Survival after mesenchymal stromal cell therapy in steroid-refractory acute graft-versus-host disease: systematic review and meta-analysis.

BACKGROUND: Graft-versus-host disease (GVHD) is the major limitation of allogeneic haemopoietic stem-cell transplantation (HSCT), for which no approved treatments are available. Use of mesenchymal stromal cells (MSCs) has become standard practice in some European countries, but controversy exists for their benefit. The aim of this meta-analysis was to analyse available evidence for the benefit of MSC treatments in steroid-resistant acute GVHD. METHODS: We did a systematic review and meta-analysis to assess response to and survival after MSC treatment in patients with steroid-refractory acute GVHD. We searched MEDLINE, Embase, Ovid, and Cochrane Central databases for published studies, and we used ClinicalTrials.gov and other websites to find unpublished studies and conference abstracts. We included prospective and retrospective studies in which MSCs were administered to patients with steroid-refractory acute GVHD. Data were extracted independently by two investigators based on strict selection criteria. A random-effects model was used to pool outcomes across studies because of anticipated heterogeneity. Our primary outcome was survival at 6 months from the first infusion of MSCs. FINDINGS: We identified 628 citations with our search, of which 610 were excluded after review and a further five did not contain pertinent data. Thus, our meta-analysis included 13 non-randomised studies at moderate risk of bias, comprising a total of 336 patients. Six studies provided data for the primary outcome analysis (119 patients). Survival at 6 months after MSC treatment was 63% (95% CI 50-74; I(2)=41%). Survival did not differ with respect to age, MSC culture medium, or dose of MSCs delivered. INTERPRETATION: Available evidence suggests that infusion of MSCs could be an acceptable treatment for patients with steroid-refractory acute GVHD. Randomised clinical trials are needed urgently to assess different treatment modalities for steroid-refractory acute GVHD. FUNDING: None.

Current variations in childhood cancer supportive care in the Netherlands.

BACKGROUND: Current treatment strategies in pediatric oncology are intensive and lead to high survival rates but also to treatment-related complications. Therefore, supportive care plays an increasingly important role. This study was designed to evaluate variations in supportive care practice in children with cancer in the Netherlands and adherence to selected existing international guidelines through an in-depth review of local guidelines and protocols at all 6 Dutch pediatric cancer centers. METHODS: Based on shared expert opinion, a questionnaire regarding current supportive care practice was compiled. For each center, the required information was extracted from local supportive care guidelines, and the list was sent to a pediatric oncologist of that center to verify its correspondence with local daily practice. Subsequently, it was determined whether clinical practice was concordant (same in ≥ 5 of 6 centers), partly concordant (highly overlapping in ≥ 5 of 6 centers), or discordant (same in < 5 of 6 centers). Local practices were compared with strong recommendations from high-quality, evidence-based guidelines. RESULTS: The questionnaire comprised 67 questions regarding supportive care practice. Concordance was observed for 11 of 67 practice items (16%), partial concordance was observed for 6 of 67 practice items (9%), and discordance was observed for 50 of 67 practice items (75%). Adherence to strong recommendations of 4 high-quality, evidence-based guidelines varied but was generally low. CONCLUSIONS: Large variations exist in pediatric oncology supportive care practice, and this could negatively influence care. Adherence to existing evidence-based guidelines and the development and implementation of new clinical practice guidelines have the potential of standardizing supportive care practice and thereby improving outcomes for children with cancer.

Determination of Eligibility in Related Pediatric Hematopoietic Cell Donors: Ethical and Clinical Considerations. Recommendations from a Working Group of the Worldwide Network for Blood and Marrow Transplantation Association.

