Activin A inhibits BMP-signaling by binding ACVR2A and ACVR2B.

BACKGROUND: Activins are members of the TGF-ß family of ligands that have multiple biological functions in embryonic stem cells as well as in differentiated tissue. Serum levels of activin A were found to be elevated in pathological conditions such as cachexia, osteoporosis and cancer. Signaling by activin A through canonical ALK4-ACVR2 receptor complexes activates the transcription factors SMAD2 and SMAD3. Activin A has a strong affinity to type 2 receptors, a feature that they share with some of the bone morphogenetic proteins (BMPs). Activin A is also elevated in myeloma patients with advanced disease and is involved in myeloma bone disease. RESULTS: In this study we investigated effects of activin A binding to receptors that are shared with BMPs using myeloma cell lines with well-characterized BMP-receptor expression and responses. Activin A antagonized BMP-6 and BMP-9, but not BMP-2 and BMP-4. Activin A was able to counteract BMPs that signal through the type 2 receptors ACVR2A and ACVR2B in combination with ALK2, but not BMPs that signal through BMPR2 in combination with ALK3 and ALK6. CONCLUSIONS: We propose that one important way that activin A regulates cell behavior is by antagonizing BMP-ACVR2A/ACVR2B/ALK2 signaling.

Melphalan and prednisone plus thalidomide or placebo in elderly patients with multiple myeloma.

In this double-blind, placebo-controlled study, 363 patients with untreated multiple myeloma were randomized to receive either melphalan-prednisone and thalidomide (MPT) or melphalan-prednisone and placebo (MP). The dose of melphalan was 0.25 mg/kg and prednisone was 100 mg given daily for 4 days every 6 weeks until plateau phase. The dose of thalidomide/placebo was escalated to 400 mg daily until plateau phase and thereafter reduced to 200 mg daily until progression. A total of 357 patients were analyzed. Partial response was 34% and 33%, and very good partial response or better was 23% and 7% in the MPT and MP arms, respectively (P < .001). There was no significant difference in progression-free or overall survival, with median survival being 29 months in the MPT arm and 32 months in the MP arm. Most quality of life outcomes improved equally in both arms, apart from constipation, which was markedly increased in the MPT arm. Constipation, neuropathy, nonneuropathy neurologic toxicity, and skin reactions were significantly more frequent in the MPT arm. The number of thromboembolic events was equal in the 2 treatment arms. In conclusion, MPT had a significant antimyeloma effect, but this did not translate into improved survival. This trial was registered at www.clinicaltrials.gov as #NCT00218855.

Effects of peripheral blood stem cell apheresis on systemic cytokine levels in patients with multiple myeloma.

BACKGROUND AIMS. Pro-angiogenic cytokines can affect myeloma cell proliferation directly and indirectly through stimulation of cancer-associated angiogenesis. METHODS. We investigated how peripheral blood stem cell (PBSC) collection affected plasma angioregulatory cytokine levels in 15 consecutive myeloma patients. RESULTS. Plasma levels of hepatocyte growth factor (HGF) were significantly increased prior to apheresis in patients compared with donors, and a further increase was detected immediately after PBSC apheresis. HGF levels decreased within 24 h, but were still higher than the levels in healthy donors, whose HGF levels were not altered by platelet apheresis. Pre-apheresis levels of other angioregulatory cytokines, angiopoietin-2 and vascular endothelial growth factor (VEGF), were also increased in patients, whereas angiopoietin-1, angiogenin and basic fibroblast growth factor levels did not differ from healthy controls. PBSC harvesting decreased angiopoietin-1 and VEGF levels, increased the microvascular endothelial cell marker endocan levels but did not affect the other mediators. CONCLUSIONS. Our results show that PBSC apheresis alters systemic angioregulatory profiles in myeloma patients. This cytokine modulation is not a general characteristic of all apheresis procedures and was not seen in healthy platelet donors.

Effect of pamidronate 30 mg versus 90 mg on physical function in patients with newly diagnosed multiple myeloma (Nordic Myeloma Study Group): a double-blind, randomised controlled trial.

