Daniel Stachel

Monitoring of Minimal Residual Disease After Allogeneic Stem-Cell Transplantation in Relapsed Childhood Acute Lymphoblastic Leukemia Allows for the Identification of Impending Relapse: Results of the ALL-BFM-SCT 2003 Trial

PURPOSE To elucidate the impact of minimal residual disease (MRD) after allogeneic transplantation, the Acute Lymphoblastic Leukemia Berlin-Frankfurt-Münster Stem Cell Transplantation Group (ALL-BFM-SCT) conducted a prospective clinical trial. PATIENTS AND METHODS In the ALL-BFM-SCT 2003 trial, MRD was assessed in the bone marrow at days +30, +60, +90, +180, and +365 after transplantation in 113 patients with relapsed disease. Standardized quantification of MRD was performed according to the guidelines of the Euro-MRD Group. RESULTS All patients showed a 3-year probability of event-free survival (pEFS) of 55%. The cumulative incidence rates of relapse and treatment-related mortality were 32% and 12%, respectively. The pEFS was 60% for patients who received their transplantations in second complete remission, 50% for patients in ≥ third complete remission, and 0% for patients not in remission (P = .015). At all time points, the level of MRD was inversely correlated with event-free survival (EFS; P < .004) and positively correlated with the cumulative incidence of relapse (P < .01). A multivariable Cox model was fitted for each time point, which showed that MRD ≥ 10(-4) leukemic cells was consistently correlated with inferior EFS (P < .003). The accuracy of MRD measurements in predicting relapse was investigated with time-dependent receiver operating curves at days +30, +60, +90, and +180. From day +60 onward, the discriminatory power of MRD detection to predict the probability of relapse after 1, 3, 6, and 9 months was more than 96%, more than 87%, more than 71%, and more than 61%, respectively. CONCLUSION MRD after transplantation was a reliable marker for predicting impending relapses and could thus serve as the basis for pre-emptive therapy.

Efficient chemokine-dependent migration and primary and secondary IL-12 secretion by human dendritic cells stimulated through Toll-like receptors.

Recent findings have demonstrated the properties of cell migration and cytokine secretion to be mutually exclusive and linked them to different functional subpopulations of dendritic cells (DCs). We studied human monocyte-derived DCs after stimulation with peptidoglycan (PGN), poly(I:C), lipopolysaccharide (LPS), and R848 (resiquimod) and found the resulting mature DCs to express CCR7, to migrate toward CCL19 and to be efficient primary interleukin (IL)-12 producers. Importantly, the potential for secondary production of large amounts of IL-12p70 in response to CD40 ligation was also preserved after stimulation by all Toll-like receptor (TLR) ligands. Differences between the TLR ligands were seen in the primary secretion of IL-12 and IL-23, in the survival of the DCs and in the expression of CD38. Finally, DCs stimulated by R848 were efficient in expanding peptide-specific CD8-positive T cells capable of peptide-specific target cell lysis. Together, our data suggest that TLR ligands induce the generation of mature DCs that integrate migratory and cytokine secretory capacity as well as cytotoxic T lymphocyte (CTL) stimulatory properties.

Incidence, Predisposing Factors, and Outcome of Engraftment Syndrome in Pediatric Allogeneic Stem Cell Transplant Recipients

