ALBERT

Beschreibung: A defect in cell trafficking and chemotaxis plays an important role in the immune deficiency observed in Wiskott-Aldrich syndrome (WAS). In this report, we show that marrow cells from WAS protein (WASP)–deficient mice also have a defect in chemotaxis. Serial transplantation and competitive reconstitution experiments demonstrated that marrow cells, including hematopoietic progenitors and stem cells (HSCs), have decreased homing capacities that were associated with a defect in adhesion to collagen. During development, HSCs migrate from the liver to the marrow and the spleen, prompting us to ask if a defect in HSC homing during development may explain the skewed X-chromosome inactivation in WAS carriers. Preliminary evidence has shown that, in contrast to marrow progenitor cells, fetal liver progenitor cells from heterozygous females had a random X-chromosome inactivation. When fetal liver cells from WASP-carrier females were injected into irradiated recipients, a nonrandom inactivation of the X-chromosome was found at the level of hematopoietic progenitors and HSCs responsible for the short- and long-term hematopoietic reconstitution. Therefore, the mechanism of the skewed X-chromosomal inactivation observed in WAS carriers may be related to a migration defect of WASP-deficient HSCs.

Beschreibung: X-linked lymphoproliferative disease (XLP1) is a rare immunodeficiency characterized by severe immune dysregulation and caused by mutations in the SH2D1A/SAP gene. Clinical manifestations are varied and include hemophagocytic lymphohistiocytosis (HLH), lymphoma and dysgammaglobulinemia, often triggered by Epstein-Barr virus infection. Historical data published before improved treatment regimens shows very poor outcome. We describe a large cohort of 91 genetically defined XLP1 patients collected from centers worldwide and report characteristics and outcome data for 43 patients receiving hematopoietic stem cell transplant (HSCT) and 48 untransplanted patients. The advent of better treatment strategies for HLH and malignancy has greatly reduced mortality for these patients, but HLH still remains the most severe feature of XLP1. Survival after allogeneic HSCT is 81.4% with good immune reconstitution in the large majority of patients and little evidence of posttransplant lymphoproliferative disease. However, survival falls to 50% in patients with HLH as a feature of disease. Untransplanted patients have an overall survival of 62.5% with the majority on immunoglobulin replacement therapy, but the outcome for those untransplanted after HLH is extremely poor (18.8%). HSCT should be undertaken in all patients with HLH, because outcome without transplant is extremely poor. The outcome of HSCT for other manifestations of XLP1 is very good, and if HSCT is not undertaken immediately, patients must be monitored closely for evidence of disease progression.

