelets with thrombin in vitro resulted in the release of large amounts of VEGF. To investigate whether VEGF is released from platelets during coagulation activation in vivo, we measured in human subjects VEGF at the site of plug formation, ie, in blood emerging from a standardized injury made to determine bleeding time (shed blood). VEGF was also determined in the same volunteers after treatment with the specific thrombin inhibitor recombinant hirudin (r-hirudin). In a double-blind, randomized, crossover study, 17 healthy male volunteers (aged 20 to 35 years) were investigated. VEGF concentrations were measured in venous blood and in shed blood by the use of an immunoassay 10 minutes after intravenous administration of r-hirudin (0.35 mg/kg of body weight) or physiological saline. Prothrombin fragment f1.2 (f1.2) and β-thromboglobulin (β-TG) were determined as indicators of coagulation and platelet activation, respectively. Concentrations of VEGF, f1.2, and β-TG in shed blood 4 minutes after injury were significantly higher than in venous blood (VEGF, 55.8±9.2 versus P P P 50% inhibition of the β-TG and f1.2 levels in shed blood. In a similar manner, much lower amounts of VEGF were detectable at the site of plug formation after r-hirudin treatment (69.0±9.5 versus 37.8±2.6 pg/mL per minute; P =0.0015). Our data indicate that substantial quantities of VEGF are released from platelets during the interaction with the injured vessel wall in vivo. This finding may be relevant with respect to wound healing and tissue repair, tumor vascularization, or arterial thrombus formation.

In order to determine the relationship between bone marrow (bm) endosteal cells (EDC) and hemopoietic progenitors, we have analyzed the immunophenotype of EDC using various antibodies (Ab) against mesenchymal antigens. The Ab were applied on paraffin sections of normal bm (iliac crest, n=17; talus, n=1; phalanx, n=1), myeloregenerative bm (after chemotherapy), and hematologic disorders (acute myeloid leukemia (AML), n=8; chronic myeloid leukemia (CML), n=6; myelodysplastic syndromes (MDS), n=14; severe aplastic anemia (SAA), n=4; essential thrombocythemia (ET), n=2; idiopathic (primary) osteomyelo-fibrosis (IMF), n=1; polycythemia vera (PV), n=1). In normal bm, EDC were found to react with Ab against vimentin, tenascin, alpha-smooth muscle actin, osteocalcin, CD51, and CD56, but did not react with Ab against CD3, CD15, CD20, CD34, CD45, CD68, or CD117. An identical phenotype of EDC was found in AML, MDS, SAA, ET, IMF, PV, myeloregenerative bm, and peripheral bones lacking active hemopoiesis (talus, phalanx). In patients with CML, EDC reacted with Ab to CD51, but did not react with Ab to CD56. Based on their unique antigen profile, EDC were enriched from normal bm by enzyme digestion and cell sorting. However, these enriched cells (CD56+, CD45-, CD34-) did not give rise to hemopoietic cells under the culture conditions used, i.e. in the presence of the growth factors IGF-1, bFGF, SCF, IL-3, and GM-CSF Together, our data do not support the hypothesis that EDC are totipotent mesenchymal progenitors giving rise to hemopoietic cells.

A 37-year-old man with a newly diagnosed chronic myelogenous leukemia received induction therapy with hydroxyurea and interferon-alpha, and maintenance therapy with low-dose ara-C and interferon-alpha. Subsequent to a rapid hematological remission, a continuously aggravating pancytopenia with bone marrow aplasia developed which persisted after withdrawal of maintenance therapy. Bone marrow examination exhibited aplastic areas and residual hematopoiesis with impaired maturation; cytogenetically, there was a 100% persistence of Philadelphia chromosome-positive metaphases. By allogeneic bone marrow transplantation, a complete reconstitution of hematopoiesis was achieved.