Engraftment syndrome (ES) has been recognized as an inflammatory condition during neutrophil recovery after hematopoietic stem cell transplantation (HSCT) characterized by noninfectious fever and skin rash. It has been reported to occur frequently after autologous HSCT in children and adults, and has been shown to be an independent risk factor for increased transplant-related mortality (TRM). However, virtually no data exist on its occurrence after allogeneic HSCT in children. To determine incidence, predisposing factors for, and complications of ES in a pediatric transplant cohort, we analyzed 61 consecutive recipients of a myeloablative allogeneic HSCT for the occurrence of ES. Diagnosis of ES was established when children presented with > or =2 of the following symptoms within 7 days before engraftment: (1) fever >38.0 degrees C, (2) skin rash, (3) weight gain and albumin drop, or (4) dyspnea, hypoxia, and pulmonary infiltrates. Incidence of ES in this cohort was 48% (29 of 61). In a univariate analysis, posttransplant granulocyte-colony stimulating factor (G-CSF) administration (P = .02), and high mononuclear cell count (MNC) (P = .002) were identified as significant risk factors predisposing for the development of ES. In a multiple logistic regression analysis, amphotericin B therapy (P = .009) and high MNC (P = .004) were significant explanatory variables for ES risk. There was a slight trend toward a higher rate of chronic GVHD (cGVHD) in patients with ES (P = .11). However, after a median follow-up of 9.5 years overall survival (OS) (P = .53) and TRM (P = .65) did not differ between the 2 groups. ES presenting with fever, rash, weight gain, and pulmonary symptoms should be recognized as a frequent complication of allogeneic HSCT after myeloablative conditioning in children. Treatment with G-CSF, amphotericin B, and a high nucleated cell count of the graft predisposed for the development of ES in this study. OS and TRM in this cohort were not affected by the occurrence of ES.

Quantification of busulfan in saliva and plasma in haematopoietic stem cell transplantation in children : validation of liquid chromatography tandem mass spectrometry method.

Background and objectiveBusulfan pharmacokinetic studies suggest that an individual dosing strategy may be necessary to optimise systemic exposure in order to decrease toxicity and improve outcome in haematopoietic stem cell transplantation. Therapeutic and toxic effects of the busulfan/cyclophosphamide regimen have been related to the area under the busulfan plasma concentration-time curve. Because of practical limitations in obtaining blood from children, saliva was evaluated as an alternative matrix for therapeutic drug monitoring, offering the advantages of a non-invasive, rapid and easy sampling procedure. Another objective was to evaluate an easy and robust liquid chromatography-tandem mass spectrometry method for plasma and saliva busulfan determination.MethodsAn online extraction cartridge with column-switching technique, analytical liquid chromatography over a Chromolith RP 18e column, and tandem mass spectrometry were used to quantify busulfan concentrations in matched plasma and saliva samples. The study population consisted of ten patients, aged 1.3–19 years (median age 11.8 years, seven females, three males), undergoing haematopoietic stem cell transplantation. All patients received busulfan 0.8–1.3 mg/kg orally every 6 hours for a total of 16 doses, followed by two doses of cyclophosphamide (60 mg/kg/day).ResultsThe lowest limit of detection was 2 μg/L and the lower limit of quantification was 10 μg/L. Only 100μL of plasma/saliva was needed. The mean recoveries (SD) of busulfan were 97.2% (2.7) in plasma and 100.4% (1.3) in saliva. Intra- and inter-assay imprecision was 2–3% and 2–4% for plasma, and 1–2% and 2–4% for saliva (concentration range 30–1500 μg/L). The bias was <4% for both plasma and saliva. The correlation between the busulfan concentration in plasma and saliva was highly significant (r = 0.958; p < 0.0001; saliva/plasma ratio = 1.09 ± 0.04; n = 69 sample pairs). The apparent plasma clearance was slightly higher than the apparent saliva clearance (202 ± 31 mL/h/kg vs 189 ±28 mL/h/kg; p = 0.001). The mean elimination half-life was found to be 2.31 ±0.46 hours for plasma and 2.30 ± 0.36 hours for saliva; these were not significantly different (p = 0.83).ConclusionThe present study demonstrated that busulfan analysis in saliva could be a valuable and reliable alternative to plasma analysis.

Treosulfan-based conditioning regimen for children and adolescents with hemophagocytic lymphohistiocytosis.