Beschreibung: Umbilical cord blood transplantation (UCBT) has been used to treat malignant and non-malignant diseases. UCBT offers the advantages of easy procurement, immediate availability, and acceptable partial HLA mismatches. Still, patients treated with UCBT show delayed hematopoietic and immunological recoveries, and have higher rates of infection. The problem of slower hematopoietic recovery post-UCBT has been addressed using different approaches: infusion of two CB units, ex-vivo expansion of CB, transplantation of a UCB combined with a selected CD34+ peripheral stem cell unit from an haplo-identical donor. Intrabone (IB) injection is a known emergency access route for infusion of fluids and drugs in children. Frassoni et al published a study on the IB injection of a single unit of CB into 32 adult patients (Frassoni F, et al. Lancet Oncol. 2008) and showed a neutrophil (0.5x10^9/L) and platelet (20x10^9/L) recoveries with a median of 23 and 36 days, respectively, with a median nucleated cell dose of only 2.6 x 10^7 cells/kg. These results have been confirmed in a retrospective study comparing IB infusion of CB with infusion of two units of CB in adult patients (Rocha V, et al. Transplantation. 2013). The only data of IB infusion in pediatric patients is a case report of 5 patients (Saglio F, et al. J Pediatr Hematol Oncol. 2012). In order to confirm the role of IB infusion of CB to improve hematopoietic reconstitution after UCBT in pediatric patients, we started a Phase II single arm, exploratory clinical trial (NCT01711788). Our primary hypothesis is that use of IB infusion of CB reduces stem cell trapping seen with IV infusion, maximizing the number of stem cells and providing a faster short- and robust long-term engraftment. Inclusion criteria includes age up to 21 years, diagnosis of hematopoietic disorders (malignant or not), availability of a single CB with at least 3 x 10^7 nucleated cells (NCs)/kg at freezing (use of two CB units is allowed provided a single CB unit fulfilling the above criteria is not available), and use of a myeloablative-conditioning regimen. On the day of UCBT, frozen CB unit is thawed, washed and re-suspended in 20 ml of a saline/albumin solution and aliquoted into four 5-ml syringes. Patient is sedated with propofol and ketamine according to the current protocol for sedation in children. After sedation, a first aliquot of CB stem cell suspension is injected into the bone marrow, followed by a of 0.5-1 ml saline solution rinse. This procedure is repeated for all remaining aliquots at different places and can be performed in one or both iliac crests, depending on patient size. Each injection is performed within a 2-minute timeframe. If patient requires two CB units, one unit is infused IB while the second unit is infused IV after recovery from sedation. G-CSF is given from D+7. A total of 12 patients has been included since Nov/2012. Eight were male, and median age was 7 (2 - 15) years. Diagnosis were AML (n=6), ALL (n=4) and sickle cell anemia (n=2). Most UCB were unrelated (n=10) and 6/6, 5/6 and 4/6 HLA matching comprised four, five and three UCBT. Busulfan-based conditioning was used in eight patients. Ten patients received a single unit UCB. Median NC infused was 3.4 (2.17-8.4) x 10^7/kg. There was no toxicity related to IB infusion. Engraftment occurred in 10 out of 12 patients and median time to engraftment (neutrophil ≥ 0.5x10^9/L) was 14.5 days. Platelet recovery (≥ 50x10^9/L) occurred in just 6/12 patients (due mainly to early relapse) and median time to recovery was 41 (28-77) days. Three patients presented a grade II aGVHD. There were three relapses. Ten out of twelve patients are alive, one patient died from UCBT complication (ARDS) and other from relapse. In conclusion, IB infusion of UCB is feasible in pediatric patients without any particular toxicity and there is a positive impact on neutrophil recovery. Inclusion of more patients are needed to confirm the impact of IB-UCBT on platelet recovery, duration of hospitalisation, and survival. IB-UCBT might be a more affordable alternative to improve hematopoietic recovery comparing to other methods of UCB expansion. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Familial hemophagocytic lymphohistiocytosis (FHL) is a rare genetic disorder associated with the onset early in life of overwhelming activation of T lymphocytes and macrophages invariably leading to death. Allogeneic bone marrow transplantation (BMT) from an HLA-identical related donor is the treatment of choice in patients with this disease. However, fewer than 20% of patients have a disease-free HLA-identical sibling. BMT from HLA-nonidentical related donors has previously met with poor results, with graft rejection a major obstacle in all cases. We describe BMTs from HLA-nonidentical related donors (n = 13) and from a matched unrelated donor (n = 1) performed in two centers in 14 consecutive cases of FHL. Remission of disease was achieved before BMT in 10 patients. Marrow was T-cell–depleted to minimize graft-versus-host disease (GVHD). Antiadhesion antibodies specific for the α chain of the leukocyte function–associated antigen-1 (LFA-1, CD11a) and the CD2 molecules were infused pre-BMT and post-BMT to help prevent graft rejection, in addition to a conditioning regimen of busulfan (BU), cyclophosphamide (CP), and etoposide (VP16) or antithymocyte globulin (ATG). Sustained engraftment was obtained in 11 of 17 transplants (3 patients had 2 transplants) and disease-free survival in 9 patients with a follow-up period of 8 to 69 months (mean, 33). Acute GVHD greater than stage I was not observed, and 1 patient had mild cutaneous chronic GVHD that resolved. Toxicity due to the BMT procedure was low. Results obtained using this protocol are promising in terms of engraftment and event-free survival within the limitations of the small sample. We conclude that an immunologic approach in terms of drugs used to obtain disease remission and a conditioning regimen that includes antiadhesion molecules in T-cell–depleted BMT from HLA genetically nonidentical donors is an alternative treatment that warrants further study in FHL patients who lack a suitable HLA genetically identical donor.

Beschreibung: The Wiskott-Aldrich syndrome (WAS) is an X-linked hereditary disease characterized by thrombocytopenia with small platelet size, eczema, and increased susceptibility to infections. The gene responsible for WAS was recently cloned. Although the precise function of WAS protein (WASP) is unknown, it appears to play a critical role in the regulation of cytoskeletal organization. The platelet defect, resulting in thombocytopenia and small platelet size, is a consistent finding in patients with mutations in the WASP gene. However, its exact mechanism is unknown. Regarding WASP function in cytoskeletal organization, we investigated whether these platelet abnormalities could be due to a defect in proplatelet formation or in megakaryocyte (MK) migration. CD34+ cells were isolated from blood and/or marrow of 14 WAS patients and five patients with hereditary X-linked thrombocytopenia (XLT) and cultured in serum-free liquid medium containing recombinant human Mpl-L (PEG-rHuMGDF) and stem-cell factor (SCF) to study in vitro megakaryocytopoiesis. In all cases, under an inverted microscope, normal MK differentiation and proplatelet formation were observed. At the ultrastructural level, there was also no abnormality in MK maturation, and normal filamentous MK were present. Moreover, the in vitro produced platelets had a normal size, while peripheral blood platelets of the same patients exhibited an abnormally small size. However, despite this normal platelet production, we observed that F-actin distribution was abnormal in MKs from WAS patients. Indeed, F-actin was regularly and linearly distributed under the cytoplasmic membrane in normal MKs, but it was found concentrated in the center of the WAS MKs. After adhesion, normal MKs extended very long filopodia in which WASP could be detected. In contrast, MKs from WAS patients showed shorter and less numerous filopodia. However, despite this abnormal filopodia formation, MKs from WAS patients normally migrated in response to stroma-derived factor-1 (SDF-1), and actin normally polymerized after SDF-1 or thrombin stimulation. These results suggest that the platelet defect in WAS patients is not due to abnormal platelet production, but instead to cytoskeletal changes occuring in platelets during circulation.

