Paul G. Schlegel

Reduced-intensity conditioning and HLA-matched haemopoietic stem-cell transplantation in patients with chronic granulomatous disease: a prospective multicentre study

BACKGROUNDnIn chronic granulomatous disease allogeneic haemopoietic stem-cell transplantation (HSCT) in adolescents and young adults and patients with high-risk disease is complicated by graft-failure, graft-versus-host disease (GVHD), and transplant-related mortality. We examined the effect of a reduced-intensity conditioning regimen designed to enhance myeloid engraftment and reduce organ toxicity in these patients.nnnMETHODSnThis prospective study was done at 16 centres in ten countries worldwide. Patients aged 0-40 years with chronic granulomatous disease were assessed and enrolled at the discretion of individual centres. Reduced-intensity conditioning consisted of high-dose fludarabine (30 mg/m(2) [infants <9 kg 1·2 mg/kg]; one dose per day on days -8 to -3), serotherapy (anti-thymocyte globulin [10 mg/kg, one dose per day on days -4 to -1; or thymoglobuline 2·5 mg/kg, one dose per day on days -5 to -3]; or low-dose alemtuzumab [<1 mg/kg on days -8 to -6]), and low-dose (50-72% of myeloablative dose) or targeted busulfan administration (recommended cumulative area under the curve: 45-65 mg/Lu2008×u2008h). Busulfan was administered mainly intravenously and exceptionally orally from days -5 to -3. Intravenous busulfan was dosed according to weight-based recommendations and was administered in most centres (ten) twice daily over 4 h. Unmanipulated bone marrow or peripheral blood stem cells from HLA-matched related-donors or HLA-9/10 or HLA-10/10 matched unrelated-donors were infused. The primary endpoints were overall survival and event-free survival (EFS), probabilities of overall survival and EFS at 2 years, incidence of acute and chronic GVHD, achievement of at least 90% myeloid donor chimerism, and incidence of graft failure after at least 6 months of follow-up.nnnFINDINGSn56 patients (median age 12·7 years; IQR 6·8-17·3) with chronic granulomatous disease were enrolled from June 15, 2003, to Dec 15, 2012. 42 patients (75%) had high-risk features (ie, intractable infections and autoinflammation), 25 (45%) were adolescents and young adults (age 14-39 years). 21 HLA-matched related-donor and 35 HLA-matched unrelated-donor transplants were done. Median time to engraftment was 19 days (IQR 16-22) for neutrophils and 21 days (IQR 16-25) for platelets. At median follow-up of 21 months (IQR 13-35) overall survival was 93% (52 of 56) and EFS was 89% (50 of 56). The 2-year probability of overall survival was 96% (95% CI 86·46-99·09) and of EFS was 91% (79·78-96·17). Graft-failure occurred in 5% (three of 56) of patients. The cumulative incidence of acute GVHD of grade III-IV was 4% (two of 56) and of chronic graft-versus-host disease was 7% (four of 56). Stable (≥90%) myeloid donor chimerism was documented in 52 (93%) surviving patients.nnnINTERPRETATIONnThis reduced-intensity conditioning regimen is safe and efficacious in high-risk patients with chronic granulomatous disease.nnnFUNDINGnNone.

Defibrotide for prophylaxis of hepatic veno-occlusive disease in paediatric haemopoietic stem-cell transplantation: an open-label, phase 3, randomised controlled trial