SummaryNormal pregnancy is associated with alterations of the hemostatic system towards a hypercoagulable state and an increased risk of venous thromboembolism. The risk of venous thrombosis is higher in pregnant women with factor V Leiden (FVL) than in those with wildtype factor V. Routine laboratory assays are not useful to detect hypercoagulable conditions. A prospective and systematic evaluation of hemostatic system activation in women with and without FVL during an uncomplicated pregnancy employing more sensitive markers of hypercoagulability, such as prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), D-Dimer, or the endogenous thrombin potential (ETP), an indicator of the plasma’s potential to generate thrombin, has not been performed. We prospectively followed 113 pregnant women with (n = 11) and without (n = 102) FVL and measured F1+2, TAT, D-Dimer and the ETP at the 12th, 22nd and 34th gestational week as well as 3 months after delivery (baseline) in each subject. None of the women developed clinical signs of venous thromboembolism during pregnancy or postpartum. Pregnant women with and without FVL exhibited substantial activation of the coagulation and fibrinolytic system as indicated by a gradual increase of F1+2, TAT and D-Dimer throughout uncomplicated pregnancy up to levels similar to those found in acute thromboembolic events (p < 0.0001 by analysis of variance for each parameters). Levels of F1+2 and TAT were comparable between women with and without FVL, but levels of D-Dimer were significantly higher in women with FVL than in those without the mutation (p = 0.0005). The ETP remained unchanged in both women with and without FVL at all timepoints. Our data demonstrate a substantial coagulation and fibrinolytic system activation in healthy women with and without FVL during uncomplicated pregnancy. An elevated F1+2, TAT or D-Dimer level during pregnancy is not necessarily indicative for an acute thromboembolic event. The normal ETP in both women with and without FVL suggests that the capacity of the plasma to generate thrombin after in vitro activation of the clotting system is not affected by pregnancy. Higher levels of D-Dimer in women with FVL than in women with wildtype factor V at baseline as well as during pregnancy indicate increased fibrinolytic system activation in carriers of the mutation.

A subset of patients with systemic mastocytosis (SM) develop acute myeloid leukaemia (AML). However, little is known about the biology of such leukaemias and their relationship to the mast cell (MC) lineage. We report on two female patients who suffered from SM and AML. According to FAB criteria, the leukaemias were classified as AML-M4 (patient 1) and AML-M0 (patient 2). The coexistence of the two distinct neoplasms (AML and SM) was demonstrable by immunostaining of serial bone marrow (BM) sections with monoclonal antibodies (mAb). In particular, the MC infiltrates were found to react with mAb against MC-tryptase and MC growth factor receptor c-kit (CD117), but not with mAb to CD15 or CD34. In contrast, the AML blasts were immunoreactive for CD15 (patient 1) or CD34 (patient 2), but did not express tryptase. The c-kit point mutation Asp Val at codon 816, considered to play a role in the transformation of MC progenitors, was detected in patient 1 in a BM cell fraction containing 4% MC. However, no c-kit mutation was found in pure AML blasts (

The WT1 gene is expressed in 73–100% of patients with acute myelogenous leukemia (AML) and is thought to play a role in maintaining the viability of leukemic cells. WT1 has been proposed as a marker for minimal residual disease in leukemia. We obtained serial blood or bone marrow samples from patients with de novo AML at diagnosis, during therapy, and up to 95 months after diagnosis and analyzed for WT1 gene expression by RT-PCR to determine whether gene expression was predictive of relapse. Forty-four patients had WT1-positive AML and achieved a complete remission (CR) following chemotherapy and 24 patients underwent unrelated donor (n = 4), sibling donor (n = 13) or autologous (n = 7) marrow transplantation. After achieving CR 62% of the patients became WT1-negative, while 38% remained WT1-positive. There was no difference in the disease-free survival (DFS) and survival from remission between WT1-positive and -negative patients (P > 0.1). Following BMT, 32% of the patients analyzed in CR within the first 100 days after transplantation were WT1 PCR positive. Detection of WT1 transcripts within 100 days following BMT did not affect DFS and overall survival (OS) after transplantation (P > 0.1). Ten of 11 patients who are in continuous CR following chemotherapy or BMT for more than 3 years were transiently WT1-positive during the observation period. Four of these patients displayed the WT1 transcript at the last examination. Thirteen of 39 patients were WT1 PCR negative within 4 months before clinical onset of relapse and eight patients were WT1 PCR negative at time of relapse. These data indicate that: (1) achievement of WT1 negativity is not associated with longer DFS, survival from remission, or OS after transplantation; (2) not all patients who relapse become WT1 positive again; (3) long-term remitters frequently display the WT1 transcript. Thus, we conclude that the monitoring of WT1 gene expression by qualitative RT-PCR during treatment and CR is of very limited value.