Beschreibung: We have retrospectively assessed the neurological manifestations in 34 patients with hemophagocytic lymphohistiocytosis (HLH) in a single center. Clinical, radiological, and cerebrospinal fluid (CSF ) cytology data were analyzed according to treatment modalities. Twenty-five patients (73%) had evidence of central nervous system (CNS) disease at time of diagnosis, stressing the frequency of CNS involvement early in the time course of HLH. Four additional patients who did not have initial CNS disease, who did not die early from HLH complications, and who were not transplanted, also developed a specific CNS disease. Therefore, all surviving and nontransplanted patients had CNS involvement. Initially, CNS manifestations consisted of isolated lymphocytic meningitis in 20 patients and meningitis with clinical and radiological neurological symptoms in nine patients. For these nine patients, neurological symptoms consisted of seizures, coma, brain stem symptoms, or ataxia. The outcome of patients treated by systemic and intrathecal chemotherapy and/or immunosuppression exclusively (n = 16) was poor, as all died following occurrence of multiple relapses or CNS disease progression in most cases. Bone marrow transplantation (BMT) from either an HLA identical sibling (n = 6) or haplo identical parent (n = 3) was performed in nine patients, once first remission of CNS and systemic disease was achieved. Seven are long-term survivors including three who received an HLA partially identical marrow. All seven are off treatment with normal neurological function and cognitive development. In four other patients, BMT performed following CNS relapses was unsuccessful. Given the frequency and the poor outcome of CNS disease in HLH, BMT appears, therefore, to be the only available treatment procedure that is capable of preventing HLH CNS disease progression and that can result in cure when performed early enough after remission induction.

Beschreibung: The Wiskott-Aldrich syndrome (WAS) is an X-linked hereditary disease characterized by thrombocytopenia with small platelet size, eczema, and increased susceptibility to infections. The gene responsible for WAS was recently cloned. Although the precise function of WAS protein (WASP) is unknown, it appears to play a critical role in the regulation of cytoskeletal organization. The platelet defect, resulting in thombocytopenia and small platelet size, is a consistent finding in patients with mutations in the WASP gene. However, its exact mechanism is unknown. Regarding WASP function in cytoskeletal organization, we investigated whether these platelet abnormalities could be due to a defect in proplatelet formation or in megakaryocyte (MK) migration. CD34+ cells were isolated from blood and/or marrow of 14 WAS patients and five patients with hereditary X-linked thrombocytopenia (XLT) and cultured in serum-free liquid medium containing recombinant human Mpl-L (PEG-rHuMGDF) and stem-cell factor (SCF) to study in vitro megakaryocytopoiesis. In all cases, under an inverted microscope, normal MK differentiation and proplatelet formation were observed. At the ultrastructural level, there was also no abnormality in MK maturation, and normal filamentous MK were present. Moreover, the in vitro produced platelets had a normal size, while peripheral blood platelets of the same patients exhibited an abnormally small size. However, despite this normal platelet production, we observed that F-actin distribution was abnormal in MKs from WAS patients. Indeed, F-actin was regularly and linearly distributed under the cytoplasmic membrane in normal MKs, but it was found concentrated in the center of the WAS MKs. After adhesion, normal MKs extended very long filopodia in which WASP could be detected. In contrast, MKs from WAS patients showed shorter and less numerous filopodia. However, despite this abnormal filopodia formation, MKs from WAS patients normally migrated in response to stroma-derived factor-1 (SDF-1), and actin normally polymerized after SDF-1 or thrombin stimulation. These results suggest that the platelet defect in WAS patients is not due to abnormal platelet production, but instead to cytoskeletal changes occuring in platelets during circulation.