Related donors for hematopoietic cell (HC) transplantation are a growing population in recent years because of expanding indications for allogeneic transplantation. The safety and welfare of the donor are major concerns for the transplantation community, especially for related sibling donors of young recipients who are children and, thus, not able to fully consent. Because donation of HC does not improve the donor's own physical health and carries a risk of side effects, careful assessment of medical risks specific to the individual donor, as well as consideration of ethical and legal aspects associated with donation from a child, must be considered. In addition, donor centers must balance the needs of both the donor and the recipient, understanding the inherent conflict parents may have as they can be overly focused on the very sick child receiving a transplant, rather than on the relatively less significant health or emotional problems that a sibling donor may have, which could impact risk with donation. Likewise, consideration must be made regarding the nature of the relationship of the sibling donor to the recipient and also aspects of performing research on pediatric HC donors. In this article, as members of the Donor Issues Committee of the Worldwide Network for Blood and Marrow Transplantation, we review key ethical concerns associated with pediatric donation and then give recommendations for screening potential child donors with underlying health conditions. These recommendations are aimed at protecting the physical and emotional well-being of childhood donors and arise out of the Third International Conference on Health and Safety of Donors sponsored by the Worldwide Network for Blood and Marrow Transplantation.

Risk-adapted approach for fever and neutropenia in paediatric cancer patients--a national multicentre study.

BACKGROUND: In this national multicentre study, we examined the safety of reducing antibiotics in selected paediatric cancer patients with febrile neutropenia. METHODS: Patients with signs of a bacterial infection and/or abnormal vital signs indicating sepsis were considered high risk and received antibiotic therapy. Remaining patients were allocated to low- or medium risk, depending on their interleukin-8 level. Low-risk patients did not receive any antibiotics and were discharged from the hospital after having been afebrile for 12 h. Medium-risk patients were re-evaluated after 72 h of antibiotic treatment and, in selected patients, antibiotics were stopped. RESULTS: Two hundred thirty-three febrile neutropenic episodes in 141 paediatric cancer patients were included in the study. Sixty-four episodes were classified high risk (28%), 122 medium risk (52%), and 47 (20%) low risk. In the medium-risk group, antibiotics were stopped after 72 h in 50 in 122 episodes (41%). Median duration of antibiotic treatment and hospital admission was significantly lower in low- and medium-risk episodes with early discharge. No failures were observed in the medium-risk group with early discharge. In the low-risk group, six failures were observed (12.8%), due to coagulase-negative staphylococci-positive blood cultures and recurrent fever. CONCLUSION: We showed that it is safe to shorten antibiotic treatment to 72 h in selected medium-risk patients with febrile neutropenia, regardless of the neutrophil count. The safety of withholding antibiotics in selected low-risk paediatric cancer patients with febrile neutropenia requires further investigation, using more suitable definitions for safety. Reduction in hospital admissions allows children with cancer more time at home and consequently improves their quality of life.

Successful treatment of fetal hemolytic disease due to glucose phosphate isomerase deficiency (GPI) using repeated intrauterine transfusions: a case report.

Hemolytic anemia due to GPI deficiency can be severe and life threatening during fetal life. When parents decline invasive testing, ultrasound monitoring of fetuses at risk is feasible. Intrauterine transfusion can be effective for the treatment of severe fetal anemia due to GPI deficiency.

Gene-expression and in vitro function of mesenchymal stromal cells are affected in juvenile myelomonocytic leukemia.

An aberrant interaction between hematopoietic stem cells and mesenchymal stromal cells has been linked to disease and shown to contribute to the pathophysiology of hematologic malignancies in murine models. Juvenile myelomonocytic leukemia is an aggressive malignant disease affecting young infants. Here we investigated the impact of juvenile myelomonocytic leukemia on mesenchymal stromal cells. Mesenchymal stromal cells were expanded from bone marrow samples of patients at diagnosis (n=9) and after hematopoietic stem cell transplantation (n=7; from 5 patients) and from healthy children (n=10). Cells were characterized by phenotyping, differentiation, gene expression analysis (of controls and samples obtained at diagnosis) and in vitro functional studies assessing immunomodulation and hematopoietic support. Mesenchymal stromal cells from patients did not differ from controls in differentiation capacity nor did they differ in their capacity to support in vitro hematopoiesis. Deep-SAGE sequencing revealed differential mRNA expression in patient-derived samples, including genes encoding proteins involved in immunomodulation and cell-cell interaction. Selected gene expression normalized during remission after successful hematopoietic stem cell transplantation. Whereas natural killer cell activation and peripheral blood mononuclear cell proliferation were not differentially affected, the suppressive effect on monocyte to dendritic cell differentiation was increased by mesenchymal stromal cells obtained at diagnosis, but not at time of remission. This study shows that active juvenile myelomonocytic leukemia affects the immune response-related gene expression and function of mesenchymal stromal cells. In contrast, the differential gene expression of hematopoiesis-related genes could not be supported by functional data. Decreased immune surveillance might contribute to the therapy resistance and progression in juvenile myelomonocytic leukemia.