BACKGROUNDnHepatic veno-occlusive disease is a leading cause of morbidity and mortality after haemopoietic stem-cell transplantation (HSCT). We aimed to assess whether defibrotide can reduce the incidence of veno-occlusive disease in this setting.nnnMETHODSnIn our phase 3 open-label, randomised controlled trial, we enrolled patients at 28 European university hospitals or academic medical centres. Eligible patients were younger than 18 years, had undergone myeloablative conditioning before allogeneic or autologous HSCT, and had one or more risk factor for veno-occlusive disease based on modified Seattle criteria. We centrally assigned eligible participants on the basis of a computer-generated randomisation sequence (1:1), stratified by centre and presence of osteopetrosis, to receive intravenous defibrotide prophylaxis (treatment group) or not (control group). The primary endpoint was incidence of veno-occlusive disease by 30 days after HSCT, adjudicated by a masked, independent review committee, in eligible patients who consented to randomisation (intention-to-treat population), and was assessed with a competing risk approach. Patients in either group who developed veno-occlusive disease received defibrotide for treatment. We assessed adverse events to 180 days after HSCT in all patients who received allocated prophylaxis. This trial is registered with ClinicalTrials.gov, number NCT00272948.nnnFINDINGSnBetween Jan 25, 2006, and Jan 29, 2009, we enrolled 356 eligible patients to the intention-to-treat population. 22 (12%) of 180 patients randomly allocated to the defibrotide group had veno-occlusive disease by 30 days after HSCT compared with 35 (20%) of 176 controls (risk difference -7·7%, 95% CI -15·3 to -0·1; Z test for competing risk analysis p=0·0488; log-rank test p=0·0507). 154 (87%) of 177 patients in the defibrotide group had adverse events by day 180 compared with 155 (88%) of 176 controls.nnnINTERPRETATIONnDefibrotide prophylaxis seems to reduce incidence of veno-occlusive disease and is well tolerated. Thus, such prophylaxis could present a useful clinical option for this serious complication of HSCT.nnnFUNDINGnGentium SpA, European Group for Blood and Marrow Transplantation.

A prospective analysis of the pattern of immune reconstitution in a paediatric cohort following transplantation of positively selected human leucocyte antigen‐disparate haematopoietic stem cells from parental donors

Transplantation of haematopoietic stem cells from human leucocyte antigen (HLA)‐disparate parental donors presents a promising new approach for the treatment of patients lacking a HLA‐matched donor. Success against major obstacles such as graft‐versus‐host disease (GvHD) and graft rejection has recently been demonstrated, so that immune reconstitution is one of the prime factors that determines the long‐term prognosis following transplantation. Twenty children transplanted with megadoses of highly purified CD34+ haematopoietic stem cells after rigorous T‐cell depletion were prospectively monitored for their immune reconstitution during the first post‐transplant year. Natural killer (NK) cells showed a marked increase on du2003+30. T and B cells began to reconstitute on du2003+72 and +68 respectively. During extended follow‐up, their numbers and proliferative capacity upon mitogen stimulation continually increased. Early reconstituting T cells were predominantly of a primed, activated phenotype with severely skewed T‐cell receptor (TCR)‐repertoire complexity. Naive T cells emerged 6u2003months post transplantation, paralleled by an increase in TCR‐repertoire diversity. All patients self‐maintained sufficient immunoglobulin levels after du2003+200. This study demonstrates that paediatric recipients of highly purified, haploidentical stem cells are able to reconstitute functioning T‐, B‐ and NK‐cell compartments within the first post‐transplant year. This, together with the absence of significant GvHD, provides a strong indication for this approach to be considered in children who lack a HLA‐matched donor.

Use of CD137 to study the full repertoire of CD8+ T cells without the need to know epitope specificities†

CD137 (4‐1BB) is a member of the TNFR‐family with costimulatory function, triggering prosurvival signals in activated T‐cells. Upregulation of CD137 upon stimulation allows identifying and isolating live, human antigen‐specific CD8+ T‐cells of all phenotypes, and therefore provides a comprehensive detection method. Furthermore responses against antigen mixtures can be easily detected, enabling antigen discovery in a stepwise deconvoluting approach. In this article, we will discuss various aspects of this methodology, including potential pitfalls as well as a variety of applications, as illustrated by examples from our laboratory.

Primed tumor-reactive multifunctional CD62L+ human CD8+T-cells for immunotherapy

T cell-mediated immunotherapy against malignancies has been shown to be effective for certain types of cancer. However, ex vivo expansion of tumor-reactive T cells has been hindered by the low precursor frequency of such cells, often requiring multiple rounds of stimulation, resulting in full differentiation, loss of homing receptors and potential exhaustion of the expanded T cells. Here, we show that when using highly purified naïve CD8+ T cells, a single stimulation with peptide-pulsed, IFNγ/LPS-matured dendritic cells in combination with the sequential use of IL-21, IL-7 and IL-15 is sufficient for extensive expansion of antigen-specific T cells. Short-term expanded T cells were tumor-reactive, multifunctional and retained a central-memory-like phenotype (CD62L+, CCR7+, CD28+). The procedure is highly reproducible and robust as demonstrated for different healthy donors and for cancer patients. Such short-term tumor-antigen-primed, multifunctional T cells may therefore serve as a platform to target different malignancies accessible to immunotherapy.