In polycythemia vera (PV) erythroid colonies that grow in vitro in the absence of exogenous erythropoietin (EPO) arise from the abnormal clone that is responsible for overproduction of red blood cells. Although the mechanism of autonomous formation of burst-forming units-erythroid (BFU-E) is not fully understood, a spontaneous release of growth regulatory molecules by PV cells and/or by accessory cells is likely to be involved. Because of its cytokine synthesis inhibiting action, interleukin-10 (IL-10) could be a potentially useful molecule to modulate abnormal erythropoiesis in PV. We studied the effect of recombinant human IL-10 on the EPO-independent growth of erythroid bursts derived from peripheral blood mononuclear cells (PBMNCs) of patients with PV. IL-10 showed a profound, dose-dependent, and specific inhibitory effect on autonomous BFU-E formation. Ten nanograms per milliliter of IL-10 significantly suppressed spontaneous growth of erythroid colonies in methylcellulose in five of five PV patients tested with a mean inhibition by 81% (range, 72-94). To elucidate the possible mechanism of the inhibitory action of IL-10 we further studied the effect of anticytokine antibodies on autonomous BFU-E growth and the ability of exogenous cytokines to restore IL-10–induced suppression of erythroid colony growth. Among a panel of growth regulatory factors tested (granulocyte-macrophage colony-stimulating factor [GM-CSF], IL-3, granulocyte colony-stimulating factor, stem cell factor, and insulin-like growth factor-1) GM-CSF was the only molecule for which both an inhibition of spontaneous BFU-E formation by its respective antibody as well as a significant restimulation of erythroid colonies in IL-10-treated cultures by exogenous addition was found. Moreover, inhibition of GM-CSF production by IL-10 was shown in PV PBMNCs at the mRNA level. Our data indicate that autonomous BFU-E growth in PV can be profoundly inhibited by IL-10 and that this inhibitory effect seems to be at least in part secondary to suppression of endogenous GM-CSF production. © 1998 by The American Society of Hematology.

Among 379 patients with AML with FAB type M1, 2 and M4-7 diagnosed between 1978 and 1997 in our institution, 19 (5%) had hypofibrinogenemia (HF), ie a fibrinogen level

By participating in a randomized safety and efficacy study of pegylated-recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) post induction and consolidation chemotherapy for de novo acute myeloid leukaemia, serial determinations of circulating haemopoietic progenitor cells were performed during 18 chemotherapy courses in eight patients (three receiving placebo; one, 2.5; and four, 5.0 μg/kg/d MGDF, respectively). Whereas failure to achieve complete remission (CR) was generally associated with poor progenitor cell increments following chemotherapy, substantial progenitor cell mobilization consistently occurred during haemopoietic recovery in patients entering, or in CR, with significantly higher peak values in patients receiving 5 μg/kg/d of MGDF as compared to controls. The median increases of progenitor cell numbers by chemotherapy alone and chemotherapy plus 5.0 μg/kg/d MGDF over that in normal individuals with steady-state haemopoiesis were 10- and 45-fold for CFU-GM, 3- and 17-fold for BFU-E, and 2- and 18-fold for CFU-mix. CFU-Mk levels were not increased above normal by chemotherapy alone but were 15-fold enhanced by chemotherapy plus MGDF. Recruitment of CD34+ cells post chemotherapy was also potentiated by MGDF. Our results suggest MGDF as a potent agent to augment progenitor cell mobilization after successful induction or consolidation chemotherapy in patients with AML.

