Martin Bornhäuser

Mesenchymal Stem Cells Can Be Differentiated Into Endothelial Cells In Vitro

Human bone marrow‐derived mesenchymal stem cells (MSCs) have the potential to differentiate into mesenchymal tissues like osteocytes, chondrocytes, and adipocytes in vivo and in vitro. The aim of this study was to investigate the in vitro differentiation of MSCs into cells of the endothelial lineage. MSCs were generated out of mononuclear bone marrow cells from healthy donors separated by density gradient centrifugation. Cells were characterized by flow cytometry using a panel of monoclonal antibodies and were tested for their potential to differentiate along different mesenchymal lineages. Isolated MSCs were positive for the markers CD105, CD73, CD166, CD90, and CD44 and negative for typical hematopoietic and endothelial markers. They were able to differentiate into adipocytes and osteocytes after cultivation in respective media. Differentiation into endothelial‐like cells was induced by cultivation of confluent cells in the presence of 2% fetal calf serum and 50 ng/ml vascular endothelial growth factor. Laser scanning cytometry analysis of the confluent cells in situ showed a strong increase of expression of endothelial‐specific markers like KDR and FLT‐1, and immunofluorescence analysis showed typical expression of the von Willebrand factor. The functional behavior of the differentiated cells was tested with an in vitro angiogenesis test kit where cells formed characteristic capillary‐like structures. We could show the differentiation of expanded adult human MSCs into cells with phenotypic and functional features of endothelial cells. These predifferentiated cells provide new options for engineering of artificial tissues based on autologous MSCs and vascularized engineered tissues.

Standard graft-versus-host disease prophylaxis with or without anti-T-cell globulin in haematopoietic cell transplantation from matched unrelated donors: a randomised, open-label, multicentre phase 3 trial

BACKGROUND Graft-versus-host disease (GVHD) is a major cause of morbidity and mortality after allogeneic haematopoietic cell transplantation from unrelated donors. Anti-T-cell globulins (ATGs) might lower the incidence of GVHD. We did a prospective, randomised, multicentre, open-label, phase 3 trial to compare standard GVHD prophylaxis with ciclosporin and methotrexate with or without anti-Jurkat ATG-Fresenius (ATG-F). METHODS Between May 26, 2003, and Feb 8, 2007, 202 patients with haematological malignancies were centrally randomly assigned using computer-generated centre-stratified block randomisation between treatment groups receiving ciclosporin and methotrexate with or without additional ATG-F. One patient in the ATG-F group did not undergo transplantation, thus 201 patients who underwent transplantation with peripheral blood (n=164; 82%) or bone marrow (n=37; 18%) grafts from unrelated donors after myeloablative conditioning were included in the full analysis set, and were analysed according to their randomly assigned treatment (ATG-F n=103, control n=98). The primary endpoint was severe acute GVHD (aGVHD) grade III-IV or death within 100 days of transplantation. The trial is registered with the numbers DRKS00000002 and NCT00655343. FINDINGS The number of patients in the ATG-F group who had severe aGVHD grade III-IV or who died within 100 days of transplantation was 12 and 10 (21.4%, 95% CI 13.4-29.3), respectively, compared with 24 and nine (33.7%, 24.3-43.0) patients, respectively, in the control group (adjusted odds ratio 0.59, 95% CI 0.30-1.17; p=0.13). The cumulative incidence of aGVHD grade III-IV was 11.7% (95% CI 6.8-19.8) in the ATG-F group versus 24.5% (17.3-34.7) in the control group (adjusted hazard ratio [HR] 0.50, 95% CI 0.25-1.01; p=0.054), and cumulative incidence of aGVHD grade II-IV was 33.0% (n=34; 95% CI 25.1-43.5) in the ATG-F group versus 51.0% (n=50; 95% CI 42.0-61.9) in the control group (adjusted HR 0.56, 0.36-0.87; p=0.011). The 2-year cumulative incidence of extensive chronic GVHD was 12.2% (n=11; 95% CI 7.0-21.3) versus 42.6% (n=34; 95% CI 33.0-55.0; adjusted HR 0.22, 0.11-0.43; p<0.0001). There were no differences between treatment groups with regard to relapse, non-relapse mortality, overall survival, and mortality from infectious causes. INTERPRETATION The addition of ATG-F to GVHD prophylaxis with ciclosporin and methotrexate resulted in decreased incidence of acute and chronic GVHD without an increase in relapse or non-relapse mortality, and without compromising overall survival. The use of ATG-F is safe for patients who are going to receive a haematopoietic cell transplantation from matched unrelated donors. FUNDING Fresenius Biotech GmbH.

