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.

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/m(2)/day, days 1-5, every 28 days) followed by DLI (from 1-5 × 10(6) to 1-5 × 10(8) 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.

Tacrolimus and mycofenolate mofetil as GvHD prophylaxis following nonmyeloablative conditioning and unrelated hematopoietic SCT for adult patients with advanced hematologic diseases.

In the current study, we evaluated a combination of tacrolimus and mycophenolate mofetil (MMF) as GvHD prophylaxis in 50 patients undergoing truly nonmyeloablative (NM; 90 mg/m(2) fludarabine, 2 Gy TBI) hematopoietic SCT (HSCT) from unrelated donors. Median patient age was 51 years (range, 25-67 years). After a median follow-up of 1123 days (range, 47-2729 days), 20 patients (40%) are alive and free from disease. The probabilities of 1-, 2- and 3-year survival were 57, 47 and 39%, respectively. Patients who achieved a remission before HSCT had a significantly better OS compared with those who had active disease (P=0.01). The incidences of grade II-IV and III-IV acute GvHD (aGvHD) were 54% (n=27) and 16% (n=8). Remarkably, using tacrolimus and MMF, the median onset of aGvHD occurred distinctly late on day +66 (range, 12-119 days). A total of 46 patients were evaluable for chronic GvHD (cGvHD). Out of these, 26 (56%) patients developed cGvHD, with 16 (34%) of them showing limited and 10 (21%) showing extensive disease. We conclude that the combination of tacrolimus and MMF as post transplant immunosuppression for patients receiving NM unrelated donor HSCT permits stable engraftment and effective prophylaxis for acute and cGvHD. In particular, the occurrence of severe early-onset aGvHD was attenuated.

A 3-day short course of palifermin before HDT reduces toxicity and need for supportive care after autologous blood stem-cell transplantation in patients with multiple myeloma.

BACKGROUND: We retrospectively determined whether a 3-day short course of palifermin could reduce the toxicity of high-dose therapy (HDT) and autologous blood stem-cell transplantation (ASCT) in patients with multiple myeloma (MM). PATIENTS AND METHODS: Sixty-seven consecutive patients received 60 mug/kg palifermin for 3 days before HDT with melphalan 200 or 140 mg/m(2) for patients with renal failure (group A). Granulocyte colony-stimulating factor (G-CSF) was applied after ASCT. Data on haematopoietic reconstitution and toxicity were compared with two previously published patient groups from our institution who had received pegfilgrastim but not palifermin (group B, n = 21) and patients who had received neither palifermin nor G-CSF (group C, n = 21). RESULTS: In group A, patients with renal failure had a significantly higher risk for severe mucositis (64% versus 16%, P < 0.002). Patients with normal renal function who received palifermin experienced significantly less days of hospitalisation (P < 0.05) and less need for narcotic analgesia (P < 0.05), parenteral nutrition (P < 0.05) and erythrocyte transfusions (P < 0.05) in comparison with groups B and C. Time to haematopoietic reconstitution was not compromised by the use of palifermin. CONCLUSIONS: In conclusion, a short 3-day course of palifermin may be able to reduce the toxicity of HDT and ASCT in patients with MM. Patients with impaired renal function at the time of HDT need additional strategies to further reduce the incidence of severe mucositis.

Bendamustine in patients with relapsed or refractory multiple myeloma.

OBJECTIVE: In patients with multiple myeloma, bendamustine monotherapy is effective as 1st and 2nd line therapy. However, data for patients with advanced multiple myeloma is rare. METHODS: In this retrospective analysis we have identified 39 patients with relapsed or refractory multiple myeloma by means of case research, who have been treated at our institution with bendamustine as salvage therapy. After in median 2 lines of prior therapy (range: 1-5) patients received in median 3 (range: 1-10) cycles of bendamustine. Bendamustine dosage was 80-150 mg on day 1+2 of a monthly cycle. Bendamustine was administered as monotherapy in 39% of patients, whereas 61% received concomitant steroids. RESULTS: Toxicity was mild to moderate. Response rates were as follows: 3% vgPR, 33% PR, 18% MR, 26% SD and 20% PD. The median event-free and overall survival were 7 and 17 months, respectively. CONCLUSIONS: In conclusion, in patients with advanced multiple myeloma bendamustine is effective and associated with mild toxicity. Therefore, the role of bendamustine in patients with multiple myeloma should be investigated in further clinical trials.