BACKGROUND: Compared with placebo, prophylactic treatment with bisphosphonates reduces risk of skeletal events in patients with multiple myeloma. However, because of toxicity associated with long-term bisphosphonate treatment, establishing the lowest effective dose is important. This study compared the effect of two doses of pamidronate on health-related quality of life and skeletal morbidity in patients with newly diagnosed multiple myeloma. METHODS: This double-blind, randomised, phase 3 trial was undertaken at 37 clinics in Denmark, Norway, and Sweden. Patients with multiple myeloma who were starting antimyeloma treatment were randomly assigned in a 1:1 ratio to receive one of two doses of pamidronate (30 mg or 90 mg) given by intravenous infusion once a month for at least 3 years. Randomisation was done by use of a central, computerised minimisation system. Primary outcome was physical function after 12 months estimated by the European Organisation for Research and Treatment of Cancer (EORTC) QLQ-C30 questionnaire (scale 0-100). All patients who returned questionnaires at 12 months and were still on study treatment were included in the analysis of the primary endpoint. This study is registered with ClinicalTrials.gov, number NCT00376883. FINDINGS: From January, 2001, until August, 2005, 504 patients were randomly assigned to pamidronate 30 mg or 90 mg (252 in each group). 157 patients in the 90 mg group and 156 in the 30 mg group were included in the primary analysis. Mean physical function at 12 months was 66 points (95% CI 62·9-70·0) in the 90 mg group and 68 points (64·6-71·4) in the 30 mg group (95% CI of difference -6·6 to 3·3; p=0·52). Median time to first skeletal-related event in patients who had such an event was 9·2 months (8·1-10·7) in the 90 mg group and 10·2 months (7·3-14·0) in the 30 mg group (p=0·63). In a retrospective analysis, eight patients in the pamidronate 90 mg group developed osteonecrosis of the jaw compared with two patients in the 30 mg group. INTERPRETATION: Monthly infusion of pamidronate 30 mg should be the recommended dose for prevention of bone disease in patients with multiple myeloma. FUNDING: Nordic Cancer Union and Novartis Healthcare.

Long-term cryopreservation of autologous stem cell grafts: a clinical and experimental study of hematopoietic and immunocompetent cells.

BACKGROUND: Autologous stem cell transplantation(ASCT) is used in the treatment of several malignancies.Harvesting sufficient peripheral blood progenitor cells (PBPCs) for a potential second autotransplantation at the time of relapse several years after diagnosis is becoming an increasingly common practice. STUDY DESIGN AND METHODS: Cryopreserved PBPCs were prepared with different concentrations of dimethyl sulfoxide (DMSO; 2, 4, 5, and 10%) and stored for at least 5 years before the recovery of CD34+ cells and various T- and natural killer (NK)-cell subsets were analyzed by flow cytometry. Furthermore,clinical variables for myeloma patients having a second autotransplantation with long-term-stored autografts were evaluated. RESULTS: The number of viable CD34+ cells in longterm-stored grafts was higher when autografts were cryopreserved with 4 or 5% than with 2 and 10%DMSO. The number of viable CD34+ cells was reduced by 13.9% after 5 years of cryostorage in 5% DMSO.Lymphocyte viability was also higher with 4 or 5%DMSO. However, the frequencies of several T-cell subsets showed DMSO-dependent differences,whereas NK-cell subsets did not. Furthermore, after a second autotransplantation with long-term-stored PBPC grafts at the time of myeloma relapse (median storage time, 42 months) all 17 patients reached neutrophil counts exceeding 0.5 x 109/L and platelet counts exceeding 20 x 109/L within 15 days. There was no difference in engraftment between patients receiving autografts preserved with 5 and 10% DMSO. CONCLUSION: PBPC autografts can safely be stored for at least 5 years in 5% DMSO and used for ASCT.

Intensive therapy for multiple myeloma in patients younger than 60 years. Long-term results focusing on the effect of the degree of response on survival and relapse pattern after transplantation.

