Maximilian Christopeit

Marked improvement of severe progressive systemic sclerosis after transplantation of mesenchymal stem cells from an allogeneic haploidentical-related donor mediated by ligation of CD137L

Marked improvement of severe progressive systemic sclerosis after transplantation of mesenchymal stem cells from an allogeneic haploidentical-related donor mediated by ligation of CD137L

C/EBPα regulated microRNA-34a targets E2F3 during granulopoiesis and is down-regulated in AML with CEBPA mutations

The transcription factor, CCAAT enhancer binding protein alpha (C/EBPα), is crucial for granulopoiesis and is deregulated by various mechanisms in acute myeloid leukemia (AML). Mutations in the CEBPA gene are reported in 10% of human patients with AML. Even though the C/EBPα mutants are known to display distinct biologic function during leukemogenesis, the molecular basis for this subtype of AML remains elusive. We have recently showed the significance of deregulation of C/EBPα-regulated microRNA (miR) in AML. In this study, we report that miR-34a is a novel target of C/EBPα in granulopoiesis. During granulopoiesis, miR-34a targets E2F3 and blocks myeloid cell proliferation. Analysis of AML samples with CEBPA mutations revealed a lower expression of miR-34a and elevated levels of E2F3 as well as E2F1, a transcriptional target of E2F3. Manipulation of miR-34a reprograms granulocytic differentiation of AML blast cells with CEBPA mutations. These results define miR-34a as a novel therapeutic target in AML with CEBPA mutations.

Haploidentical allogeneic hematopoietic cell transplantation in adults using CD3/CD19 depletion and reduced intensity conditioning: a phase II study

Background We report a prospective multicenter phase II study of haploidentical hematopoietic stem cell transplantation using CD3/CD19-depleted grafts after reduced intensity conditioning with fludarabine, thiotepa, melphalan and OKT-3. Design and Methods Sixty-one adults with a median age of 46 years (range 19-65 years) have been enrolled. Diagnoses were acute myeloid leukemia (n=38), acute lymphoblastic leukemia (n=8), non-Hodgkins lymphoma (n=6), myeloma (n=4), chronic myeloid leukemia (n=3), chronic lymphatic leukemia (n=1) and myelodysplastic syndrome (n=1). Patients were considered high risk because of refractory disease (n=18), cytogenetics (n=6), complete remission (≥2) (n=9), chemosensitive relapse in partial remission (n=4) or relapse after prior hematopoietic stem cell transplantation (n=15 allogeneic, n=8 autologous, n=1 both). At haploidentical hematopoietic stem cell transplantation, 30 patients were in complete remission and 31 in partial remission. Grafts contained a median of 7.0×106 (range 3.2-22) CD34+ cells/kg, 4.2×104 (range 0.6-44) CD3+ T cells/kg and 2.7×107 (range 0.00-37.3) CD56+ cells/kg. Results Engraftment was rapid with a median of 12 days to granulocytes more than 0.5×109/L (range 9-50 days) and 11 days to platelets more than 20×109 (range 7-38 days). Incidence of grade IIIV acute graft-versus-host-disease and chronic graft-versus-host-disease was 46% and 18%, respectively. Non-relapse mortality on Day 100 was 23% and 42% at two years. Cumulative incidence of relapse/progression at two years was 31%. Kaplan-Meier estimated 1-year and 2-year overall survival with median follow up of 869 days (range 181-1932) is 41% and 28%, respectively. Conclusions This regimen allows successful haploidentical hematopoietic stem cell transplantation with reduced intensity conditioning in high-risk patients lacking a suitable donor. (clinicaltrials.gov identifier:NCT00202917).

