Ingmar Bruns

Azacitidine and donor lymphocyte infusions as first salvage therapy for relapse of AML or MDS after allogeneic stem cell transplantation.

The combination of azacitidine and donor lymphocyte infusions (DLI) as first salvage therapy for relapse after allogeneic transplantation (allo-HSCT) was studied in 30 patients with acute myeloid leukemia (AML; n=28) or myelodysplastic syndromes (MDS; n=2) within a prospective single-arm multicenter phase-II trial. Treatment schedule contained up to eight cycles azacitidine (100 mg/m2/day, days 1–5, every 28 days) followed by DLI (from 1–5 × 106 to 1–5 × 108 CD3+cells/kg) after every second azacitidine cycle. A median of three courses azacitidine (range 1–8) were administered, and 22 patients (73%) received DLI. Overall response rate was 30%, including seven complete remissions (CRs, 23%) and two partial remissions (7%). Five patients remain in CR for a median of 777 days (range 461–888). Patients with MDS or AML with myelodysplasia-related changes were more likely to respond (P=0.011), and a lower blast count (P=0.039) as well as high-risk cytogenetics (P=0.035) correlated with the likelihood to achieve CR. Incidence of acute and chronic graft-versus-host disease was 37% and 17%, respectively. Neutropenia and thrombocytopenia grade III/IV occurred during 65% and 63% of treatment cycles, while infections were the most common grade III/IV non-hematological toxicity. Azacitidine and DLI as salvage therapy is safe, induces long-term remissions and may become an alternative for patients with AML or MDS relapsing after allo-HSCT.

Molecular signature of CD34 + hematopoietic stem and progenitor cells of patients with CML in chronic phase

In this study, we provide a molecular signature of highly enriched CD34+ cells from bone marrow of untreated patients with chronic myelogenous leukemia (CML) in chronic phase in comparison with normal CD34+ cells using microarrays covering 8746 genes. Expression data reflected several BCR-ABL-induced effects in primary CML progenitors, such as transcriptional activation of the classical mitogen-activated protein kinase pathway and the phosphoinositide-3 kinase/AKT pathway as well as downregulation of the proapoptotic gene IRF8. Moreover, novel transcriptional changes in comparison with normal CD34+ cells were identified. These include upregulation of genes involved in the transforming growth factorβ pathway, fetal hemoglobin genes, leptin receptor, sorcin, tissue inhibitor of metalloproteinase 1, the neuroepithelial cell transforming gene 1 and downregulation of selenoprotein P. Additionally, genes associated with early hematopoietic stem cells (HSC) and leukemogenesis such as HoxA9 and MEIS1 were transcriptionally activated. Differential expression of differentiation-associated genes suggested an altered composition of the CD34+ cell population in CML. This was confirmed by subset analyses of chronic phase CML CD34+ cells showing an increase of the proportion of megakaryocyte-erythroid progenitors, whereas the proportion of HSC and granulocyte–macrophage progenitors was decreased in CML. In conclusion, our results give novel insights into the biology of CML and could provide the basis for identification of new therapeutic targets.

Insufficient stromal support in MDS results from molecular and functional deficits of mesenchymal stromal cells

Ineffective hematopoiesis is a major characteristic of myelodysplastic syndromes (MDS) causing relevant morbidity and mortality. Mesenchymal stromal cells (MSC) have been shown to physiologically support hematopoiesis, but their contribution to the pathogenesis of MDS remains elusive. We show that MSC from patients across all MDS subtypes (n=106) exhibit significantly reduced growth and proliferative capacities accompanied by premature replicative senescence. Osteogenic differentiation was significantly reduced in MDS-derived MSC, indicated by cytochemical stainings and reduced expressions of Osterix and Osteocalcin. This was associated with specific methylation patterns that clearly separated MDS–MSC from healthy controls and showed a strong enrichment for biological processes associated with cellular phenotypes and transcriptional regulation. Furthermore, in MDS–MSC, we detected altered expression of key molecules involved in the interaction with hematopoietic stem and progenitor cells (HSPC), in particular Osteopontin, Jagged1, Kit-ligand and Angiopoietin as well as several chemokines. Functionally, this translated into a significantly diminished ability of MDS-derived MSC to support CD34+ HSPC in long-term culture-initiating cell assays associated with a reduced cell cycle activity. Taken together, our comprehensive analysis shows that MSC from all MDS subtypes are structurally, epigenetically and functionally altered, which leads to impaired stromal support and seems to contribute to deficient hematopoiesis in MDS.