One hundred and five cases of factor V inhibitors were published between 1955 and 1997. According to pathogenesis, factor V inhibitor patients can be divided into five groups: patients exposed to bovine thrombin; patients after surgery without exposure to bovine proteins; miscellaneous associated conditions; 'idiopathic' inhibitors; inhibitors in congenital factor V deficiency. The clinical and biochemical properties are described. The overall prognosis of factor V inhibitors is good, but there are differences among the five groups with the best prognosis in patients exposed to bovine thrombin and the worst prognosis in 'idiopathic' inhibitors. Only a few treatment options are available. Immunoadsorption and plasmapheresis seem to be the most effective methods for therapy of acute bleeding. Many inhibitors disappear spontaneously and it is uncertain whether an immunosuppressive treatment hastens the disappearance of the inhibitor.

Mastocytosis is a term used for a spectrum of disorders characterized by abnormal growth and accumulation of mast cells. The cutaneous variants of the disease have to be distinguished from systemic mastocytosis (SM), in which at least one extracutaneous organ is involved. In contrast to cutaneous mastocytosis, SM is often associated with another hematologic neoplasm. In most cases clonal myeloid malignancies such as a myeloproliferative or myelodysplastic syndrome occur. In a few cases of SM, however, clonal lymphoid disorders have been described. We here report on a case of SM associated with multiple myeloma. At first presentation, the 48-year old female patient showed monoclonal IgGlambda gammopathy and bone marrow (BM) mastocytosis, but no BM plasma cell infiltrates. Eight years later, the patient presented with BM mastocytosis and overt multiple myeloma. The co-existence of myeloma and mastocytosis was demonstrable by staining serial BM sections with antibodies against mast cell tryptase, CD68R, and the plasma cell marker VS38c. Interphase FISH analysis of BM sections revealed a numeric gain of chromosome 5 and chromosome 7 in the plasma cells but not in the mast cell infiltrates, thereby confirming the presence of two different neoplastic cell populations. To our knowledge, this is the first report describing the co-existence of multiple myeloma and mastocytosis.

The 110-kD lung resistance protein (LRP) is overexpressed in P-glycoprotein–negative multidrug-resistant cell lines and most likely involved in the multidrug resistance (MDR) of these cell lines. To determine the clinical significance of LRP, we have studied LRP expression of leukemic blasts and its association with clinical outcome in patients with de novo acute myeloid leukemia (AML). LRP expression of leukemic blasts obtained from peripheral blood or bone marrow of previously untreated patients (n = 86) was determined by immunocytochemistry by means of monoclonal antibody LRP-56. LRP expression at diagnosis was detected in 31 (36%) patients. LRP expression was independent of age and sex of the patients, French-American-British subtype, cytogenetic abnormalities, and lactate dehydrogenase levels, but correlated with white blood cell count (P = .01). Eighty-two patients received standard induction chemotherapy that included cytarabine and MDR drugs (daunorubicin in most patients, additional etoposide in the majority of patients). The complete remission rate of induction chemotherapy was 72% (95% confidence interval [CI] = 61% to 82%) for the total study population. The complete remission rate was 81% (95% CI = 67% to 91%) for patients without LRP expression but only 55% (95% CI = 36% to 74%) for patients with LRP expression (P = .01). Overall survival and disease-free survival were estimated according to Kaplan-Meier in 82 and 59 patients, respectively. Overall survival was significantly longer in patients without LRP expression than in patients with LRP expression. At a median follow-up of 16 months, median overall survival was 17 months (95% CI = 12 to 38 months) for LRP-negative patients but only 8 months (95% CI = 4 to 12 months) for -positive patients (P = .006). Disease-free survival was 9 months (95% CI = 7 to 11 months) for LRP-negative patients and 6 months (95% CI = 5 to 8 months) for -positive patients (P = .078). Outcome was best in patients lacking both LRP and P-glycoprotein expression. In conclusion, LRP predicts for poor outcome and thus theLRP gene appears to be another clinically relevant drug resistance gene in AML.