Beschreibung: Familial hemophagocytic lymphohistiocytosis (FHL) is a rare genetic disorder associated with the onset early in life of overwhelming activation of T lymphocytes and macrophages invariably leading to death. Allogeneic bone marrow transplantation (BMT) from an HLA-identical related donor is the treatment of choice in patients with this disease. However, fewer than 20% of patients have a disease-free HLA-identical sibling. BMT from HLA-nonidentical related donors has previously met with poor results, with graft rejection a major obstacle in all cases. We describe BMTs from HLA-nonidentical related donors (n = 13) and from a matched unrelated donor (n = 1) performed in two centers in 14 consecutive cases of FHL. Remission of disease was achieved before BMT in 10 patients. Marrow was T-cell–depleted to minimize graft-versus-host disease (GVHD). Antiadhesion antibodies specific for the α chain of the leukocyte function–associated antigen-1 (LFA-1, CD11a) and the CD2 molecules were infused pre-BMT and post-BMT to help prevent graft rejection, in addition to a conditioning regimen of busulfan (BU), cyclophosphamide (CP), and etoposide (VP16) or antithymocyte globulin (ATG). Sustained engraftment was obtained in 11 of 17 transplants (3 patients had 2 transplants) and disease-free survival in 9 patients with a follow-up period of 8 to 69 months (mean, 33). Acute GVHD greater than stage I was not observed, and 1 patient had mild cutaneous chronic GVHD that resolved. Toxicity due to the BMT procedure was low. Results obtained using this protocol are promising in terms of engraftment and event-free survival within the limitations of the small sample. We conclude that an immunologic approach in terms of drugs used to obtain disease remission and a conditioning regimen that includes antiadhesion molecules in T-cell–depleted BMT from HLA genetically nonidentical donors is an alternative treatment that warrants further study in FHL patients who lack a suitable HLA genetically identical donor.

Beschreibung: Background: The original use of immune globulin (IG) was for replacement in patients with primary immune deficiency (PID). The evidence supporting this stems from the 19650s; however there have not been any rigorously developed evidence based clinical practice guidelines (CPGs) for the use of IG for PID. In 2007, Canadian Blood Services and the National Advisory Committee on Blood and Blood Products in Canada convened a panel of Canadian immunologists and a methodologist to develop a CPG for the use of IG in patients with PID. The objectives of this guideline are to examine the evidence for the use of IG in patients who have PID and to provide guidance for practitioners involved in the care of PID patients and transfusion medicine specialists on the use of IG. Methods: The panel identified key clinical questions and a systematic, expert and bibliography literature search up to July 2008 was conducted to ensure all relevant publications were included. The panel generated recommendations based on the evidence. The levels of evidence and grading of recommendations were adapted from the Canadian Task Force on Preventative Health Care. To validate conclusions and recommendations, the practice guideline will be sent to immunologists internationally and to a patient representative in September 2008. The guideline will be disseminated to all immunologists, internists and pediatricians in Canada to aid implementation of the guideline. The National Advisory Committee of Blood and Blood Products in Canada will assess the performance of the guideline and will renew the guideline at timely intervals. Results and Conclusions: The panel identified the following key clinical questions: what is the prevalence of the PIDs that require IG; does treatment with IG improve morbidity and mortality; and what criteria should be used to monitor the effectiveness of IVIG. The literature search was conducted in August 2007 and updated to July 2008. 1087 citations were reviewed. 101 reports, 1 systematic review and 3 consensus documents/guidelines were included. Current estimates suggest PID is under-diagnosed. Although more than 75% of patients require IG at some point in their treatment, the actual incidence of requiring IG is unknown. There is ample evidence that IG reduces infections, hospitalization and days lost from work/school. There is some evidence to suggest that IG may ameliorate chronic illnesses in patients with PID however, there is no data of the effect of IG on malignancy and insufficient data on the effect of IG on autoimmune disease in this patient population to make a recommendation. Quality of life (QoL) has not been adequately assessed in the literature; however the reduction in infections and hospitalizations likely translates to an improved QoL. Although, there is no evidence to suggest clinical superiority of one IVIG formulation over another, for patients with autoimmune manifestations and PID, there is insufficient evidence that subcutaneous IG is equivalent to IVIG. The vaccination of these patients is an evolving field. Due to the complex nature of the management of patients with PID, the panel recommended assessment by an immunologist prior to the initiation of IG and monitoring by a comprehensive care clinic. Clinical outcomes such as frequency of infections, hospitalization, days missed from school/work should be used to monitor the effectiveness of IVIG. Specific recommendations for dosing and interval of IG therapy were made within the guideline based on the literature The development of a national registry for patients with primary immune deficiency, and the development of a surveillance system for adverse events from IG particularly infections were considered priorities for future development.