BACKGROUND AND OBJECTIVES: From 1994 to 1997 we conducted a population-based, prospective study on intensive therapy in newly diagnosed symptomatic myeloma patients younger than 60 years, comparing their survival to that of a conventionally treated historic population. Long-term results are presented, including the impact of the degree of response on survival and relapse pattern after transplantation. DESIGN AND METHODS: The prospective population was formed of 397 patients and the historic population of 313 patients. Both populations were calculated to comprise more than 75% of the expected number of new cases. RESULTS: After a median follow-up of 7 years survival was longer in the prospective population than in the historic one (median 60 versus 39 months; p=0.0002). When comparing only patients eligible for intensive therapy the median survival was 63 versus 44 months (p<0.0001). Attaining a complete response was associated with prolonged event-free survival but not overall survival. The pattern of relapse after transplantation was heterogeneous but could be divided into four major groups; insidious, classical, plasmacytoma form and transformed disease. The median survival after relapse was 29 months. The relapse pattern and time to relapse predicted outcome. Patients relapsing with an insidious or classical form of disease with skeletal events only, or after a long lasting first response were likely to respond well to conventional salvage therapy. In contrast, relapse with multiple symptoms, transformed disease or a short duration of first response implied bad prognosis. INTERPRETATION AND CONCLUSIONS: The relapse pattern after autologous transplantation is heterogeneous and response to salvage therapy is variable. The degree of response and event-free survival after transplantation are not reliable surrogate markers for survival.

Cellular immune responses in multiple myeloma patients with treatment-induced cytopenia early after high-dose chemotherapy and autologous peripheral blood stem cell transplantation.

Proliferative T cell responses were compared for two patient groups with severe treatment-induced leukopenia (white blood cell counts < 0.5 x 10(9)/l): (i). multiple myeloma patients receiving high-dose melphalan and autologous peripheral blood stem cell transplantation; (ii). patients receiving conventional intensive chemotherapy for acute leukemia or myelodysplasia. Although the majority of circulating leukocytes were CD2(+)TCRalphabeta(+) in both groups, the myeloma patients showed significantly lower T cell proliferation in responses to several activation signals (anti-CD3, anti-CD3 + IL2, anti-CD3 + anti-CD28, anti-CD3 + anti-CD28+IL2. Our results suggest that myeloma patients with post-transplant cytopenia have a more severe cellular immune defect than patients with other hematological malignancies and severe cytopenia due to conventional intensive chemotherapy.

A phase I/II study of the MDR modulator Valspodar (PSC 833) combined with daunorubicin and cytarabine in patients with relapsed and primary refractory acute myeloid leukemia.

The cyclosporine analog Valspodar (PSC 833, Novartis Pharma) is a strong inhibitor of the mdr1 gene product p-glycoprotein (pgp). A phase I/II study was conducted in order to evaluate if addition of Valspodar to treatment with daunorubicin and cytarabine, given to patients with primary refractory or relapsed acute myeloid leukemia, could increase the complete remission rate.Fifty-three patients were treated in cohorts of three to six patients. Twelve patients reached a complete remission in bone marrow, five of whom also normalized their peripheral blood values. Three patients experienced treatment-related deaths from pneumonia, liver failure and cerebral hemorrhage, respectively. It is concluded that Valspodar 10 mg/kg per 24 h in combination with daunorubicin 45 mg/m(2) for 3 days and cytarabine 1 g/m(2) twice daily for 4 days is tolerable in this heavily pre-treated group of patients. Due to the moderate treatment results, the phase II part of the study was ended prematurely. The modulation of only pgp did not give an obvious improvement of the treatment results in this group of patients.

[Blood transfusion and pretransfusion testing in patients with warm autoimmune haemolytic anaemia]. / Blodtransfusjon og pretransfusjonsutgreiing ved autoimmun hemolytisk anemi av varmetype.

BACKGROUND: In warm autoimmune haemolytic anaemia, patients have red cell autoantibodies that complicate the serological compatibility testing before transfusion. Different autoadsorption techniques are utilised for more extensive pretransfusion testing. We reviewed the literature on blood transfusion in these patients. We also present our experience with autoadsorption by papain and polyethylene glycol methods. MATERIAL AND METHODS: Blood samples from 13 patients with warm autoantibodies were analysed in parallel. After autoadsorption, antibody screening and identification tests were performed to detect possible red cell alloantibodies. If the serological testing is inadequate, clinically important alloantibodies can be ignored before transfusion, which can lead to haemolytic transfusion reactions. RESULTS: Autoantibodies were completely adsorbed in six of the 13 patients. In five, alloantibodies were detected. In three patients, different results were obtained with the two different techniques. INTERPRETATION: Restrictive transfusion practice in warm autoimmune haemolytic anaemia is recommended. Adequate pretransfusion testing in a specialised laboratory is required before transfusion. Autoadsorption with polyethylene glycol and papain can supplement each other in pretransfusion testing.

[Survival after high-dose therapy with autologous stem cell support]. / Overlevelse etter høydosebehandling med autolog stamcellestøtte.