The safety and efficacy of filgrastim as an adjunct to acute myeloid leukemia (AML) induction and consolidation therapy was assessed in this prospective double-blind, randomized, placebo-controlled, multicenter trial. A total of 521 consecutive de novo AML patients aged 16 or more years were randomized to receive filgrastim (5 μg/kg/d subcutaneously) or placebo after standard induction as well as consolidation chemotherapy. Blinded study drug was given from 24 hours after chemotherapy until the absolute neutrophil count was ≥1.0 × 109/L for 3 consecutive days. The overall complete remission rate was 68%. After a median follow-up of 24 months (range 5 to 40) the median disease-free survival was 10 months (95% confidence interval [CI], 8.7 to 10.8) and the median overall survival was 13 months (95%CI, 12.2 to 14.6). These did not differ between treatment groups. Patients receiving filgrastim experienced neutrophil recovery 5 days earlier after induction 1 than those receiving placebo (P < .0001). This was accompanied by reductions in the duration of fever (7 v 8.5 days; P = .009), parenteral antibiotic use (15 v 18.5 days; P = .0001), and hospitalization (20 v 25 days; P = .0001). Similar reductions were seen after induction 2 and the consolidation courses. There was a significant reduction in the number of patients requiring systemic antifungal therapy in the filgrastim group during induction treatment (34% v 43%; P = .04). In conclusion, filgrastim is safe in that it had no negative impact on the prognosis of the AML patients. In addition, it effectively reduced the duration of neutropenia, leading to significant clinical benefits by reducing the duration of fever; requirement for parenteral anti-infectives, specifically amphotericin B; and the duration of hospitalization.

Because of the recommendation to avoid the concomitant administration of growth factors and chemotherapy, there is only limited information on colony-stimulating factor (CSF ) therapy in acute lymphoblastic leukemia (ALL) induction protocols, in which cytotoxic drugs are administered in divided doses over a prolonged period of time, thus requiring a simultaneous administration of growth factors and chemotherapy. We conducted a prospective, randomized, controlled study to determine the safety and efficacy of granulocyte colony-stimulating factor (G-CSF; filgrastim) as an adjunct to phase I of induction chemotherapy for adult ALL. Patients (n = 53) were randomized to receive no growth factor or G-CSF (5 μg/kg/d subcutaneously) starting on day 2 of chemotherapy consisting of daunorubicin (45 mg/m2) and vincristine (1.5 mg/m2) on days 1, 8, 15, and 22; L-asparaginase (2500 U/m2) on days 1 through 14; and prednisone (60 mg/m2) on days 1 through 28. A total of 25 patients in the G-CSF group and 26 patients in the control arm fulfilled the inclusion criteria of the study. G-CSF markedly ameliorated neutropenia because the median proportion of days with neutropenia less than 1,000/μL was 29% in the G-CSF group as compared with 84% in the control arm (P < .00005). The median time to reach absolute neutrophil counts (ANC) ≥ 1,000/μL was 16 days in G-CSF patients and 26 days in controls (P < .001). More importantly, G-CSF significantly reduced the incidence of febrile neutropenia (12% v 42% in controls, P < .05) and documented infections (40% v 77%, P < .05). No significant differences were found with regard to requirements for red blood cell transfusions and platelet concentrates. A total of 24 of 25 (96%) patients in the G-CSF group and 20 of 25 (80%) evaluable control patients had complete remission after phase I of induction therapy. We conclude that G-CSF can be safely administered as an adjunct to induction therapy of ALL and is clinically beneficial by ameliorating neutropenia and reducing infectious complications.

