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Isolation of mesenchymal stromal cells (MSCs) from pretreated, hematologic patients is challenging. Especially after allogeneic hematopoietic cell transplantation (HCT), standard protocols using bone marrow aspirates fail to reliably recover sufficient cell numbers. Because MSCs are considered to contribute to processes that mainly affect the outcome after transplantation, such as an efficient lymphohematopoietic recovery, extent of graft-versus-host disease as well as the occurrence of leukemic relapse, it is of great clinical relevance to investigate MSC function in this context. Previous studies showed that MSCs can be isolated by collagenase digestion of large bone fragments of hematologically healthy patients undergoing hip replacement or knee surgeries. We have now further developed this procedure for the isolation of MSCs from hematologic patients after allogeneic HCT by using trephine biopsy specimens obtained during routine examinations. Comparison of aspirates and trephine biopsy specimens from patients after allogeneic HCT revealed a significantly higher frequency of clonogenic MSCs (colony-forming unit-fibroblast [CFU-F]) in trephine biopsy specimens (mean, 289.8 ± standard deviation 322.5 CFU-F colonies/1 × 10 6 total nucleated cells versus 4.2 ± 9.9; P < 0.0001). Subsequent expansion of functional MSCs isolated from trephine biopsy specimen was more robust and led to a significantly higher yield compared with control samples expanded from aspirates (median, 1.6 × 10 6 ; range, 0-2.3 × 10 7 P0 MSCs versus 5.4 × 10 4 ; range, 0-8.9 × 10 6 ; P < 0.0001). Using trephine biopsy specimens as MSC source facilitates the investigation of various clinical questions., Competing Interests: Declaration of Competing Interest The project was funded by the Deutsche Forschungsgemeinschaft (Clinician Scientist position and Seed grant within the Centre for Regenerative Therapies Dresden, www.crt-dresden.de). The authors have no conflicts of interest to disclose., (Copyright © 2019 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.)

The role of allogeneic stem cell transplantation (HSCT) as compared to chemotherapy in acute myeloid leukaemia (AML) patients with abnormalities of chromosome 17p [abnl(17p)] has not yet been defined. Therefore, we analysed 3530 AML patients treated in three randomized, prospective, controlled clinical trials and compared post-remission therapies using a multivariate Cox regression analysis to determine whether allogeneic HSCT is superior than chemotherapy in overcoming the detrimental impact of patients with abnl(17p) AML. One hundred and forty-three patients (4%) were identified with abnl(17p) AML. All patients had received intensive induction chemotherapy. Forty-seven patients with a median age of 54 years (18-69 years) proceeded to allogeneic HSCT in first or second remission. The 3-year overall survival (OS) rate for the entire cohort of patients was 4% [95% confidence interval (CI), 1-7%]. OS and event-free survival at 3 years, calculated from the day of HSCT, was 11% (95% CI, 2-20%) and 6% (95% CI, 0-13%), respectively. Multivariate Cox regression analysis showed no benefit of allogeneic HSCT compared to chemotherapy (Hazard Ratio 0·97, 95% CI 0·56-1·67, P = 0·9). In conclusion, allogeneic HSCT does not improve survival in patients with abnl(17p) AML as compared to other adverse cytogenetic risk abnormalities., (© 2013 Blackwell Publishing Ltd.)

Purpose: Gemtuzumab ozogamicin (GO) has been associated with an increased risk of liver sinusoidal obstruction syndrome (SOS) when applied within 3 months of allogeneic hematopoietic cell transplantation (HCT). We hypothesized that GO might be safe and effective as part of a reduced-intensity conditioning regimen as salvage therapy of CD33+ acute myeloid leukemia., Experimental Design: Thirty-one patients with acute myeloid leukemia which relapsed following conventional therapy (n=15), autologous (n=3), or allogeneic (n=13) HCT were included in a prospective phase I/II trial. The preparative regimen contained 6 and 3 mg/m(2) of GO on days -21 and -14 before transplantation, leading to a reduction of marrow blasts in 18 patients (58%). Eight patients received further cytoreductive chemotherapy before conditioning therapy was initiated. Fludarabine-based reduced-intensity (n=11) or nonmyelablative (n=16) conditioning and peripheral blood stem cell infusion from related (n=6) or unrelated (n=21) donors could be done in 27 patients during cytopenia., Results: Primary engraftment occurred in all evaluable patients. Only one case of reversible hepatic sinusoidal obstruction syndrome was documented. Non-relapse mortality until day 100 was 22% (n=6). The probabilities of overall and disease-free survival at 24 months were 39% and 35%, respectively. Relapse of leukemia occurring between 2 and 24 months after transplantation (median, 8 months) was the major reason for treatment failure and death., Conclusion: These data suggest that GO can be combined with reduced-intensity conditioning even after previous autologous or allogeneic HCT.