BACKGROUND: High-dose therapy with autologous stem cell support has been carried out in our hospital since 1996. We have recently collected survival data on patients who have undergone this procedure. MATERIAL AND METHODS: The study population comprised 111 patients, 58 of whom had been diagnosed with multiple myeloma, 38 with various forms of malignant lymphoma, 11 with sarcoma and 4 with testicular cancer. RESULTS: Median survival from reinfusion of stem cells was 74.8 months for patients with myeloma, 47.8 months for patients with malignant lymphoma and 11.7 months for patients with sarcoma. Three-year survival was 72.2 % for myeloma patients and 54.8 % for lymphoma patients. While the survival slope decreased steadily throughout the study period for patients with multiple myeloma, it seemed to level out after approximately 18 months for patients with malignant lymphomas. INTERPRETATION: Our data on myeloma patients show survival comparable to previously published data. For malignant lymphoma patients, our data support the assumption that high-dose therapy has the potential for cure.

Autologous peripheral blood progenitor cells cryopreserved with 5 and 10 percent dimethyl sulfoxide alone give comparable hematopoietic reconstitution after transplantation.

BACKGROUND: Previous in vitro studies have demonstrated decreased apoptosis and necrosis in peripheral blood progenitor cells (PBPCs) cryopreserved with 5 percent instead of 10 percent dimethyl sulfoxide (DMSO). This study was carried out to investigate whether these in vitro findings were supported by clinical data concerning hematopoietic engraftment after autologous stem cell transplantations with PBPCs cryopreserved with 5 and 10 percent DMSO. STUDY DESIGN AND METHODS: During a 6-year period, 103 consecutive patients with newly diagnosed multiple myeloma (MM; n = 58) and lymphoma (n = 45) were transplanted with autologous PBPCs. Throughout the first part of the period cells were cryopreserved with 10 percent DMSO and later with 5 percent. A retrospective comparison was carried out of the clinical results for these two groups. RESULTS: No significant difference in median time to neutrophil and platelet (PLT) engraftment was demonstrated for MM and lymphoma patients transplanted with PBPCs cryopreserved with 5 or 10 percent DMSO. Time until neutrophil counts of more than 0.5 x 10(9) per L was 10 days both for the 5 and 10 percent MM groups and 12 days for both the 5 and the 10 percent lymphoma patients. Median time until stable PLT counts of more than 20 x 10(9) per L was 11 days in all four groups. In addition, transfusion requirements and duration of days admitted to hospital did not differ between the groups. CONCLUSION: The routines for cryopreservation of autografts vary considerably between transplantation centers, and this makes it difficult to compare different clinical studies. Our results suggest that cryopreservation with 5 percent DMSO alone followed by storage in nitrogen is a simple, highly standardized, and safe procedure for cryopreservation of autologous stem cell graft.

Impact of age on survival after intensive therapy for multiple myeloma: a population-based study by the Nordic Myeloma Study Group.

The value of intensive therapy, including autologous stem cell transplantation, in newly diagnosed myeloma patients >60 years is not clear. We evaluated the impact of age (<60 years vs. 60-64 years) on survival in a prospective, population-based setting and compared survival with conventionally treated historic controls. The prospective population comprised 452 patients registered between 1998 and 2000. Of these, 414 received intensive therapy. The historic population, derived from our most recent population-based study on conventional therapy, comprised 281 patients. Of these, 243 fulfilled our eligibility criteria for intensive therapy. For patients undergoing intensive therapy it was found that two factors, beta-2-microglobulin and age <60 years vs. 60-64 years, had independent prognostic impact on survival. However, compared with the historic controls a survival advantage was found both for patients <60 (median 66 months vs. 43 months, P < 0.001) and 60-64 years (median 50 months vs. 27 months; P = 0.001). We conclude that in a population-based setting higher age adversely influences outcome after intensive therapy. Our results indicate that intensive therapy prolongs survival also at age 60-64 years but with less superiority than in younger patients.

A multi-centre prospective study of febrile neutropenia in Norway: microbiological findings and antimicrobial susceptibility.