Effect of the nonpeptide thrombopoietin receptor agonist Eltrombopag on bone marrow cells from patients with acute myeloid leukemia and myelodysplastic syndrome

Thrombocytopenia is a frequent symptom and clinical challenge in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Eltrombopag is a small molecule thrombopoietin receptor agonist that might be a new option to treat thrombocytopenia in these diseases, provided that it does not stimulate malignant hematopoiesis. In this work, we studied the effects of Eltrombopag on proliferation, apoptosis, differentiation, colony formation, and malignant self-renewal of bone marrow mononuclear cells of patients with AML and MDS. Malignant bone marrow mononuclear cells did not show increased proliferation, or increased clonogenic capacity at concentrations of Eltrombopag ranging from 0.1 to 30 microg/mL. On the contrary, we observed a moderate, statistically nonsignificant (P = .18), decrease of numbers of malignant cells (mean, 56%; SD, 28%). Eltrombopag neither led to increased 5-bromo-2-deoxyuridine incorporation, decreased apoptosis, an increase of malignant self-renewal, nor enhanced in vivo engraftment in xenotransplantations. Furthermore, we found that Eltrombopag was capable of increasing megakaryocytic differentiation and formation of normal megakaryocytic colonies in patients with AML and MDS. These results provide a preclinical rationale for further testing of Eltrombopag for treatment of thrombocytopenia in AML and MDS.

5-Azacytidine for the treatment of patients with acute myeloid leukemia or myelodysplastic syndrome who relapse after allo-SCT: a retrospective analysis

Patients with AML or myelodysplastic syndrome who relapse after allo-SCT have a poor prognosis. In the search for novel treatment strategies for these patients, we conducted a multicenter retrospective analysis and identified 22 patients treated with the DNA-methylation inhibitor 5-azacytidine (5-Aza). Patients received a median number of two cycles 5-Aza (range 1–8) at a dose of 100 mg/m2 over 5 days following relapse. Eighteen patients (82%) also received a median number of two donor lymphocyte infusions (DLI, range 1–5). Sixteen patients (72%) responded to 5-Aza treatment and five patients (23%) achieved a CR. 5-Aza-induced CR lasted for 433 days (median, range 114–769). Median survival and the estimated 2-year survival rate were 144 days and 23%, respectively. Acute GVHD after DLI was seen in six patients (33%) and four of these patients developed chronic GVHD of the skin. There were no treatment-related deaths. Patients who achieved halving of leukocyte counts after the first 5-Aza cycle had a superior median survival of 802 days compared with 135 days (P=0.0025) in all other patients. On univariate analysis, the achievement of this halving of leukocyte counts was identified as a significant predictor of survival.

Successful transplantation of peripheral blood stem cells mobilized by chemotherapy and a single dose of pegylated G-CSF in patients with multiple myeloma

Summary:Following induction therapy and 4 g/m2 cyclophosphamide, a single dose of 12 mg polyethyleneglycol-conjugated G-CSF (pegfilgrastim; n=12) or daily doses of unconjugated G-CSF (8.5 μg/kg/day) (n=12) were administered to myeloma patients. Pegfilgrastim was associated with an earlier leukocyte recovery (12 vs 14 days) and peripheral blood CD34+ cell peak (12 vs 15 days). The peripheral blood CD34+ cell peak was lower in the pegfilgrastim group (78 vs 111/μl). Following high-dose melphalan (200 mg/m2) and autografting, leukocyte and platelet reconstitution was similar in both groups and stable blood counts were observed 100 days post transplant. In summary, a single dose of pegfilgrastim after chemotherapy is capable of mobilizing a sufficient number of CD34+ cells for successful autografting with early engraftment and sustained hematological reconstitution in patients with myeloma. These data provide the basis for randomized studies evaluating the optimal dose and time of pegfilgrastim as well as long-term outcome in larger cohorts of patients.