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/m(2) 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.

Fludarabine, amsacrine, high-dose cytarabine and 12 Gy total body irradiation followed by allogeneic hematopoietic stem cell transplantation is effective in patients with relapsed or high-risk acute lymphoblastic leukemia.

In this prospective study, we examined the toxicity and efficacy of an intensified conditioning regimen for treatment of patients with relapsed or high-risk acute lymphoblastic leukemia who undergo allogeneic hematopoietic stem cell transplantation. Fifteen patients received fludarabine 30 mg/m(2), cytarabine 2000 mg/m(2), amsacrine 100 mg/m(2) on days -10, -9, -8 and -7, anti-thymocyte globulin (ATG-Fresenius) 20 mg/kg body weight on days -6, -5 and -4 and fractionated total body irradiation 2 x 2 Gy on days -3, -2 and -1 (FLAMSA-ATG-TBI) before allogeneic hematopoietic stem cell transplantation. At the time of hematopoietic stem cell transplantation, 10 patients were in complete remission (8 CR1; 2 CR2), 3 with primary refractory and 2 suffered from refractory relapse. All patients achieved a complete remission after hematopoietic stem cell transplantation; and after a median follow-up time of 1091 days (range, 334-1554 days), nine patients (60%) are alive and free from disease, including three patients with prior refractory disease. Three patients died due to treatment-related mortality. The most frequent and severe conditioning-related toxicities observed in 9 out of 15 patients were grade III/IV infections according to common toxicity criteria. Thus, conditioning with the FLAMSA-ATG-TBI regimen is a feasible and effective alternative for patients with relapsed or high-risk acute lymphoblastic leukemia.

Pegfilgrastim for PBSC mobilization and autologous haematopoietic SCT.

To date, G-CSF is the most favoured cytokine administered for PBSC mobilization because of its great efficacy and lack of serious toxicity. Recently, a pegylated filgrastim (pegfilgrastim) has been introduced. Attachment of the polyethylene glycol (PEG) moiety reduces renal excretion and masks proteolytic cleavage sites resulting in elevated G-CSF serum levels for up to 14 days after a single injection. As single-dose pegfilgrastim had similar effects in the prophylaxis of chemotherapy-induced neutropenia as the daily administration of the unconjugated drug, its capability for the mobilization of haematopoietic stem and progenitor cells has been assessed and presented to be at least equal to that of conventional G-CSF. Administration of pegfilgrastim following high-dose therapy and autologous blood SCT (BSCT) shortened the time to myeloid recovery as seen in conventional G-CSF. Plasma G-CSF levels were about 1 log higher with pegfilgrastim, but in the setting of autologous BSCT this did not translate into a faster haematopoietic recovery. Only few data exist on the biological effects of pegfilgrastim. Still, these data suggest that pegfilgrastim-stimulation results in different functional properties of haematopoietic stem and progenitor cells compared with G-CSF.

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.

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.

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 factorbeta 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.

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

Following induction therapy and 4 g/m(2) cyclophosphamide, a single dose of 12 mg polyethyleneglycol-conjugated G-CSF (pegfilgrastim; n=12) or daily doses of unconjugated G-CSF (8.5 mug/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/mul). Following high-dose melphalan (200 mg/m(2)) 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.

[Evaluation of the therapeutic benefit of 41.8 degrees C whole body hyperthermia plus ifosfamide, carboplatin and etoposide (ICE) for patients with malignant pleural mesothelioma using the Modified Brunner-Score (MBS)]. / Bewertung des therapeutischen Gewinns von Patienten mit Pleuramesotheliom unter Therapie mit 41,8 degrees C Ganzkörperhyperthermie plus Ifosfamid, Carboplatin und Etoposid (ICE) anhand des Modifizierten Brunner-Score (MBS).