Background: Infections remain a major cause of morbidity and mortality in patients undergoing autologous or allogeneic hematopoietic stem cell transplantation (HSCT). About 80% of patients experience fever during aplasia and early engraftment despite oral antibacterial chemoprophylaxis., Methods: In a pilot study, 50 patients undergoing autologous or allogeneic HSCT received a prophylactic antibacterial treatment with intravenous piperacillin/tazobactam beginning on day of stem cell or bone marrow transfusion. They were analyzed retrospectively for frequencies of fever of unknown origin (FUO), documented infection, bacteremia and death because of infection. Furthermore, data from microbiological monitoring and tolerability were evaluated., Results: Among 28 autologous transplanted patients, 10 (36%) developed fever more than 38.5 degrees C; 9/10 FUO, 1/28 pulmonary infiltrates. Eighteen patients (64%) remained without any symptom of infection. In the allogeneic group (n = 22), there were eight patients (36%) with FUO, and five patients (23%) with documented infections (pneumonia 2, enteritis 1, pyelonephritis 1, Escherichia coli bacteremia 1). In nine patients (41%), escalation of antimicrobial treatment was not necessary. The majority of detected microbes in cultures of throat and nose secretions, blood, urine and stool were gram-positive bacteria (77.8%), among them Staphylococcus epidermidis (23.5%), streptococci (group A, B, C; 21.0%) and enterococci (10.6%). Incidence of gram-negative bacteria and fungi was similar with 11.8% and 10.4%, respectively. The most frequent gram-negative strains were Escherichia coli (6.5%) and Pseudomonas aeruginosa (1.7%). There was no severe toxicity or hypersensitivity., Conclusion: Compared to oral decontamination and chemoprophylaxis, an intravenous prophylactic regimen as described above could be an effective and well-tolerated approach in prevention of bacterial infections and related complications, with a higher acceptance in recipients of bone marrow or stems cell grafts. Further evaluation in comparison with fluoroquinolone prophylaxis regarding efficacy, development of resistances as well as cost-benefit analyses is warranted.

This study was undertaken to evaluate the toxicity and pharmacokinetics of a dimethyl sulphoxide (DMSO)-based intravenous formulation of busulphan in the conditioning of 45 patients undergoing allogeneic or autologous stem cell transplantation (SCT). Busulphan was given as a single daily dose. In 15 patients a single dose of intravenous busulphan, given over 3 h in 1 d, was combined with additional oral (single daily) doses. Thirty patients received all four daily doses intravenously. Busulphan plasma levels were analysed using high performance liquid chromatography. There was no major acute toxicity with daily intravenous doses of 2.8-3.1 mg/kg infused over 3 h. No veno-occlusive disease (VOD) was seen in 30 patients receiving busulphan as an intravenous formulation over 4 d. In the group of 15 patients receiving three oral doses and one intravenous single daily dose, one patient experienced mild VOD. Pharmacokinetic samples were taken over at least 2 d of treatment in 44 patients. The area under the concentration time curve (AUC) values normalized for a dose of 1 mg/kg were 7000 ng/ml x h on d 1 and 5890 ng/ml x h on d 4, thus showing a moderate decrease over time. This was accompanied by a moderate increase of the clearance from 2.6 to 3.0 ml/min/kg. Administration of busulphan as a DMSO-based intravenous formulation was well tolerated. The total dose of busulphan can be given in four (rather than the typical 16) doses. With such a regimen, the intravenous administration becomes feasible on an outpatient basis.