Donor Lymphocyte Infusion in the Treatment of First Hematological Relapse After Allogeneic Stem-Cell Transplantation in Adults With Acute Myeloid Leukemia: A Retrospective Risk Factors Analysis and Comparison With Other Strategies by the EBMT Acute Leukemia Working Party

PURPOSE To evaluate the role of donor lymphocyte infusion (DLI) in the treatment of relapsed acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (HSCT). PATIENTS AND METHODS We retrospectively analyzed the data of 399 patients with AML in first hematological relapse after HSCT whose treatment did (n = 171) or did not (n = 228) include DLI. After correction for imbalances and established risk factors, the two groups were compared with respect to overall survival. Further, a detailed analysis of risk factors for survival among DLI recipients was performed. RESULTS Median follow-up was 27 and 40 months, respectively. Estimated survival at 2 years (+/- standard deviation) was 21% +/- 3% for patients receiving DLI and 9% +/- 2% for patients not receiving DLI. After adjustment for differences between the groups, better outcome was associated with age younger than 37 years (P = .008), relapse occurring more than 5 months after HSCT (P < .0001), and use of DLI (P = .04). Among DLI recipients, a lower tumor burden at relapse (< 35% of bone marrow blasts; P = .006), female sex (P = .02), favorable cytogenetics (P = .004), and remission at time of DLI (P < .0001) were predictive for survival in a multivariate analysis. Two-year survival was 56% +/- 10%, if DLI was performed in remission or with favorable karyotype, and 15% +/- 3% if DLI was given in aplasia or with active disease. CONCLUSION Although further evidence for a graft-versus-leukemia effect by DLI is provided, our results confirm, that the clinical benefit is limited to a minority of patients. Strategies to reduce tumor burden before DLI, as well as alternative treatment options should be investigated in adults with relapsed AML after HSCT.

Evidence of a Graft-Versus-Leukemia Effect in Chronic Lymphocytic Leukemia After Reduced-Intensity Conditioning and Allogeneic Stem-Cell Transplantation: The Cooperative German Transplant Study Group

PURPOSE To study whether hematopoietic stem-cell transplantation (HSCT) after reduced-intensity conditioning is effective and tolerable in patients with advanced chronic lymphocytic leukemia (CLL). PATIENTS AND METHODS Thirty patients with advanced B-cell CLL were included into the study. After reduced-intensity conditioning with fludarabine, busulfan, and antithymocyte globulin, patients received a transplant from related (n = 15) or unrelated donors (n = 15). Minimal residual disease (MRD) was monitored with a clone-specific polymerase chain reaction. RESULTS After a median follow-up of 2 years, 23 patients are alive (to date). Neutrophil and platelet engraftment occurred after a median of 17.5 and 15 days, respectively. Acute graft-versus-host disease (GVHD) grade 2 to 4 was observed in 17 patients (56%), and chronic GVHD was observed in 21 patients (75%). Twelve patients (40%) achieved a complete remission (CR), and 16 patients (53%) achieved a partial remission. Late CR occurred up to 2 years after transplantation. MRD was monitored in eight patients with CR. All patients achieved a molecular CR. At last follow-up, six patients were in ongoing molecular CR. Causes of death were treatment-related complications in four patients and progressive disease in three patients. The probability of overall survival, progression-free survival, and nonrelapse mortality at 2 years was 72% (95% confidence interval [CI], 54% to 90%), 67% (95% CI, 49% to 85%), and 15% (95% CI, 1% to 29%), respectively. CONCLUSION Treatment-related mortality after reduced-intensity conditioning followed by allogeneic HSCT was low. The procedure induced molecular remissions in patients with advanced CLL. The observation of late remissions provided evidence of a graft-versus-leukemia effect.