Role of NKG2D, DNAM-1 and natural cytotoxicity receptors in cytotoxicity toward rhabdomyosarcoma cell lines mediated by resting and IL-15-activated human natural killer cells.

Children with advanced stages (relapsed/refractory and stage IV) of rhabdomyosarcoma (RMS) have a poor prognosis despite intensive chemotherapy and autologous stem cell rescue, with 5-year survival rates ranging from 5 to 35 %. Development of new, additional treatment modalities is necessary to improve the survival rate. In this preclinical study, we investigated the potential of resting and cytokine-activated natural killer (NK) cells to lyse RMS cell lines, as well as the pathways involved, to explore the eventual clinical application of (activated) NK cell immunotherapy. RMS cell lines (n = 3 derived from embryonal RMS and n = 2 derived from alveolar RMS) were susceptible to cytolysis mediated by resting NK cells, and this susceptibility was significantly increased using IL-15-activated NK cells. Flow cytometry and cytolytic assays were used to define the activating and inhibitory pathways of NK cells involved in recognizing and lysing RMS cells. NKG2D and DNAM-1 receptor-ligand interactions were essential in cytolysis by resting NK cells, as simultaneous blocking of both pathways resulted in almost complete abrogation of the cytotoxicity. In contrast, combined blocking of DNAM-1 and NKG2D only led to partial reduction of the lytic activity of IL-15-activated NK cells. In this respect, residual lysis was, at least partly, mediated by pathways involving the natural cytotoxicity receptors NKp30 and NKp46. These findings support further exploration of NK cell-based immunotherapy as adjuvant modality in current treatment strategies of RMS.

The effect of cidofovir on adenovirus plasma DNA levels in stem cell transplantation recipients without T cell reconstitution.

Cidofovir is frequently used to treat life-threatening human adenovirus (HAdV) infections in immunocompromised children after hematopoietic stem cell transplantation (HSCT). However, the antiviral effect irrespective of T cell reconstitution remains unresolved. Plasma HAdV DNA levels were monitored by real-time quantitative PCR during 42 cidofovir treatment episodes for HAdV viremia in 36 pediatric allogeneic HSCT recipients. HAdV load dynamics were related to T and natural killer (NK) cell reconstitution measured by flow cytometry. To evaluate the in vivo antiadenoviral effect of cidofovir, we focused on 20 cidofovir treatment episodes lacking concurrent T cell reconstitution. During 2 to 10 weeks of follow-up in the absence of T cells, HAdV load reduction (n = 7) or stabilization (n = 8) was observed in 15 of 20 treatments. Although HAdV load reduction was always accompanied by NK cell expansion, HAdV load stabilization was measured in 2 children lacking both T and NK cell reconstitution. In cases with T cell reconstitution, rapid HAdV load reduction (n = 14) or stabilization (n = 6) was observed in 20 of 22 treatments. In the absence of T cells, cidofovir treatment was associated with HAdV viremia control in the majority of cases. Although the contribution of NK cells cannot be excluded, cidofovir has the potential to mediate HAdV load stabilization in the time pending T cell reconstitution.

Ovarian insufficiency and pubertal development after hematopoietic stem cell transplantation in childhood.