Background: Cidofovir (CDV) is a nucleotide analogue with proven in vitro effects against cytomegalovirus (CMV) and adenovirus and has been successfully used in the treatment of CMV retinitis in AIDS patients., Methods: We performed a prospective study to evaluate the efficacy of CDV in 17 patients with hematological malignancies after allogeneic blood stem cell transplantation from related (n=3) and unrelated (n=14) donors. Dose-reduced conditioning (DRC) regimen consisted of busulfan (Bu)/fludarabine (Flu) (n=9) and idarubicin/cytosine arabinoside/Flu (n=1). Myeloablative conditioning (MC) was performed with Bu/cyclophosphamide (Cy)/etoposide (Eto) (n=4), Bu/Cy (n=2), and total body irradiation (TBI)/Cy/Eto (n=1). Antithymocyte globulin (ATG) was used in seven patients with DRC and in six patients with MC. In all patients, either the donor, host, or both were CMV IgG positive pretransplant. Indication for therapy was preemptive treatment of primary CMV antigenemia defined as two consecutive positive tests of pp65 antigenemia assay after transplant. In case of response with a decreasing number of pp65-positive leukocytes, CDV was scheduled in a dosage of 5 mg/kg body weight once a week for 2 weeks followed by maintenance therapy every 2 weeks in an outpatient setting. All patients received probenecid and prehydration as recommended. Patients were monitored using an immunostaining assay for pp65 antigen and a qualitative and quantitative CMV polymerase chain reaction (PCR). Success of treatment was defined as negativity for the pp65 antigen., Results: After DRC, nine of ten patients (90%) showed a response with seven of nine revealing a complete clearance of the virus (pp65 negative, qualitative PCR negative). In the remaining two responders, treatment was changed to ganciclovir because of either renal impairment or slow clearance of antigenemia. Only one of seven patients in the MC group experienced a temporary clearance of pp65 antigen. After MC, two patients experienced CMV disease. Treatment-related toxicity rate was moderate with four patients developing reversible renal impairment (creatinine 133-180 micromol/L); one patient with proteinuria and three patients with complaints of nausea and vomiting., Conclusion: Our data suggest the feasibility of CDV administration in patients after allogeneic transplantation. In the recommended dose, it might be used successfully for low-risk patients, e.g., after DRC or organ transplantation, in an outpatient setting.

Between February 1998 and October 1999, 24 patients with advanced leukemia, lymphoma or solid tumors received G-CSF mobilized peripheral blood stem cells (PBSC) from HLA-matched sibling donors after dose-reduced conditioning therapy. Only patients with reduced performance status or major infectious complications, not eligible for standard transplant procedures, were included. The 5-day conditioning therapy consisted of 3.3 mg/kg intravenous busulphan x 2 days and 30 mg/m2 fludarabine x 5 days. GVHD prophylaxis was performed with either CsA alone (n = 5), CsA combined with short course methotrexate (n = 5) or mycophenolate mofetil (n = 14). The day 100 survival was 95.2% for the whole group. All patients engrafted after a median of 15 days (range, 11-19) and 12.5 days (range, 10-19) for neutrophils and platelets, respectively. The median time to a neutrophil count of <0.5 x 109/l was 7 days (range, 2 to 12). Acute GVHD >I was observed in six patients, whereas eight patients have signs of chronic GVHD. The prospective 12 month overall survival with a median follow-up of 7 months is 63%. Relapse of disease and toxicity associated with chronic GVHD were the main causes of death. The treatment-related mortality was 12.5%. Dose-reduced conditioning using intravenous busulphan and fludarabine allows stable engraftment without ATG in related transplants and leads to a reduction of transplant-related mortality.

