Roland Fenk

Hyperfractionated cyclophosphamide in combination with pulsed dexamethasone and thalidomide (HyperCDT) in primary refractory or relapsed multiple myeloma

Summary. Sixty patients with advanced multiple myeloma received 2–6 monthly treatment courses combining hyperfractionated cyclophosphamide (300 mg/m2 i.v. over 3 h q 12 h × 6, d 1–3) with pulsed dexamethasone (20 mg/m2/d p.o., d 1–4, 9–12, 17–20) and once daily thalidomide at individually escalating doses (100–400 mg/d) depending on tolerability (HyperCDT). Responding patients were maintained on daily thalidomide and monthly dexamethasone pulses. Complete, partial and minor response rates were 4%, 68% and 12% respectively; overall response rate was 84% (efficacy analysis). Median event‐free and overall survival was 11 and 19 months respectively. During at least one treatment cycle, 67% of patients experienced grade 4 neutropenia resulting in 17% grade 3 and 9% grade 4 infections. Side‐effects, presumably related to thalidomide, included neuropathy (40% grade 2, 16% grade 3), constipation (17%), oedema (5%), bradycardia (5%), skin reactions (3%), cerebrovascular events (5%) and deep vein thromboses (8%). Thromboses were not related to known thrombophilic risk factors. Four patients with prior myeloma therapy > 50 months developed myelodysplastic syndrome or secondary acute myeloid leukaemia 2–4 months after study entry. HyperCDT is a highly active and reasonably well‐tolerated salvage regimen in advanced or refractory multiple myeloma.

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.

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.

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.

Cyclosporine A and Mycophenolate Mofetil vs Cyclosporine A and Methotrexate for graft-versus-host disease prophylaxis after stem cell transplantation from HLA-identical siblings

Summary:The combination of Cyclosporin A (CSA) and Methotrexate (MTX) is considered to be the standard regimen for the prevention of graft-versus-host disease (GVHD) after stem cell transplantation (SCT) from HLA-identical siblings. Mycophenolate Mofetil (MMF) has been widely used for GVHD prophylaxis after nonmyeloablative SCT, but experience following myeloablative therapy is still limited. We retrospectively compared CSA/MTX and CSA/MMF in 93 patients (median age 35 years, range 17–59 years, male subjects 48, female subjects 45) with acute myeloid leukemia (n=33), myelodysplastic syndrome (MDS) (n=3), acute lymphoblastic leukemia (ALL) (n=20) or chronic myeloid leukemia (n=37) who received CSA/MMF (n=26) or CSA/MTX (n=67) as GVHD prophylaxis following high-dose therapy and allogeneic SCT from HLA-identical siblings. No statistically significant differences were found in overall survival, relapse rate, treatment-related mortality and acute or chronic GVHD. Time to myeloid recovery was significantly shorter in patients who received CSA/MMF. We conclude that the combination of CSA/MMF appears equivalent to CSA/MTX for GVHD prophylaxis in patients receiving conventional-intensity SCT from HLA-identical siblings.

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.

IFN-alpha skews monocytes into CD56+-expressing dendritic cells with potent functional activities in vitro and in vivo.

The antitumor effect of IFN-α is mediated by the activation of CTLs, NK cells, and the generation of highly potent Ag-presenting dendritic cells (IFN-DCs). In this study, we show that IFN-DCs generated in vitro from monocytes express CD56 on their surface, a marker which has been thought to be specific for NK cells. FACS analyses of CD56+ and CD56− IFN-DCs showed a nearly identical pattern for most of the classical DC markers. Importantly, however, only CD56+ IFN-DCs exhibited cytolytic activity up to 24% that could almost completely be blocked (−81%) after coincubation with anti-TRAIL. Intracytoplasmatic cytokine staining revealed that the majority of IFN-DCs independently of their CD56 expression were IFN-γ positive as well. In contrast, CD56+ IFN-DCs showed stronger capacity in stimulating allogenic T cells compared with CD56− IFN-DC. Based on these results, five patients with metastasized medullary thyroid carcinoma were treated for the first time with monocyte-derived tumor Ag-pulsed IFN-DCs. After a long term follow-up (in mean 37 mo) all patients are alive. Immunohistochemical analyses of delayed-type hypersensitivity skin reaction showed a strong infiltration with CD8+ cells. In two patients no substantial change in tumor morphology was detected. Importantly, by analyzing PBMCs, these patients also showed an increase of Ag-specific IFN-γ-secreting T cells. In summary, we here describe for the first time that cytotoxic activity of IFN-DCs is mainly mediated by an IFN-DC subset showing partial phenotypic and functional characteristics of NK cells. These cells represent another mechanism of the antitumor effect induced by IFN-α.