Varicella-zoster virus infections in immunocompromised patients - a single centre 6-years analysis

BackgroundInfection with varicella-zoster virus (VZV) contemporaneously with malignant disease or immunosuppression represents a particular challenge and requires individualized decisions and treatment. Although the increasing use of varicella-vaccines in the general population and rapid initiation of VZV-immunoglobulins and acyclovir in case of exposure has been beneficial for some patients, immunocompromised individuals are still at risk for unfavourable courses.MethodsIn this single center, 6-year analysis we review incidence, hospitalization and complication rates of VZV-infections in our center and compare them to published data. Furthermore, we report three instructive cases.ResultsHospitalization rate of referred children with VZV-infections was 45%, among these 17% with malignancies and 9% under immunosuppressive therapy. Rate of complications was not elevated in these two high-risk cohorts, but one ALL-patient died due to VZV-related complications. We report one 4-year old boy with initial diagnosis of acute lymphoblastic leukemia who showed a rapidly fatal outcome of his simultaneous varicella-infection, one 1.8-year old boy with an identical situation but a mild course of his disease, and an 8.5-year old boy with a steroid-dependent nephrotic syndrome. This boy developed severe hepatic involvement during his varicella-infection but responded to immediate withdrawl of steroids and administration of acyclovir plus single-dose cidofovir after nonresponse to acyclovir after 48 h.ConclusionOur data show that patients with malignant diseases or immunosuppressive therapy should be hospitalized and treated immediately with antiviral agents. Despite these measures the course of VZV-infections can be highly variable in these patients. We discuss aids to individual decision-making for these difficult situations.

TH1 predominance is associated with improved survival in pediatric medulloblastoma patients

Medulloblastoma, a primitive neuro-ectodermal tumor that arises in the posterior fossa, is the most common malignant brain tumor occurring in childhood. Even though 60–70% of children with medulloblastoma will be cured with intensive multimodal therapy, including surgery, radiotherapy, and chemotherapy, a significant proportion of surviving patients may suffer from long-term treatment-related sequelae. Therapeutic success is limited especially in younger children by radiotherapy-induced neurocognitive longterm deficits. In order to avoid or delay craniospinal radiotherapy, high-dose chemotherapy followed by autologous stem cell transplantation (HSCT) has become an established treatment modality. Data on the host immunologic environment in medulloblastoma patients are rare, notably data on cytokine expression and immune reconstitution in patients with medulloblastoma undergoing HSCT are lacking. In this present study, we therefore decided to prospectively assess immune function following 24 consecutive autologous HSCT in 17 children with medulloblastoma treated according to the German-Austrian-Swiss HIT-2000-protocol. TH1 predominance was found to be the most important factor for probability of survival. Already before HSCT, survivors showed higher IFNγ levels in sera as well as higher numbers of IFNγ-positive T-cells. After transplantation, this effect was even more pronounced. Patients with higher numbers of IFNγ- and TNFα-positive T-cells had a more favorable outcome at all analyzed time points. In addition, patients in complete remission (CR) before transplantation, known to have a better prognosis a priori, showed higher expression of IFNγ in T-cells. Taken together, this is the first report to demonstrate that high expression of IFNγ and TNFα in T-cells of medulloblastoma patients in the early post-transplant period correlates with a better prognosis. Our data point toward a potentially important influence of TH1-cytokine expression before and after transplantation on the survival of pediatric medulloblastoma patients.

Src-kinase inhibitors sensitize human cells of myeloid origin to Toll-like-receptor-induced interleukin 12 synthesis.