Spontaneous growth of myeloid colonies (colony-forming unit–granulocyte-macrophage [CFU-GM]) can be observed in methylcellulose cultures containing peripheral blood mononuclear cells (PB-MNCs) and is supposedly caused by the release of colony-stimulating factors (CSF ) by accessory cells. Because of its cytokine synthesis-inhibiting effects on T lymphocytes and monocytes, interleukin-10 (IL-10) may be a potential candidate for indirect modulation of hematopoiesis. We studied the effect of recombinant human IL-10 (rhIL-10) on spontaneous growth of myeloid colonies derived from human PB-MNCs. A total of 10 ng/mL of IL-10 almost completely inhibited spontaneous CFU-GM proliferation (by 95.1%; P < .001, n = 7) in unseparated PB-MNCs. This effect was dose-dependent and specific, because a neutralizing anti–IL-10 antibody was able to prevent IL-10–induced suppression of CFU-GM growth. Spontaneous CFU-GM growth, which required the presence of both monocytes (CD14+ cells) and T lymphocytes (CD3+ cells), was also greatly suppressed by a neutralizing anti–granulocyte-macrophage CSF (GM-CSF ) antibody but was only slightly or not at all inhibited by antibodies against G-CSF or IL-3. Moreover, IL-10–suppressed colony growth could be completely restored by the addition of exogenous GM-CSF. Using semiquantitative polymerase chain reaction, we were able to show that GM-CSF transcripts that spontaneously increased in PB-MNCs within 48 hours of culture were markedly reduced by the addition of IL-10. Inhibiton of GM-CSF production in PB-MNCs by IL-10 was also confirmed at the protein level by measuring GM-CSF levels in suspension cultures. Our findings suggest that autonomous CFU-GM growth, resulting from an interaction of monocytes and T lymphocytes, is mainly caused by endogenous GM-CSF release and can be profoundly suppressed by the addition of exogenous IL-10. Considering the strong inhibitory action of IL-10 on GM-CSF production and spontaneous cell growth in vitro, this cytokine may be useful in myeloid malignancies in which autocrine and/or paracrine mechanisms involving GM-CSF are likely to play a pathogenetic role.

SummaryCoumarin-induced skin necrosis is believed to be due to a transient hypercoagulable state resulting from a more rapid decline of the protein C activity relative to that of coagulation factors (F) II, IX and X during initiation of oral anticoagulant therapy. We studied hemostatic system activation during early oral anticoagulant treatment with a technique that investigates coagulation activation in the microcirculation.We determined in 10 healthy volunteers the concentrations of prothrombin fragment F1+2 (f1.2) and thrombin-antithrombin complex (TAT) in blood emerging from an injury of the microvasculature (bleeding time incision) before and after initiation of both high-inten- sity and low-intensity coumarin therapy. In addition, f1.2, TAT, activated F VII (F Vila) and the activities of FII, F VII, F X and protein C were measured in venous blood.A rapid decline of F VII and protein C was observed in venous blood with activities at 24 h of 7 ± 1% and 43 ± 2%, respectively, during the high-intensity regimen. A 20 to 30% reduction of f1.2 and TAT was seen in venous blood at 72 h with no major difference between the high- and the low-intensity regimen. F Vila levels were substantially affected by anticoagulation with a >90% reduction at 48 h during the high-intensity regimen. Following high-intensity coumarin, a >50% decrease in the fl.2 and TAT levels was found in shed blood at 48 h suggesting substantial inhibition of thrombin generation during early oral anticoagulation. An increase in the f1.2 and TAT levels was seen neither in shed blood nor in venous blood.Our data do not support the concept of a transient imbalance between generation and inhibition of thrombin as the underlying pathomechanism of coumarin-induced skin nekrosis.