Plerixafor with and without chemotherapy in poor mobilizers: results from the German compassionate use program

The CXCR4-inhibitor plerixafor mobilizes hematopoietic stem cells amplifying the effects of granulocyte-CSF (G-CSF). Before approval plerixafor was used in a compassionate use program (CUP) for patients who failed a previous mobilization. In the German CUP 60 patients from 23 centers (median age 56.5 years (2–75)) were given 240 μg/kg plerixafor SC 9–11 h before apheresis. A total of 78.3% (47/60) received G-CSF for 4 days before plerixafor administration; 76.6% of those (36/47) yielded at least 2.0 × 106 CD34+ cells/μL. The median cell yield was 3.35 × 106 CD34+ cells/kg (0–29.53). Nine patients received plerixafor alone or with G-CSF for less than 4 days mobilizing a median of 3.30 × 106 CD34+ cells/kg (1.6–5.6). There was no significant difference between G-CSF application for 4 days and for a shorter period of time (P=0.157). A total of 47 patients received plerixafor plus G-CSF combined with chemotherapy yielding a median of 3.28 × 106 CD34+ cells/kg (0–24.79). In all, 40 of 60 patients (66.7%) proceeded to transplantation, and achieved a timely and stable engraftment. Side effects were rare and manageable. In conclusion, mobilization with plerixafor in poor mobilizers is safe and results in a sufficient stem cell harvest in the majority of patients.

New IDH1 mutant inhibitors for treatment of acute myeloid leukemia

Neomorphic mutations in isocitrate dehydrogenase 1 (IDH1) are driver mutations in acute myeloid leukemia (AML) and other cancers. We report the development of new allosteric inhibitors of mutant IDH1. Crystallographic and biochemical results demonstrated that compounds of this chemical series bind to an allosteric site and lock the enzyme in a catalytically inactive conformation, thereby enabling inhibition of different clinically relevant IDH1 mutants. Treatment of IDH1 mutant primary AML cells uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block and induction of granulocytic differentiation at the level of leukemic blasts and more immature stem-like cells, in vitro and in vivo. Molecularly, treatment with the inhibitors led to a reversal of the DNA cytosine hypermethylation patterns caused by mutant IDH1 in the cells of individuals with AML. Our study provides proof of concept for the molecular and biological activity of novel allosteric inhibitors for targeting different mutant forms of IDH1 in leukemia.

Second Allograft for Hematologic Relapse of Acute Leukemia After First Allogeneic Stem-Cell Transplantation From Related and Unrelated Donors: The Role of Donor Change

PURPOSE To evaluate the role of a second allogeneic hematopoietic stem-cell transplantation (HSCT2) given for relapsed acute leukemia (AL) after related or unrelated first hematopoietic stem-cell transplantation (HSCT1) and to analyze the role of donor change for HSCT2 in both settings. PATIENTS AND METHODS We performed a retrospective registry study on 179 HSCT2s given for relapse after HSCT1 from matched related donors (n = 75) or unrelated donors (n = 104), using identical or alternative donors for HSCT2. Separate analyses were performed according to donor at HSCT1. RESULTS Independent of donor, 74% of patients achieved complete remission after HSCT2, and half of these patients experienced relapse again. Overall survival (OS) at 2 years was 25% ± 4% (39% ± 7% after related HSCT2; 19% ± 4% after unrelated HSCT2). Long-term survivors were observed even after two unrelated HSCT2s. Multivariate analysis for OS from HSCT2 confirmed established risk factors (remission duration after HSCT1: hazard ratio [HR], 2.37; 95% CI, 1.61 to 3.46; P < .001; stage at HSCT2: HR, 0.53; 95% CI, 0.34 to 0.83; P = .006). Outcome of HSCT2 was better after related HSCT1 than after unrelated HSCT1 (2-year OS: 37% ± 6% v 16% ± 4%, respectively; HR, 0.68; 95% CI, 0.47 to 0.98; P = .042, multivariate Cox regression). After both related and unrelated HSCT1, selecting a new donor for HSCT2 did not result in a relevant improvement in OS compared with HSCT2 from the original donor; however, donor change was not detrimental either. CONCLUSION After relapse from allogeneic HSCT1, HSCT2 can induce 2-year OS in approximately 25% of patients. Unrelated HSCT2 is feasible after related and unrelated HSCT1. Donor change for HSCT2 is a valid option. However, a clear advantage in terms of OS could not be demonstrated.

Management of sepsis in neutropenic patients: guidelines from the infectious diseases working party of the German Society of Hematology and Oncology

Sepsis is a leading cause of mortality in neutropenic cancer patients. Early initiation of effective causative therapy as well as intensive adjunctive therapy is mandatory to improve outcome. We give recommendations for the management of adults with neutropenia and sepsis. The guidelines are written for clinicians involved in care of cancer patients and focus on pathophysiology, diagnosis and treatment of sepsis during neutropenia.