In hematopoietic stem cell transplantation for hemophagocytic lymphohistiocytosis, high transplant-related mortality after busulfan-based myeloablative regimens has been observed. Conditioning regimens with reduced toxicity based on melphalan or treosulfan are promising alternatives. We retrospectively analyzed hematopoietic stem cell transplantations in 19 hemophagocytic lymphohistiocytosis patients after conditioning with fludarabine, treosulfan, alemtuzumab, with or without thiotepa. Overall and disease-free survivals were 100% (follow up 7–31 months). Two patients required second transplant (1 after haploidentical transplantation). In 6 patients, overall donor chimerism dropped below 75% and prompted donor lymphocyte infusions. Administration of donor lymphocytes or second transplantation were significantly more frequent after transplantation from a human leukocyte antigen mismatched (9/10) versus matched (10/10) donor (P=0.018). The toxicity profile was favorable, with one veno-occlusive disease, one grade 3 graft-versus-host disease after donor lymphocyte infusion, and 2 severe viral infections (1 influenza, 1 Epstein Barr virus). In conclusion, the treosulfan-based regimen in hemophagocytic lymphohistiocytosis is effective with low toxicity and gives excellent overall and disease-free survival rates. In the future, the incidence of mixed chimerism, particularly after human leukocyte antigen mismatched donor transplants, needs to be addressed.

Genotype, Clinical Course, and Therapeutic Decision Making in 76 Infants with Severe Generalized Junctional Epidermolysis Bullosa

Severe generalized junctional epidermolysis bullosa, a lethal hereditary blistering disorder, is usually treated by palliative care. Allogeneic stem cell transplantation (SCT) has been proposed as a therapeutic approach, yet without clinical evidence. Decision making was evaluated retrospectively in 76 patients with severe generalized junctional epidermolysis bullosa born in the years 2000-2015. The diagnosis was based on the absence of laminin-332 in skin biopsies. With an incidence of 1 of 150,000, severe generalized junctional epidermolysis bullosa occurred more often than published previously. Eleven as yet unreported mutations in the laminin-332 genes were detected. Although patients homozygous for the LAMB3 mutation c.1903C>T lived longer than the others, life expectancy was greatly diminished (10.8 vs. 4.6 months). Most patients failed to thrive. In two patients with initially normal weight gain, the decision for SCT from haploidentical bone marrow or peripheral blood was made. Despite transiently increasing skin erosions, the clinical status of both subjects stabilized for several weeks after SCT, but finally deteriorated. Graft cells, but no laminin-332, were detected in skin biopsies. The patients died 96 and 129 days after SCT, respectively, one of them after receiving additional skin grafts. Treatment of severe generalized junctional epidermolysis bullosa by SCT is a last-ditch attempt still lacking proof of efficacy.

Platelet function in variable platelet split products intended for neonatal transfusion

BACKGROUND:  Only a few systematic studies have examined the effect of variable produced small platelet (PLT) aliquots on PLT function before transfusion to neonates. Although neonatal transfusion could be critical, no standardization of production or systematic quality controls have been introduced so far.

Enhanced lymphocyte proliferation responses in pediatric patients early after myelosuppressive chemotherapy

It has long been known that patients both after myelosuppressive chemotherapy (ChTh) and after myeloablative bone marrow transplantation (BMT) show a long lasting impairment of cellular immune functions. However, recent reports have revealed that early after BMT a passing state of augmented immune responsiveness exists. Adoptive T cell therapy in this period of lymphopenia‐induced (homeostatic) proliferation has shown better results than in steady state in murine studies.