Treatment of Acute Myeloid Leukemia or Myelodysplastic Syndrome Relapse after Allogeneic Stem Cell Transplantation with Azacitidine and Donor Lymphocyte Infusions—A Retrospective Multicenter Analysis from the German Cooperative Transplant Study Group

To expand the current knowledge about azacitidine (Aza) and donor lymphocyte infusions (DLI) as salvage therapy for relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and to identify predictors for response and survival, we retrospectively analyzed data of 154 patients with acute myeloid leukemia (AML, n = 124), myelodysplastic (MDS, n = 28), or myeloproliferative syndrome (n = 2). All patients received a median number of 4 courses of Aza (range, 4 to 14) and DLI were administered to 105 patients (68%; median number of DLI, 2; range, 1 to 7). Complete and partial remission rates were 27% and 6%, respectively, resulting in an overall response rate of 33%. Multivariate analysis identified molecular-only relapse (hazard ratio [HR], 9.4; 95% confidence interval [CI], 2.0 to 43.5; P = .004) and diagnosis of MDS (HR, 4.1; 95% CI, 1.4 to 12.2; P = .011) as predictors for complete remission. Overall survival (OS) at 2 years was 29% ± 4%. Molecular-only relapse (HR, .14; 95% CI, .03 to .59; P = .007), diagnosis of MDS (HR, .33; 95% CI, .16 to .67; P = .002), and bone marrow blasts <13% (HR, .54; 95% CI, .32 to .91; P = .021) were associated with better OS. Accordingly, 2-year OS rate was higher in MDS patients (66% ± 10%, P = .001) and correlated with disease burden in patients with AML. In summary, Aza and DLI is an effective and well-tolerated treatment option for patients with relapse after allo-HSCT, in particular those with MDS or AML and low disease burden. The latter finding emphasizes the importance of stringent disease monitoring and early intervention.

Monocyte derived dendritic cells generated by IFN-α acquire mature dendritic and natural killer cell properties as shown by gene expression analysis

BackgroundDendritic cell (DC) vaccines can induce antitumor immune responses in patients with malignant diseases, while the most suitable DC culture conditions have not been established yet. In this study we compared monocyte derived human DC from conventional cultures containing GM-CSF and IL-4/TNF-α (IL-4/TNF-DC) with DC generated by the novel protocol using GM-CSF and IFN-α (IFN-DC).MethodsTo characterise the molecular differences of both DC preparations, gene expression profiling was performed using Affymetrix microarrays. The data were conformed on a protein level by immunophenotyping, and functional tests for T cell stimulation, migration and cytolytic activity were performed.ResultsBoth methods resulted in CD11c+ CD86+ HLA-DR+ cells with a typical DC morphology that could efficiently stimulate T cells. But gene expression profiling revealed two distinct DC populations.Whereas IL-4/TNF-DC showed a higher expression of genes envolved in phagocytosis IFN-DC had higher RNA levels for markers of DC maturity and migration to the lymph nodes like DCLAMP, CCR7 and CD49d. This different orientation of both DC populations was confined by a 2.3 fold greater migration in transwell experiments (p = 0.01).Most interestingly, IFN-DC also showed higher RNA levels for markers of NK cells such as TRAIL, granzymes, KLRs and other NK cell receptors. On a protein level, intracytoplasmatic TRAIL and granzyme B were observed in 90% of IFN-DC. This translated into a cytolytic activity against K562 cells with a median specific lysis of 26% at high effector cell numbers as determined by propidium iodide uptake, whereas IL-4/TNF-DC did not induce any tumor cell lysis (p = 0.006). Thus, IFN-DC combined characteristics of mature DC and natural killer cells.ConclusionOur results suggest that IFN-DC not only stimulate adaptive but also mediate innate antitumor immune responses. Therefore, IFN-DC should be evaluated in clinical vaccination trials. In particular, this could be relevant for patients with diseases responsive to a treatment with IFN-α such as Non-Hodgkin lymphoma or chronic myeloid leukemia.