Sorafenib (Nexavar®) induces molecular remission and regression of extramedullary disease in a patient with FLT3-ITD+ acute myeloid leukemia

The fms-related tyrosine kinase 3 internal tandem duplication (FLT3-ITD) can be found in about one quarter of patients with acute myeloid leukemia (AML) [Small D. FLT3 mutations: biology and treatment. Hematology Am Soc Hematology. Educ. Program 2006;178-84 [Review]]. Patients who carry this mutation have a high risk of relapse even after allogeneic stem cell transplantation [Sheikhha MH, Awan A, Tobal K, Liu Yin JA. Prognostic significance of FLT3 ITD and D835 mutations in AML patients. Hematol J 2003;4:41-6; Meshinchi S, Arceci RJ, Sanders JE, Smith FO, Woods WB, Radich JP, et al. Role of allogeneic stem cell transplantation in FLT3/ITD-positive AML. Blood 2006;108(1):400-1]. Recent reports show that Sorafenib, a multikinase inhibitor has significant activity against FLT3-ITD(+) blasts in vitro [Auclair D, Miller D, Yatsula V, Pickett W, Carter C, Chang Y, et al. Antitumor activity of sorafenib in FLT3-driven leukemic cells. Leukemia 2007;21(3):439-45]. We here report the first clinical case of molecular remission induced by Sorafenib in a patient with FLT3-ITD(+) AML and extramedullary disease after allogenic stem cell transplantation.

The hematopoietic stem cell in chronic phase CML is characterized by a transcriptional profile resembling normal myeloid progenitor cells and reflecting loss of quiescence.

We found that composition of cell subsets within the CD34+ cell population is markedly altered in chronic phase (CP) chronic myeloid leukemia (CML). Specifically, proportions and absolute cell counts of common myeloid progenitors (CMP) and megakaryocyte–erythrocyte progenitors (MEP) are significantly greater in comparison to normal bone marrow whereas absolute numbers of hematopoietic stem cells (HSC) are equal. To understand the basis for this, we performed gene expression profiling (Affymetrix HU-133A 2.0) of the distinct CD34+ cell subsets from six patients with CP CML and five healthy donors. Euclidean distance analysis revealed a remarkable transcriptional similarity between the CML patients’ HSC and normal progenitors, especially CMP. CP CML HSC were transcriptionally more similar to their progeny than normal HSC to theirs, suggesting a more mature phenotype. Hence, the greatest differences between CP CML patients and normal donors were apparent in HSC including downregulation of genes encoding adhesion molecules, transcription factors, regulators of stem-cell fate and inhibitors of cell proliferation in CP CML. Impaired adhesive and migratory capacities were functionally corroborated by fibronectin detachment analysis and transwell assays, respectively. Based on our findings we propose a loss of quiescence of the CML HSC on detachment from the niche leading to expansion of myeloid progenitors.

Complete remission in a patient with relapsed angioimmunoblastic T-cell lymphoma following treatment with bevacizumab

Complete remission in a patient with relapsed angioimmunoblastic T-cell lymphoma following treatment with bevacizumab

A single dose of 6 or 12 mg of pegfilgrastim for peripheral blood progenitor cell mobilization results in similar yields of CD34+ progenitors in patients with multiple myeloma

BACKGROUND:  Current regimens for peripheral blood progenitor cell (PBPC) mobilization in patients with multiple myeloma are based on daily subcutaneous injections of granulocyte–colony‐stimulating factor (G‐CSF) starting shortly after cytotoxic therapy. Recently a polyethylene glycol–conjugated G‐CSF (pegfilgrastim) was introduced that has a substantially longer t1/2 than the original formula.