Src-kinase inhibitors hold great potential as targeted therapy against malignant cells. However, such inhibitors may also affect nonmalignant cells and cause pronounced off-target effects. We investigated the role of the dual kinase inhibitor dasatinib on human myeloid cells. Dasatinib is clinically used for the treatment of bcr/abl⁺ leukemias because it blocks the mutated tyrosine kinase abl. To understand its effect on the development of antigen-specific T-cell responses, we assessed antigen-specific priming of human, naïve T cells. In surprising contrast to the direct inhibition of T-cell activation by dasatinib, pretreatment of maturing dendritic cells (DCs) with dasatinib strongly enhanced their stimulatory activity. This effect strictly depended on the activating DC stimulus and led to enhanced interleukin 12 (IL-12) production and T-cell responses of higher functional avidity. Src-kinase inhibitors, and not conventional tyrosine kinase inhibitors, increased IL-12 production in several cell types of myeloid origin, such as monocytes and classical or nonclassical DCs. Interestingly, only human cells, but not mouse or macaques DCs, were affected. These data highlight the potential immunostimulatory capacity of a group of novel drugs, src-kinase inhibitors, thereby opening new opportunities for chemoimmunotherapy. These data also provide evidence for a regulatory role of src kinases in the activation of myeloid cells.

Thrombosis as a complication of central venous access in pediatric patients with malignancies: a 5-year single-center experience

BackgroundReliable central venous access (CVC) is essential for hematology–oncology patients since frequent puncture of peripheral veins—e.g., for chemotherapy, antibiotic administration, repeated blood sampling, and monitoring—can cause unacceptable pain and psychological trauma, as well as severe side effects in cases of extravasation of chemotherapy drugs. However, CVC lines still carry major risk factors, including thrombosis, infection (e.g., entry site, tunnel, and luminal infections), and catheter dislocation, leakage, or breakage.MethodsHere we performed a retrospective database analysis to determine the incidence of CVC-associated thrombosis in a single-center cohort of 448 pediatric oncologic patients, and to analyze whether any subgroup of patients was at increased risk and thus might benefit from prophylactic anticoagulation.ResultsOf the 448 patients, 269 consecutive patients received a CVC, and 55 of these 269 patients (20%) also had a thrombosis. Of these 55 patients, 43 had at least one CVC-associated thrombosis (total number of CVC-associated thrombosis: nu2009=u200952). Among all patients, the median duration of CVC exposure was 464xa0days. Regarding exposure time, no significant difference was found between patients with and without CVC-associated thrombosis. Subclavia catheters and advanced tumor stages seem to be the main risk factors for the development of CVC-associated thrombosis, whereas pharmacologic prophylaxis did not seem to have a relevant impact on the rate of thrombosis.ConclusionsWe conclude that pediatric surgeons and oncologists should pay close attention to ensuring optimal and accurate CVC placement, as this appears the most effective tool to minimize CVC-associated complications.

Pre‐differentiated human committed T‐lymphoid progenitors promote peripheral T‐cell re‐constitution after stem cell transplantation in immunodeficient mice

T‐cell re‐constitution after allogeneic stem cell transplantation (alloSCT) is often dampened by the slow differentiation of human peripheral blood CD34+ (huCD34+) hematopoietic stem cells (HSCs) into mature T cells. This process may be accelerated by the co‐transfer of in vitro‐pre‐differentiated committed T/NK‐lymphoid progenitors (CTLPs). Here, we analysed the developmental potential of huCD34+ HSCs compared with CTLPs from a third‐party donor in a murine NOD‐scid IL2Rγnull model of humanised chimeric haematopoiesis. CTLPs (CD34+lin−CD45RA+CD7+) could be generated in vitro within 10 days upon co‐culture of huCD34+ or cord blood CD34+ (CB‐CD34) HSCs on murine OP9/N‐DLL‐1 stroma cells but not in a novel 3‐D cell‐culture matrix with DLL‐1low human stroma cells. In both in vitro systems, huCD34+ and CB‐CD34+ HSCs did not give rise to mature T cells. Upon transfer into 6‐wk‐old immune‐deficient mice, CTLPs alone did not engraft. However, transplantation of CTLPs together with huCD34+ HSCs resulted in rapid T‐cell engraftment in spleen, bone marrow and thymus at day 28. Strikingly, at this early time point mature T cells originated exclusively from CTLPs, whereas descendants of huCD34+ HSCs still expressed a T‐cell‐precursor phenotype (CD7+CD5+CD1a+/−). This strategy to enhance early T‐cell re‐constitution with ex vivo‐pre‐differentiated T‐lymphoid progenitors could bridge the gap until full T‐cell recovery in severely immunocompromised patients after allogeneic stem cell transplantation.