Autonomous release of hematopoietic growth factors may play a crucial role in the pathogenesis of certain hematological malignancies. Because of its cytokine synthesis-inhibiting action, interleukin 10 (IL-10) could be a potentially useful molecule to affect leukemic cell growth in such disorders. Chronic myelomonocytic leukemia (CMML) cells spontaneously form myeloid colonies (colony-forming units-granulocyte/macrophage) in methylcellulose, suggesting an autocrine growth factor-mediated mechanism. We studied the effect of recombinant human IL-10 (rhIL-10) on the in vitro growth of mononuclear cells obtained from peripheral blood or bone marrow of patients with CMML. IL-10 specifically binding to leukemic cells had a profound and dose-dependent inhibitory effect on autonomous in vitro growth of CMML cells. IL-10 significantly inhibited the spontaneous growth of myeloid colonies in methylcellulose in 10/11 patients, and autonomous CMML cell growth in suspension in 5/5 patients tested. Spontaneous colony growth from CMML cells was also markedly reduced by addition of antigranulocyte/macrophage colony-stimulating factor (GM-CSF) antibodies, but not by addition of antibodies against G-CSF, IL-3, or IL-6, IL-10-induced suppression of CMML cell growth was reversed by the addition of exogenous GM-CSF and correlated with a substantial decrease in GM-CSF production by leukemic cells, both at the mRNA and protein levels. Our data indicate that IL-10 profoundly inhibits the autonomous growth of CMML cells in vitro most likely through suppression of endogenous GM-CSF release. This observation suggests therapeutic evaluation of rhIL-10 in patients with CMML.

Stem cell factor (SCF) is a key regulator of human mast cells (MCs) and a potential mediator of allergy. In this study the effects of cyclosporin A (CSA) and FK-506, two potent immunosuppressive drugs, on SCF-dependent histamine release and growth of human MCs were analyzed. Preincubation of tissue MCs with CSA (3 micrograms/ml) resulted in inhibition of histamine release provoked by either recombinant human (rh) SCF (70.3% +/- 20.6% inhibition, p0.001) or anti-IgE (76.7% +/- 21.9%, p0.001) or by rhSCF+ anti-IgE (77.4% +/- 13.9%, p0.001). Almost the same inhibition was produced by FK-506 (rhSCF: 82.0% +/- 18.9% inhibition, p0.001; anti-IgE: 71.5% +/- 16.7%, p0.001; rhSCF+ anti-IgE: 70.0% +/- 7.3%, p0.001). The effects of CSA and FK-506 on SCF-dependent release of histamine were dose-dependent (IC50: CSA, 1 to 10 ng/ml; FK-506, 0.3 to 3 ng/ml). IC50 values about three to 10 times higher were found for MCs preincubated with rhSCF before anti-IgE activation, compared with anti-IgE or SCF alone. SCF-dependent differentiation of human MCs was analyzed in a long-term suspension culture system (n = 6). Unexpectedly, CSA and FK-506 were unable to suppress, but even enhanced SCF-dependent growth of MCs and formation of MC tryptase in long-term culture. Together, CSA and FK-506 inhibit SCF-dependent release of histamine from human MCs and even augment SCF-dependent growth of human MCs in long-term culture.

It has been speculated that an impairment in anticoagulant pathways (protein C (PC), protein S (PS), resistance to activated protein C (APC)), may contribute to the thrombotic tendency in lupus anticoagulant (LA) patients. Increased plasma levels of fibrinogen are predictive for arterial thrombosis and increased molecular markers of thrombosis are indicative for activation of the clotting cascade. We investigated 25 patients (20 women) with LA. All patients were stratified according to their thromboembolic history and women according to their history of fetal loss. Eighteen patients had a history of venous or arterial thrombosis, or both. Seven of 16 women with at least one pregnancy had a history of fetal loss. The interrelation among the levels of fibrinogen, PC and PS, and resistance to APC, thrombin-antithrombin III complexes (TAT), prothrombin fragment F1 + 2, D-dimer and the history of thrombotic events and obstetric complications in patients with LA were evaluated. LA patients with a history of venous or arterial thrombosis had a significantly higher fibrinogen level than LA patients without (mean 366 versus 304 mg/dl; P = 0.018). Among 16 women a slightly lower mean TAT level in women with fetal loss was found (2.4 versus 4.3 ng/ml; P = 0.02). No other statistically significant difference in the remaining parameters was yielded in both analyzed subgroups. The results of the study suggest an association between increased fibrinogen levels and the history of venous or arterial thrombosis, or both, in patients with LA. In the other investigated parameters, no relationship to the thrombotic or obstetric history was found.