Haemorrhagic cystitis (HC) after allogeneic haematopoietic stem cell transplantation (HSCT) or high-dose cyclophosphamide (CP) chemotherapy is a severe side-effect and can cause significant morbidity and mortality. In this report, we describe the clinical courses of 10 patients with HC and review the literature. The patients were treated with oral conjugated estrogen in an attempt to improve severe haemorrhagic cystitis. In seven patients positive effects were seen, haematuria resolved in all, but residual symptoms of dysuria remained for longer periods. In one patient application of estrogen was interrupted because of hepatotoxicity. Two patients failed all treatment modalities including oral estrogen because of terminal illness. We conclude that in the management of HC the administration of oral conjugated estrogen should be considered.

The German bone marrow donor center (DKMS) hasrecruited over 732 500 donors during the first 9 years of its existence. Initially, donors were typed for HLA-A and B, and DR typing was only done on request for a patient-initiated search. In 1994, a project was started which led to the donor center-initiated DR typing (DCI-DRT) of >35,000 donors. These donors were selected by donor-specific criteria (age, sex, height and weight) and according to HLA-A and B phenotypes. The latter was done to avoid unnecessary DR typing of the most common A, B phenotypes With a follow up of >6 years, this strategy has led to a number of confirmatory typings (CT) (n=4588) and stem cell harvests (n=568), which is at least comparable to those ensuing after patient-initiated HLA-DR typing (126 000 DR typings, 8,213 CTs, 888 resulting in stem-cell donation). DCI-DRT seems to be a cost-effective strategy which may help to reduce search times and improve search outcome, and improve the overall efficiency of donor center operations

The simultaneous measurement of DNA content and myeloma-related antigens (B-B4 or CD38) by flow cytometry is proposed as a method for the detection of aneuploid plasma cells in peripheral blood stem cell (PBSC) harvests and in bone marrow after therapy. In 30 patients with initially detected aneuploid myeloma cells we evaluated the bone marrow after therapy and in eight of these patients 23 PBSC harvests were analyzed. In 13 of 23 PBSC harvests aneuploid myeloma cells were detectable (range: 0.02-0.63%). In the bone marrow of the 30 patients aneuploid plasma cells were detectable in all samples after chemotherapy (range: 0.12-35.70%) and after autologous PBSC transplantation in two of three patients (0.21% and 0.03%). Furthermore the relationship between diploid and aneuploid plasma cells can be evaluated. In the PBSC harvests the percentage of aneuploid plasma cells is significantly lower than that of diploid plasma cells (P=0.006). In contrast, in bone marrow the aneuploid plasma cells are predominant in most patients even after therapy (24 of 30 patients; P=0.0055). In the case of initially detected aneuploid myeloma cells, a contamination with malignant cells can be estimated with a simple flow cytometric method in PBSC harvests and in bone marrow after therapy.

Background: Mycophenolate mofetil (MMF) is an inhibitor of purine nucleotide de novo synthesis leading to impaired proliferation of activated lymphocytes. Studies in animals show a synergistic effect of MMF and cyclosporine (CsA) in preventing acute graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation. We performed a pilot study evaluating the feasibility of the combined application of MMF and CsA as GVHD prophylaxis after allogeneic blood stem cell transplantation. Toxicity and the bioavailability of MMF in this setting were investigated., Methods: Fourteen patients who had received grafts from HLA-compatible siblings received 2 g of oral MMF from day 1 to 14 combined with intravenous CsA at 4 mg/kg starting at day-1. Plasma levels of mycophenolic acid (MPA) and its glucoronide were measured by high-performance liquid chromatography. Fifteen patients treated with a combination of CsA and methotrexate at the same institution were referred to as the control group., Results: Trilineage engraftment was achieved in all study and control patients. Acute GVHD > or = grade II was observed in 46.5% and 60% of the study and control patients, respectively. No major differences in the rate of acute toxicities were detectable. The mean trough blood level of MPA in 10 patients was 0.28 microg/ml, and 5.7 microg/ml for MPA glucoronide. Reduced peak levels of MPA indicate a reduced absorption rate of MMF in the early posttransplant phase., Conclusions: The combined administration of MMF and CsA was shown to be feasible in patients after allogeneic blood stem cell transplantation. Because of the decreased bioavailability of MMF, dose-finding studies for an intravenous formulation are warranted.