Treatment of severe progressive systemic sclerosis with transplantation of mesenchymal stromal cells from allogeneic related donors: Report of five cases

Systemic sclerosis (SSc; scleroderma) is a connective tissue disorder that is characterized by an obstructive vasculopathy and by the excessive deposition of collagen in the skin and internal organs. The prognosis is poor in individuals with lung, cardiac, or renal involvement. A safe and effective treatment has not yet been developed. Mesenchymal stromal cells (MSCs) may contribute to the pathogenesis of autoimmune diseases (1). MSCs can differentiate into mesenchymal cells, such as bone or muscle cells, but also into endothelial cells. Significantly, MSCs from patients with SSc have an impaired ability to differentiate into endothelial progenitor cells and to contribute to vasculogenesis (2). MSCs are capable of suppressing both the innate and the adaptive immune systems. In vitro, they inhibit the proliferation of T lymphocytes (3) and the function of B lymphocytes (4). MSCs are not recognized by alloreactive T cells in vitro (5) and exert immunosuppressive effects regardless of HLA and species boundaries (6). A specific mechanism that could explain these effects has not been defined. Even though conclusive evidence is still missing, MSCs have shown promising activity in refractory graft-versus-host disease (GVHD), especially in cases with a scleroderma-like course (7), and may offer a new treatment approach (at least in subgroups of patients with GVHD). Here we report the first data regarding MSC transplantation from allogeneic related donors into SSc patients. MSC transplantation was performed in 5 white patients with progressive SSc who were positive for Scl-70 antibodies (except patient 5, who was positive for anti–RNA polymerase III antibodies). In each case, the treatment was approved by the local ethics committee, and patients and donors or the respective legal representative provided written informed consent. After administration of a local anesthetic, 20–120 ml bone marrow was aspirated from the iliac crest of each donor, using sodium citrate as an anticoagulant. All subsequent procedures were performed at a certified facility. After centrifugation at 300g, cells were resuspended with phosphate buffered saline (PBS), overlaid on leukocyte separation medium (1,077 gm/liter), and centrifuged for 30 minutes at 300g. Bone marrow mononuclear cells (BMMCs) were harvested and resuspended with growth medium. After centrifugation at 300g, cells were plated in growth medium at 1–2.5 10 BMMCs/cm. Cultivation was performed with 5% CO2 and 98% humidity, at 36°C. For patient 1, growth medium consisted of low-glucose Dulbecco’s modified Eagle’s medium (DMEM), 100 units/ml penicillin, 100 g/ml streptomycin, and 2% fetal calf serum. For patients 2–5, growth medium consisted of low-glucose DMEM, 2.7% human platelet apheresis lysate, 4.6% fresh frozen plasma, and 1.8 units/ml unfractionated heparin. At 50–80% confluence, cells were detached with trypsin, counted, and seeded at 2 10–2 10 BMMCs/cm. Medium was changed every 2–3 days. Upon reaching passage 4 or a cell number of 1 10 cells/kg donor body weight, cells were harvested. For final packaging of the noncryopreserved product, cells were centrifuged at 300g and resuspended in PBS containing 5% human serum albumin (HSA). For packaging for subsequent cryopreservation, cells were resuspended in PBS containing 10% DMSO and 5% HSA. Cryopreservation was performed automatically (Biosafe 200; MesserCryotherm). Microbiologic analysis was performed at the passage before final packaging and from a probe of the final packed product. MSCs were characterized in patients 1, 4, and 5 as CD34 , CD45 , CD14 , CD19 , HLA–DR–negative, CD105 , CD90 , and CD73 , using a 4-color flow cytometer. Cytogenetic testing of MSCs was not performed. Intravenous infusion was used to transplant MSCs. Patients 1 and 2 received freshly prepared MSCs, whereas patients 3–5 received cryopreserved MSCs (which was necessary for logistic reasons). Vital functions were monitored for 60 minutes. After MSC transplantation, patients were monitored weekly for 1 month, biweekly for 2 subsequent months, and then once per month after that point. Beginning 6 months after MSC transplantation, patients were seen once every 3 months. The modified Rodnan skin thickness score (MRSS) (8) was determined before MSC transplantation and every 3 months thereafter. The functional ability was assessed by the Health Assessment Questionnaire (HAQ) (9). The number and size of acral ulcerations were documented by digital photography before treatment, and at 6 weeks, 3 months, and 6 months after treatment. Lymphocyte subpopulations (i.e., B cells [CD19 ], T cells [CD3 ], T helper cells [CD4 ], cytotoxic T cells [CD8 ], and natural killer cells [CD16 CD56 ]) were determined by flow cytometry. Pulmonary function analyses, including forced vital capacity (FVC) and diffusing capacity for carbon monoxide (DLCO), as well as echocardiography, vascular ultrasound examination of the fingers, and determination of transcutaneous partial oxygen pressure of the skin of the forearms, were performed before MSC transplantation and every 3 months afterward. Patients who had pulmonary involvement underwent high-resolution computed tomography (HRCT) every 6 months. All patients tolerated MSC transplantation without immediate toxicity. Mild respiratory tract infections that did not require hospitalization occurred during followup (Table 1). The levels of antinuclear antibodies remained unchanged. Of the 5 patients, 4 had at least temporary improvements in the MRSS (Table 1). Acral necroses improved or healed in all 3 affected patients. In these cases, a tendency toward improved oxygen saturation over the involved tissue was seen 3 months after MSC transplantation. Pulmonary function remained stable in all patients except in patient 2 (data not shown). Lymphocyte subpopulations did not change significantly. No significant alterations in HAQ scores were seen. Additional details on the patients’ courses are available online at http:// www.medizin.uni-halle.de/rheumazentrum/index.php?id 341. Patient 1 presented with progressive involvement of