BACKGROUND: Ovarian insufficiency (OI) and infertility are common and devastating late effects of cancer treatment and hematopoietic stem cell transplantation (HSCT). In children, gonadal insufficiency may subsequently lead to abnormal pubertal development. The aim of this study was to assess the cumulative incidence of OI and the need for hormonal induction of pubertal development after HSCT in childhood. We additionally assessed HSCT-related risk factors for OI. PROCEDURES: A single center cohort study was undertaken of female patients transplanted during childhood, surviving at least 2 years post-HSCT and who were at least 10 years old at initiation of the study. Of 141 eligible patients, 109 were included and hormone levels and clinical data of these patients during follow-up were collected. Risk factors for OI were analyzed by multivariate Cox regression analysis. RESULTS: Cumulative incidence of OI was 56% at a median follow-up of 7.2 years. Eight patients, initially diagnosed with OI, showed recovery of ovarian function over time. Hormonal induction of puberty was necessary in 44% of females who were pre-pubertal or pubertal at HSCT. In multivariate analysis, more advanced pubertal stage at HSCT was associated with OI. We found a trend for an association of busulfan with OI in patients conditioned with chemotherapy only. CONCLUSIONS: The incidence of OI after HSCT was high and associated with more advanced pubertal stage at HSCT. Almost half of the females who were pre-pubertal or pubertal at HSCT required hormonal induction of pubertal development.

Mesenchymal stromal cell therapy is associated with increased adenovirus-associated but not cytomegalovirus-associated mortality in children with severe acute graft-versus-host disease.

Beneficial effects of mesenchymal stromal cells (MSCs) in patients with severe steroid-refractory acute graft-versus-host disease (aGvHD) have been reported. However, controversy exists about the effect of MSCs on virus-specific T cells. We evaluated 56 patients with grade II-IV aGvHD who responded to steroids (n = 21) or were steroid refractory receiving either MSCs (n = 22) or other second-line therapy (n = 13). Although the overall incidence of cytomegalovirus (CMV), Epstein-Barr virus, and human adenovirus (HAdV) infections was not significantly increased, HAdV infection was associated with decreased survival in children treated with MSCs. Thus, we investigated in vitro the effects of MSCs on virus-specific T cells. Both CMV-specific and, to a lesser extent, HAdV-specific T-cell activation and proliferation were negatively affected by MSCs either after induction of a response in peripheral blood mononuclear cells (PBMCs) or after restimulation of virus-specific T-cell lines. In patient-derived PBMCs, CMV-specific proliferative responses were greatly decreased on first-line treatment of aGvHD with systemic steroids and slowly recovered after MSC administration and tapering of steroids. HAdV-specific T-cell proliferation could not be detected. In contrast, the proportion of CMV- and HAdV-specific effector T cells, measured as interferon-γ-secreting cells, remained stable or increased after treatment with MSCs. In conclusion, although in vitro experimental conditions indicated a negative impact of MSCs on CMV- and HAdV-specific T-cell responses, no solid evidence was obtained to support such an effect of MSCs on T-cell responses in vivo. Still, the susceptibility of steroid-refractory severe aGvHD patients to viral reactivation warrants critical viral monitoring during randomized controlled trials on second-line treatment including MSCs.

Gastrointestinal acute graft-versus-host disease in children: histology for diagnosis, mesenchymal stromal cells for treatment, and biomarkers for prediction of response.

Steroid-nonresponsive acute graft-versus-host disease (aGVHD) after hematopoietic stem cell transplantation carries a poor prognosis. Various groups have reported beneficial effects of mesenchymal stromal cell (MSC) infusion as salvage treatment. Response to treatment is typically evaluated using the diagnostic clinical criteria for aGVHD. In this study, we evaluated the usefulness of additional gastrointestinal biopsy specimens paired with serum biomarkers. In a cohort of 22 pediatric patients, persistent or recurrent diarrhea was seen in 18 children treated with MSC infusion for steroid-refractory aGVHD. To exclude other causes of gastrointestinal pathology, patients were biopsied for histological analysis. Eight of 12 patients exhibited residual tissue damage and villous atrophy, but no active aGVHD. Serum biomarkers have been identified as an alternative tool for monitoring the response to aGVHD treatment. The value of biomarkers for inflammation, tissue, and endothelial cell damage was evaluated in our cohort. Although predictive of response to treatment and survival, these markers lack the necessary specificity and sensitivity to predict response to MSC therapy. Objective endpoints for clinical trials on the treatment of steroid-refractory aGVHD remain to be defined. Thus, we recommend including biopsies and biomarkers to discriminate between ongoing aGVHD and postinflammatory malabsorption.