Drug resistance often results in failure of anticancer chemotherapy in leukemias. Several mechanisms of drug resistance are known with multidrug resistance (MDR) being the best characterized one. MDR can be due to enhanced expression of certain genes (MDR1, MRP or LRP), alterations in glutathione-S-transferase activity or GSH levels and to reduction of the amount or the activity of topoisomerase II. Here we review the current status of the clinical significance of the various mechanisms of MDR in leukemias and also discuss possibilities for the reversal of MDR. MDR1 gene expression has been seen in many leukemias, notably in acute myeloid leukemia (AML) and blast crisis of chronic myeloid leukemia. Both MDR1 RNA and P-glycoprotein expression of the leukemic cells have been shown to correlate with poor clinical outcome in AML. However, preliminary results indicate that the MRP gene as well as the LRP gene can be expressed in AML. Thus, drug resistance in leukemias appears to be multifactorial. P-glycoprotein-mediated MDR can be reversed by several drugs. These resistance modifiers are currently evaluated with regard to their clinical efficacy. Despite some encouraging results, reversal of drug resistance and subsequent improvement in clinical outcome remains to be shown.

Mast cells and blood basophils are distinct hemopoietic cells. They can be distinguished from each other and from all other lymphohemopoietic cells using antibodies against surface receptors or stored cytoplasmic molecules. In patients with myelodysplastic syndromes (MDS) or myeloproliferative syndromes (MPS), an elevation of metachromatically granulated cells (MCS) is frequently seen. These cells can be classified as basophils or mast cells using monoclonal antibodies (mAbs) against leukocyte antigens, including mast cell tryptase, c-kit (= mast cell growth factor [MGF] receptor), interleukin-3 receptor alpha chain (IL-3R alpha = CD123), and CD11b (C3biR). In a stable phase of MDS or MPS, the circulating MCS usually are basophils (histamine+, tryptase-, c-kit-, IL-3R alpha +, CD11b+). In an accelerated or terminal phase of disease, however, mast cell lineage involvement and circulating mast cell precursors (histamine+, tryptase+, c-kit+, IL-3R alpha-, CD11b-) are found in a subset of patients. The use of mAbs against mast cell antigens and granulocyte antigens is diagnostic in these patients.

We report on the rapid elimination of a potent spontaneous factor V antibody of undetermined etiology by extracorporeal immunoadsorption on sepharose-bound polyclonal sheep antibodies to human immunoglobulins (Ig-Therasorb, Baxter) in combination with immunosuppressive treatment. A 68-year-old woman presented with severe hematuria. Severe factor V deficiency (< 1%) caused by an antibody to factor V (26 BU/ml) was found. Extracorporeal immunoadsorption (8.245 +/- 553 ml plasma processed per session) led to an average reduction of the antibody titer by 75% per session. The procedure was well tolerated without any side effects. Hematuria ceased after three immunoadsorptions and complete elimination of the antibody was achieved after seven sessions (day 15), followed by a rapid increase of the factor V activity to normal levels. Treatment with cyclophosphamide and prednisone was started on day 6 and continued for 2 months. The patient remains in remission at 6 months. Extracorporeal immunoadsorption is a highly effective method for eliminating antibodies to factor V (or other clotting factors) in selected cases, i.e., in patients with severe bleeding tendency, high antibody titer, and low probability of a rapid spontaneous remission.