Genome-wide hydroxymethylation tested using the HELP-GT assay shows redistribution in cancer

5-hydroxymethylcytosine (5-hmC) is a recently discovered epigenetic modification that is altered in cancers. Genome-wide assays for 5-hmC determination are needed as many of the techniques for 5-methylcytosine (5-mC) determination, including methyl-sensitive restriction digestion and bisulfite sequencing cannot distinguish between 5-mC and 5-hmC. Glycosylation of 5-hmC residues by beta-glucosyl transferase (β-GT) can make CCGG residues insensitive to digestion by MspI. Restriction digestion by HpaII, MspI or MspI after β-GT conversion, followed by adapter ligation, massive parallel sequencing and custom bioinformatic analysis allowed us determine distribution of 5-mC and 5-hmC at single base pair resolution at MspI restriction sites. The resulting HpaII tiny fragment Enrichment by Ligation-mediated PCR with β-GT (HELP-GT) assay identified 5-hmC loci that were validated at global level by liquid chromatography-mass spectrometry (LC-MS) and the locus-specific level by quantitative reverse transcriptase polymerase chain reaction of 5-hmC pull-down DNA. Hydroxymethylation at both promoter and intragenic locations correlated positively with gene expression. Analysis of pancreatic cancer samples revealed striking redistribution of 5-hmC sites in cancer cells and demonstrated enrichment of this modification at many oncogenic promoters such as GATA6. The HELP-GT assay allowed global determination of 5-hmC and 5-mC from low amounts of DNA and with the use of modest sequencing resources. Redistribution of 5-hmC seen in cancer highlights the importance of determination of this modification in conjugation with conventional methylome analysis.

Management of sepsis in neutropenic patients: 2014 updated guidelines from the Infectious Diseases Working Party of the German Society of Hematology and Medical Oncology (AGIHO)

Sepsis is a major cause of mortality during the neutropenic phase after intensive cytotoxic therapies for malignancies. Improved management of sepsis during neutropenia may reduce the mortality of cancer therapies. Clinical guidelines on sepsis treatment have been published by others. However, optimal management may differ between neutropenic and non-neutropenic patients. Our aim is to give evidence-based recommendations for haematologist, oncologists and intensive care physicians on how to manage adult patients with neutropenia and sepsis.