Multiple infusions of mesenchymal stromal cells induce sustained remission in children with steroid-refractory, grade III-IV acute graft-versus-host disease.

Mesenchymal stromal cell (MSC) infusions have been reported to be effective in patients with steroid-refractory, acute graft-versus-host disease (aGvHD) but comprehensive data on paediatric patients are limited. We retrospectively analysed a cohort of 37 children (aged 3 months-17 years) treated with MSCs for steroid-refractory grade III-IV aGvHD. All patients but three received multiple MSC infusions. Complete response (CR) was observed in 24 children (65%), while 13 children had either partial (n = 8) or no response (n = 5). Cumulative incidence of transplantation-related mortality (TRM) in patients who did or did not achieve CR was 17% and 69%, respectively (P = 0.001). After a median follow-up of 2.9 years, overall survival (OS) was 37%; it was 65% vs. 0% in patients who did or did not achieve CR, respectively (P = 0.001). The median time from starting steroids for GvHD treatment to first MSC infusion was 13 d (range 5-85). Children treated between 5 and 12 d after steroid initiation showed a trend for better OS (56%) and lower TRM (17%) as compared with patients receiving MSCs 13-85 d after steroids (25% and 53%, respectively; P = 0.22 and 0.06, respectively). Multiple MSC infusions are safe and effective for children with steroid-refractory aGvHD, especially when employed early in the disease course.

Mesenchymal stromal cells isolated from children with systemic juvenile idiopathic arthritis suppress innate and adaptive immune responses.

BACKGROUND AIMS: Infusion of mesenchymal stromal cells (MSCs) has been reported to be an effective treatment modality for acute graft-versus-host disease, and MSCs have been considered for use in the treatment of patients with autoimmune diseases. Before contemplating clinical studies with MSCs in patients with systemic juvenile idiopathic arthritis (sJIA), the immunomodulatory capacity of MSCs in this setting needs to be explored. A comparative analysis of bone marrow-derived MSCs from children with sJIA and healthy pediatric controls was performed. METHODS: MSCs were successfully expanded from 11 patients with sJIA and 10 controls. The phenotype, differentiation and immunomodulatory capacity of these MSCs were compared. The effect of immunosuppressive drugs on MSC function was also investigated. RESULTS: MSCs from patients with sJIA and controls showed no differences in their suppressive effect using control peripheral blood mononuclear cells. Furthermore, the suppression of the response of peripheral blood mononuclear cells from patients with sJIA by autologous sJIA MSCs and allogeneic control MSCs was comparable. The immunosuppressive effect of both groups of MSCs was diminished in the presence of indomethacin (P < 0.05). MSCs from patients with sJIA and controls suppressed interleukin-2-induced natural killer cell activation to a similar extent. In addition, MSCs of patients with sJIA and controls inhibited the differentiation of monocytes to dendritic cells. CONCLUSIONS: This is the first explorative study in a significant cohort of patients with sJIA to evaluate the effect of MSCs on adaptive and innate immune responses. The comparable immunosuppressive characteristics of MSCs derived from patients with sJIA to age-matched controls support the potential use of patient-derived MSCs in the treatment of sJIA.

Immunotherapy in the context of hematopoietic stem cell transplantation: the emerging role of natural killer cells and mesenchymal stromal cells.

Immunotherapy in the context of hematopoietic stem cell transplantation has been dominated for many years by T-cell- and dendritic-cell-based treatment modalities. During the last decade, insight into the biology of natural killer (NK) cells and mesenchymal stromal cells (MSC) has rapidly increased and resulted in NK- and MSC-based therapeutic strategies in clinical practice. This article reviews current knowledge of the biology and clinical aspects of NK cells and MSC.