Childhood Polyarteritis Nodosa in Autoimmune Lymphoproliferative Syndrome

Autoimmune lymphoproliferative syndrome (ALPS) is an uncommon disorder of Fas-mediated apoptosis that results in impaired lymphocyte death and, therefore, disturbed immune homeostasis. Besides presentation with lymphadenopathy and splenomegaly, patients with ALPS have a high incidence of autoimmune phenomena. To our knowledge, this is the first description of polyarteritis nodosa that includes numerous arterial aneurysms in a child with ALPS. Active vasculitis resolved after allogeneic hematopoietic stem cell transplantation. This report of polyarteritis nodosa associated with human ALPS supports previous findings in Fas-deficient mouse models that frequently develop vasculitic manifestations and suggests that apoptotic defects of lymphocytes may play a role in the pathophysiology of systemic vasculitis. Thus, patients with ALPS might be more susceptible to autoimmune vessel inflammation. This case furthermore emphasizes that even rare autoimmune manifestations should be considered and investigated in patients with immunodeficiencies, because that might help in planning treatment strategies for these patients.

Successful stem cell mobilization with stem cell factor and granulocyte colony-stimulating factor in patients with solid tumors failing conventional mobilization with chemotherapy and G-CSF.

HIGH-DOSE CHEMOTHERAPY with stem cell support is a treatment option increasingly used for solid tumors in childhood and adolescence (1). However, after repeated cycles of myelosuppressive chemotherapy with or without radiotherapy, poor stem cell mobilization has been reported in up to 20% of patients (2,3). Risk factors have been identified as preceding cumulative high doses of alkylating agents and radiation therapy (4,5). In these cases, mobilization with granulocyte colony-stimulating factor (G-CSF) from steady state, mobilization following high-dose cyclophosphamide plus G-CSF, or a bone marrow harvest have been proposed with variable success. With regard to alternative stem cell mobilization protocols using new hematopoietic growth factors the combination of stem cell factor (SCF) plus G-CSF seems to have strong mobilization potential for first-line mobilization in patients with breast cancer, multiple myeloma and non-Hodgkin lymphoma (NHL) (6–10). It is unclear, however, whether SCF plus G-CSF would reliably mobilize stem cells in clinical situations where a conventional mobilization regime has already failed. Additionally, little information exists regarding the use, safety, and efficacy of the SCF plus G-CSF approach in a pediatric or adolescent setting. Here we report 3 patients with solid tumors who were successfully mobilized by SCF and G-CSF stimulation after failing mobilization with chemotherapy and G-CSF alone. The first patient, aged 17 years at initial diagnosis, suffered from relapsed stage IV B Hodgkin’s disease, which was treated initially with radiochemotherapy according to the GPOH-HD-95 protocol. Following relapse, she received additional IEP-ABVD-IEP-ABVD chemotherapy. Mobilization of CD34 progenitor cells with G-CSF after the second IEP chemotherapy cycle was not successful (Table 1). As an alternative, mobilization with SCF plus G-CSF was undertaken after the second ABVD cycle. Mobilization consisted of subcutaneous SCF (Ancestim, StemgenTM, Amgen Canada Inc., Mississauga, Ontario, Canada) (10 mg/kg s.c.) for 3 days alone, followed by 6 days of the combination of SCF (10 mg/kg s.c.) and GCSF (Filgrastim, Neupogen®, Amgen, Munich, Germany) (10–12 mg/kg s.c.) as described (7,8) under appropriate antiallergic premedication, as recommended by the drug manufacturer. Leukapheresis was performed on the 5th, 6th, and 7th day of the G-CSF stimulation with a total yield of 5.13 106 CD34pos cells/kg b.w. At this time, the cumulative chemotherapy dose had reached 9 g/m2 procarbazin, 21 mg/m2 vincristin, 260 mg/m2 adriamycin, 4 g/m2 cyclophosphamide, 20 g/m2 ifosfamide, 1.25 g/m2 etoposide, 40 mg/m2 bleomycin, 24 mg/m2 vinblastin, and 1.5 g/m2 dacarbazin. The second patient, aged 14 years at diagnosis, suffered from relapsed Ewing’s sarcoma of the right humerus and the brain. Firstline therapy consisted of 14 EVAIA cycles plus local radiation with 44.8 Gy according to the EICESS 92 protocol; second-line treatment was 4 Cyc-ETO cycles