The urgent need to treat presumptive bacterial or fungal infections in neutropenic patients has meant that initial therapy is empiric and based on the pathogens most likely to be responsible, and drug resistance. The traditional empirical treatment in Norway has been penicillin G and an aminoglycoside, and this combination has been criticized over recent y. We wished to analyse the microbiological spectrum and susceptibility patterns of pathogens causing bacteraemia in febrile neutropenic patients. This was a prospective multicentre study. During the study period of 2 y, a total of 282 episodes of fever involving 243 neutropenic patients was observed. In 34% of episodes bacteraemia was documented. Overall, 40% of the episodes were caused by Gram-positive organisms, 41% by Gram-negative organisms and 19% were polymicrobial. The most frequently isolated bacteria were Escherichia coli (25.6%), a- and non-haemolytic streptococci (15.6%), coagulase-negative staphylococci (12.4%) and Klebsiella spp. (7.4%). None of the Gram-negative isolates was resistant to gentamicin, meropenem, ceftazidime or ciprofloxacin. Only 5 coagulase-negative staphylococci isolates were resistant to both penicillin G and aminoglycoside. The overall mortality rate was 7%, and 1.2% due to confirmed bacteraemic infection.

Functional characterization of T lymphocytes derived from patients with acute myelogenous leukemia and chemotherapy-induced leukopenia.

T-cell-targeting immunotherapy is now considered in acute myelogenous leukemia (AML). Immunotherapy seems most effective for patients with a low AML cell burden, and a possible strategy is therefore to administer immunotherapy early after intensive chemotherapy when patients have a low leukemia cell burden and severe treatment-induced cytopenia. To further investigate this possible therapeutic approach we used a whole blood assay to characterize the proliferative responsiveness (3H-thymidine incorporation) of circulating T cells from AML patients with severe treatment-induced leukopenia, i.e., peripheral blood leukocyte counts < 0.5x10(9)/l. This assay will reflect both quantitative and qualitative differences. Responses were compared for 17 AML patients, 6 patients with acute lymphoblastic leukemia (ALL), and a group of 21 healthy controls. Most circulating leukocytes in the AML patients were T lymphocytes, whereas B lymphocytes and monocytes usually constituted < 10%. Anti-CD3-stimulated proliferation was significantly lower for AML patients compared with healthy controls. However, proliferation in response to anti-CD3 + anti-CD28 did not differ for AML patients and healthy controls, an observation suggesting that T cells from AML patients have an increased responsiveness in the presence of optimal costimulation that compensates for the quantitative T-cell defect. In contrast, the responses were significantly lower for ALL than for AML patients. We conclude that the remaining T-cell population in AML patients with severe chemotherapy-induced cytopenia show an increased proliferative responsiveness and may represent a therapeutic target when antileukemic immunotherapy is tried in combination with intensive chemotherapy.

Malignancy: Insulin-like Growth Factor-1 (IGF-1) is a Costimulator of the Expansion of Lineage Committed Cells Derived from Peripheral Blood Mobilized CD34+ Cells in Multiple Myeloma Patients.

The effect of insulin-like growth factor-1 (IGF-1) on highly enriched human apheresis CD34(+) progenitor cells was investigated in vitro. The progenitor cells were mobilized by treatment with cyclophosphamide + granulocyte - colony stimulating factor (G-CSF) in patients with multiple myeloma. CD34(+) cells were cultured for 7 days in serumfree medium containing stem cell factor (SCF), granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3), and this is referred to as cytokine-dependent proliferation. After 7 days of cytokine-dependent proliferation the total number of viable cells increased 1.6-8.2 times, and subsets of cells expressing the granulocyte marker CD15, the myelomonocytic marker CD64 and the erythrocyte phenotype CD71(high) /CD64(-) were detected among the in vitro cultured cells. Addition of G-CSF together with SCF + IL-3 + GM-CSF increased the number of CD15(+) and CD64(+) cells, but without altering the number of erythroid cells. IGF-1 caused a dose-dependent increase in the number of CD15(+), CD64(+) and CD71(high) /CD64(-) cells, and this increase was detected when cells were cultured in both SCF + IL-3 + GM-CSF alone and G-CSF + SCF + IL-3 + GM-CSF. A minor subset of CD34(+) cells could still be detected among in vitro cultured cells and the number of CD34(+) cells was not altered by adding G-CSF and/or IGF-1. Morphologically recognizable mature granulocytes or erythroid cells could not be detected for any of the combinations investigated. We conclude that IGF-1 can enhance the in vitro proliferation of committed progenitor cells derived from apheresis CD34(+) cells.