Delayed immune recovery following sequential orthotopic liver transplantation and haploidentical stem cell transplantation in erythropoietic protoporphyria.

A nine-yr-old boy with EPP suffered from severe skin burns and liver failure caused by progressive cholestasis and fibrosis. OLT was performed without major complications. Four months following liver transplantation he underwent parental haploidentical HSCT. The myeloablative conditioning regimen was relatively well tolerated and hematological engraftment was rapid (on day 10). Protoporphyrin concentrations returned to normal following HSCT. However, immune recovery was significantly delayed. Varicella zoster virus reactivation resulted in impaired vision, prolonged hospitalization and eventually in multiorgan failure and death. Sequential liver and haploidentical HSCT proved feasible though a high risk procedure in this EPP patient. The management of post-IST after these combined transplantations remains a challenge and needs to be further established.

Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors.

BACKGROUND: Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However, relatively little is known about the mechanisms responsible for their immunomodulatory effects, which could be influenced by both the cell source and culture conditions. DESIGN AND METHODS: We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells, in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells. RESULTS: The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology, immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency, umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition, these cells do not express hTERT and telomerase activity, do express p16(ink4a) protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses, umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation, (ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10, while decreasing interferon-gamma secretion, in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects, a prostaglandin E(2)-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA-G. CONCLUSIONS: Umbilical cord blood- and bone marrow-mesenchymal stromal cells may differ in terms of clonogenic efficiency, proliferative capacity and immunomodulatory properties; these differences may be relevant for clinical applications.

Atypical varicella zoster infection associated with hemophagocytic lymphohistiocytosis.

Two adolescents, on immunosuppressive therapy for graft-versus-host disease, developed hemophagocytic lymphohistiocytosis (HLH) after varicella zoster virus (VZV) reactivation. In the absence of dermatome restricted characteristic skin lesions, VZV reactivation was not immediately recognized and treatment with acyclovir was delayed. The first patient developed optical neuritis and died 2 months after the VZV episode due to massive intracranial hemorrhage. The second patient presented with severe abdominal pain and pancreatitis, followed by atypical skin eruptions, which prompted a faster diagnosis. Both patients recovered from their HLH, the first patient being successfully treated with immunosuppressive agents and the second with VZV treatment only. These two cases demonstrate the difficulties in recognizing VZV reactivation, and in order to start adequate and timely treatment, the need to consider VZV as a possible cause of HLH in severely immunocompromised patients.

Mesenchymal stem cells exert differential effects on alloantigen and virus-specific T-cell responses.

Mesenchymal stem cells (MSCs) suppress alloantigen-induced T-cell functions in vitro and infusion of third-party MSCs seems to be a promising therapy for graft-versus-host disease (GVHD). Little is known about the specificity of immunosuppression by MSCs, in particular the effect on immunity to pathogens. We have studied how MSCs affect T-cell responses specific to Epstein-Barr virus (EBV) and cytomegalovirus (CMV). We found that EBV- and CMV-induced proliferation and interferon-gamma (IFN-gamma) production from peripheral blood mononuclear cells (PBMCs) was less affected by third-party MSCs than the response to alloantigen and that MSCs had no effect on expansion of EBV and CMV pentamer-specific T cells. Established EBV-specific cytotoxic T cells (CTL) or CMV-CTL cultured with MSCs retained the ability to proliferate and produce IFN-gamma in response to their cognate antigen and to kill virally infected targets. Finally, PBMCs from 2 patients who received MSCs for acute GVHD showed persistence of CMV-specific T cells and retained IFN-gamma response to CMV after MSC infusion. In summary, MSCs have little effect on T-cell responses to EBV and CMV, which contrasts to their strong immunosuppressive effects on alloreactive T cells. These data have major implications for immunotherapy of GVHD with MSCs and suggest that the effector functions of virus-specific T cells may be retained after MSC infusion.