BACKGROUND: We performed a phase-II-study combining 41.8 degrees C whole body hyperthermia with ICE chemotherapy, i. e., ifosfamide (5 g/m (2) on day 1), carboplatin (300 mg/m (2) on day 1) and etoposide (150 mg/m (2) on days 2 and 3), administered every 4 weeks, to assess the treatment benefit for patients with malignant pleural mesothelioma. To date this is mainly done by measurement of response rates and overall survival, as it can be widely found in the literature. In fewer cases there is also a quality of life assessment. Here we describe an instrument well-capable for a more comprehensive statement on the therapeutic benefit by linking several study end points including quality of life assessment, the Modified Brunner-Score (MBS). MATERIAL AND METHODS: The Modified Brunner Score (MBS) was used for this assessment. MBS integrates progression free survival, change of physical performance (WHO-index), a quality of life self-assessment by the patient and toxicity. A positive score means a therapy benefit and vice versa. RESULTS AND CONCLUSIONS: Of 27 chemonäive, non-metastatic patients enrolled, 22 were evaluable for assessment. Overall survival and progression free survival for all patients was 76 weeks (95 % CI 65.4 weeks - 87.8 weeks) and 29.6 weeks (95 % CI 24.4 weeks - 34.7 weeks) respectively. Major treatment toxicities included grade 3/4 neutropenia and thrombocytopenia and affection of the GI tract, like mucositis, nausea and vomiting. Mean Improvement of Performance Index (WHO) was 0.29 points. The MBS showed a score of 4.21 points (- 4.43 - 16.45 range) for the overall study group. 16 of 22 evaluable patients achieved a positive score. MBS is a suitable tool to evaluate the treatment benefit especially in non-standard therapy approaches. For WBH plus ICE, it showed a beneficial effect on overall quality of life in the majority of patients.

Ifosfamide, carboplatin and etoposide combined with 41.8 degrees C whole body hyperthermia for malignant pleural mesothelioma.

We performed a phase II study combining 41.8 degrees C whole body hyperthermia with ICE chemotherapy, i.e. ifosfamide (5 g/m(2)), carboplatin (300 mg/m(2)) and etoposide (150 mg/m(2) on days 2 and 3), administered every 4 weeks, for patients with malignant pleural mesothelioma. Of 27 chemonäive, non-metastatic patients enrolled, 25 patients were evaluable for response. Overall response rate was 20% (five partial remissions; 95% CI 8.9-39.1%). Median survival time from the start of treatment for all patients was 76.6 weeks (95% CI 65.4-87.8 weeks). Progression free survival for all patients measured 29.6 weeks (95% CI 24.4-34.7 weeks). One year overall survival was 68% and 2 year overall survival was 20%. Major treatment toxicities included grade 3/4 neutropenia and thrombocytopenia in 74 and 33% of treatment cycles, respectively. One patient died due to sepsis. These promising results are consistent with continued clinical investigation; a phase III clinical trial with whole body hyperthermia as the independent variable has been initiated.

[Chemotherapy in combination with whole-body hyperthermia in advanced malignant pleural mesothelioma]. / Chemotherapie in Kombination mit Ganzkörperhyperthermie bei fortgeschrittenem malignem Pleuramesotheliom.

HISTORY AND CLINICAL FINDINGS: A 57-year-old man presented with dyspnoea, cough, fatigue and weight loss. He had been exposed to asbestos 30 years ago. Physical examination was unremarkable apart from a suspected pleural effusion. INVESTIGATIONS: Computed tomography (CT) of the thorax showed multiple pleural masses with pleural effusion on the left side. CT of the abdomen and bronchoscopy were normal. The patient underwent explorative thoracoscopy; biopsies were taken, and diffuse malignant pleural mesothelioma was demonstrated. TREATMENT AND COURSE: The patient was evaluated at the University Hospital Lübeck for Phase II experimental therapy with whole-body hyperthermia (WBH). The pretreatment evaluation revealed normal cardiorespiratory function and a normal contrast-enhanced CT of the brain. The patient's haematologic profile and electrolytes were normal. The WBH-radiant heat device (RHD) used for therapy was Aquatherm provided by the Cancer Research Institute (CRI, New York, USA). The patient received ifosfamide (5 g/m2, day 1), carboplatin (300 mg/m2, day 1), etoposide (150 mg/m2, days 2-3) combined with WBH at 41.8 degrees C (for 60 minutes). Two cycles were applied without complications and a partial remission of the disease was observed. CONCLUSION: Radiant heat whole body hyperthermia, in conjunction with a defined anticancer treatment and pharmacological approach to sedation, was a safe and effective palliative treatment in this patient.