Busulfan (BU) is included in many conditioning protocols for haematopoietic stem cell transplantation (HSCT). Pharmacokinetic parameters in individual patients have been related to short-term toxicity and risk of relapse after HSCT. In a series of 11 patients receiving the usual 16 x 1 mg/kg schedule over 4 days, we investigated the pharmacokinetics of replacing one dose with an intravenous formulation (BU in DMSO) which we had previously investigated in dogs. A dose of 0.5-0.6 mg/kg was used. No acute side-effects of BU/DMSO infusions administered over 1 h were observed. Bioavailability of BU powder capsules was on average 70% (range, 44-94%). Interindividual variability of the resulting AUC after intravenous doses was still substantial. Further studies are under way to define the possible role of BU/DMSO infusions in conditioning before HSCT.

Allogeneic peripheral blood stem cell transplantation leads to an earlier engraftment compared to BMT. The feasibility, acceptance and long-term side-effects of G-CSF mobilisation of PBSC in unrelated healthy donors needs to be evaluated. Forty unrelated healthy donors received G-CSF in a dose of 10 microg/kg bodyweight for 5 days and two aphereses were performed. The donors were monitored prospectively. The data were compared to bone marrow harvests from unrelated donors. Almost all stem cell donors reported some side-effects due to Filgrastim application. Bone pain (32), headache (20), chest pain (two) and night sweats (one) were complained of. By taking analgesics, the pain was relieved in most cases. No donor discontinued the filgrastim application. Bone pain and headache resolved within 2-4 days after termination of Filgrastim application. There was, as expected, a seven-fold increase in the number of total WBCs. There were no significant changes of platelet counts during G-CSF application. After 4 weeks haemoglobin concentration and platelet counts showed no significant differences compared to baseline values. The aphereses were mostly tolerated very well. Eighteen donors reported paraesthesia, one donor developed dizziness, two complained of nausea and vomiting. There was a significant decrease in platelet count (242 before, 98 x 10(9)/l after aphereses). Autologous platelets were transfused after the second aphereses in four donors. These data were compared to data from 245 unrelated bone marrow donors, who had on average, 14 days bone pain and tiredness after donation. The G-CSF mobilisation and apheresis of peripheral blood stem cells is an alternative to traditional bone marrow harvesting in unrelated healthy donors. It is well tolerated and the duration of side-effects on average is shorter than after the surgical procedure. So far no long-term effects have been observed in the follow-up.

High-dose chemotherapy with autologous and allogeneic blood stem cell transplantation has become a standard therapy for hematological malignancies and solid tumors. To ensure quality of treatment and adherence to internationally approved protocols, the use of computer systems has been introduced in some oncology centers. Most software packages cannot be used automatically by other institutions because of different treatment strategies and medications. To overcome this problem, we designed a software system based on an Oracle database, which allows the user to develop his own therapy blocks and assemble them to generate specific therapy plans. The system supports the daily work of the physicians by suggesting medications based on changes in the vital parameters or the clinical chemistry. Parameters can be given upper and lower limits whose surpassing leads to treatment suggestions by the system. All quantitative variables may be depicted in a graphical way. The system is connected to external laboratory devices. Export of data to international registries is possible. Until now, 43 patients have been transplanted with the help of this system. There is a high degree of acceptance among the nursing staff and the physicians. The system can be transferred to other centers, if the local hardware and network capacities meet the minimal requirements.

A patient with CML in accelerated phase received G-CSF-mobilized PBPC from an unrelated HLA genotypically matched donor. The blood groups of the patient and donor were bidirectionally incompatible. Hematologic recovery was rapid with > 500 PMN/microliter on day +9. Starting on day +5 bilirubin levels increased from 1.3 mg/dl up to a maximum of 18 mg/dl on day +14. Clinical signs and laboratory tests supported major hemolysis. Blood typing on day +16 revealed early blood-group change, consistent with donor-derived antibodies produced by passenger-lymphocytes which may have mediated severe hemolysis. The early onset and strong intensity of the hyperbilirubinemia could be a specific feature of ABO-incompatible allogeneic PBPC transplantation which would be difficult to differentiate from GVHD or VOD.