Rapid quantification of mixed chimerism using multiplex amplification of short tandem repeat markers and fluorescence detection

Monitoring the engraftment of donor cells after allogeneic blood stem cell transplantation (BSCT) may be important for the early diagnosis of graft failure or relapse of disease. Several techniques have been reported for this purpose. PCR-based assays analyzing polymorphic short tandem repeat (STR) markers are attractive because they are sensitive and can be performed rapidly. The intent of the present study was to test a novel approach for the quantification of mixed chimerism using a commercial multiplex STR assay with fluorescence-based detection for forensic purposes. The feasibility of this assay and the accuracy of quantitative results was tested using serial cell mixtures of unrelated individuals. Sample preparation was optimized to obtain information from minute amounts of starting material, eg from patients with aplasia or from sorted cell populations. Using the STR-PCR, discrimination between donor and recipient was possible in all patients analyzed (n = 25). Cell dilution experiments showed a linear correlation between the cell numbers added and the proportions found, with the limit of detection for a minor cell population being 5%. Comparison of values obtained with standard FISH analysis in patients transplanted from sex-mismatched donors showed an excellent correlation with the STR-PCR results. Taken together, this procedure allows the rapid, versatile and accurate quantification of mixed chimerism, even with minuscule numbers of cells.

Treatment of refractory acute GVHD with third-party MSC expanded in platelet lysate-containing medium

Mesenchymal stem cells have been shown to mediate immunomodulatory effects. They have been used in patients with steroid-refractory acute GVHD (aGVHD), but their relevance as a therapeutic agent targeting aGVHD has still to be defined. In this case series, we report 13 patients with steroid-refractory aGVHD who received BM-derived MSC expanded in platelet lysate-containing medium from unrelated HLA disparate donors. MSC were characterized by their morphological, phenotypical and functional properties. All tested preparations suppressed the proliferation of in vitro activated CD4+ T cells. MSC were transfused at a median dosage of 0.9 × 106/kg (range 0.6–1.1). The median number of MSC applications was 2 (range 1–5). Only two patients (15%) responded and did not require any further escalation of immunosuppressive therapy. Eleven patients received additional salvage immunosuppressive therapy concomitant to further MSC transfusions, and after 28 days, five of them (45%) showed a response. Four patients (31%) are alive after a median follow-up of 257 days, including one patient who initially responded to MSC treatment. In our patient cohort, response to MSC transfusion was lower than in the series reported earlier. However, our experience supports the potential efficacy of MSC in the treatment of steroid-refractory aGVHD.

Allogeneic stem cell transplantation after reduced-intensity conditioning in patients with myelofibrosis: a prospective, multicenter study of the Chronic Leukemia Working Party of the European Group for Blood and Marrow Transplantation.

From 2002 to 2007, 103 patients with primary myelofibrosis or postessential thrombocythemia and polycythemia vera myelofibrosis and a median age of 55 years (range, 32-68 years) were included in a prospective multicenter phase 2 trial to determine efficacy of a busulfan (10 mg/kg)/fludarabine (180 mg/m(2))-based reduced-intensity conditioning regimen followed by allogeneic stem cell transplantation from related (n = 33) or unrelated donors (n = 70). All but 2 patients (2%) showed leukocyte and platelet engraftment after a median of 18 and 22 days, respectively. Acute graft-versus-host disease grade 2 to 4 occurred in 27% and chronic graft-versus-host disease in 43% of the patients. Cumulative incidence of nonrelapse mortality at 1 year was 16% (95% confidence interval, 9%-23%) and significantly lower for patients with a completely matched donor (12% vs 38%; P = .003). The cumulative incidence of relapse at 3 years was 22% (95% confidence interval, 13%-31%) and was influenced by Lille risk profile (low, 14%; intermediate, 22%; and high, 34%; P = .02). The estimated 5-year event-free and overall survival was 51% and 67%, respectively. In a multivariate analysis, age older than 55 years (hazard ratio = 2.70; P = .02) and human leukocyte antigen-mismatched donor (hazard ratio = 3.04; P = .006) remained significant factors for survival. The study was registered at www.clinicaltrials.gov as #NCT 00599547.

Azacitidine for treatment of imminent relapse in MDS or AML patients after allogeneic HSCT: results of the RELAZA trial

This study evaluated azacitidine as treatment of minimal residual disease (MRD) determined by a sensitive donor chimerism analysis of CD34+ blood cells to pre-empt relapse in patients with CD34+ myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (HSCT). At a median of 169 days after HSCT, 20/59 prospectively screened patients experienced a decrease of CD34+ donor chimerism to <80% and received four azacitidine cycles (75 mg/m2/day for 7 days) while in complete hematologic remission. A total of 16 patients (80%) responded with either increasing CD34+ donor chimerism to ⩾80% (n=10; 50%) or stabilization (n=6; 30%) in the absence of relapse. Stabilized patients and those with a later drop of CD34+ donor chimerism to <80% after initial response were eligible for subsequent azacitidine cycles. A total of 11 patients (55%) received a median of 4 (range, 1–11) additional cycles. Eventually, hematologic relapse occurred in 13 patients (65%), but was delayed until a median of 231 days (range, 56–558) after initial decrease of CD34+ donor chimerism to <80%. In conclusion, pre-emptive azacitidine treatment has an acceptable safety profile and can substantially prevent or delay hematologic relapse in patients with MDS or AML and MRD after allogeneic HSCT.

The European LeukemiaNet AML Working Party consensus statement on allogeneic HSCT for patients with AML in remission: an integrated-risk adapted approach

Allogeneic haematopoietic stem-cell transplantation (HSCT) is frequently applied as part of the treatment in patients with acute myeloid leukaemia (AML) in their first or subsequent remission. Allogeneic HSCT reduces relapse, but nonrelapse mortality and morbidity might counterbalance this beneficial effect. Here, we review recent studies reporting new disease-specific prognostic markers, in addition to allogeneic-HSCT-related risk factors, which can be assessed at specific time points during treatment. We propose risk assessment as a dynamic process during treatment, incorporating both disease-related and transplant-related factors for the decision to proceed either to allogeneic HSCT or to apply a nontransplant strategy. We suggest that allogeneic HSCT might be favoured if the projected disease-free survival is expected to improve by at least 10% based on an individuals risk assessment. The approach requires initial disease risk assessment, identifying a sibling or unrelated donor soon after diagnosis and the incorporation of time-dependent risk factors, all within the context of an integrated therapeutic management approach.

Sequential monitoring of chimerism and detection of minimal residual disease after allogeneic blood stem cell transplantation (BSCT) using multiplex PCR amplification of short tandem repeat-markers.

Sequential analysis of chimerism after allogeneic blood stem cell transplantation (BSCT) has been shown to be predictive for graft failure and relapse. We have explored the impact of a novel approach for the quantitative determination of chimerism using a commercial PCR assay with multiplex amplification of nine STR-loci and fluorescence detection. The feasibility was studied in 121 patients transplanted from related or unrelated donors. Follow-up investigation was performed in 88 patients. Twenty-eight of these patients had received a transplantation after dose-reduced conditioning therapy. Results were compared to data obtained by FISH analysis in a subgroup of patients receiving grafts from sex-mismatched donors. The analysis was possible in all patients, the median number of informative alleles was 4 (range 1–8) compared to 7 (range 1–9) in the related and unrelated situation, respectively. A good correlation was seen in 84 samples from 14 patients analyzed in parallel with STR-PCR and FISH. Decreasing values of donor chimerism were detected prior to or concomitantly with the occurrence of graft failure and relapse of disease in all patients investigated prospectively. Using FACS-sorted material, eg peripheral blood CD34+ cells, the assay permitted the detection of residual recipient cells with high sensitivity (down to one CD34+ Kasumi cell in 40 000 normal WBC). Evaluation of the inter-laboratory reproducibility revealed that in 20 samples analyzed in three different centers, the median coefficient of variation was 2.1% (range 0.7–9.6%). Taken together, the results support the use of the test as a valuable tool in the follow-up of patients undergoing allogeneic BSCT. In cases lacking PCR-detectable disease-specific gene products, this assay may represent an alternative to recently established real-time PCR methods.