ALBERT

Description: Following allogeneic hematopoietic stem cell transplantation (alloHSCT), children are at risk of life-threatening pneumococcal infections. Whereas vaccination with polysaccharide vaccines fails to elicit protective immunity in most alloHSC transplant recipients, pneumococcal conjugate vaccines may effectively prevent invasive disease by eliciting T-cell–dependent antibody responses. Here, we report safety and immunogenicity in 53 children immunized with a regimen of 3 consecutive doses of a heptavalent pneumococcal conjugate vaccine (7vPCV) in monthly intervals starting 6 to 9 months after alloHSCT. Immunization was well tolerated with no vaccine-related serious adverse events. Serologic response rates evaluable in 43 patients ranged from 41.9% to 86.0% and 58.1% to 93.0% after 2 and 3 vaccinations, respectively, with 55.8% and 74.4% of patients achieving protective antibody levels to all 7 vaccine serotypes. Our study provides the first evidence that vaccination with 7vPCV is safe and elicits protective antipneumococcal antibody responses in pediatric recipients of related or unrelated donor alloHSC transplants within the first year following transplantation. This trial was registered at www.clinicaltrials.gov as NCT00169728.

Description: Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disease presenting with isolated thrombocytopenia in infancy and developing into a pancytopenia in later childhood. Thrombopoietin (TPO) is the main regulator of thrombocytopoiesis and has also been demonstrated to be an important factor in early hematopoiesis. We analyzed 9 patients with CAMT for defects in TPO production and reactivity. We found high levels of TPO in the sera of all patients. However, platelets and hematopoietic progenitor cells of patients with CAMT did not show any reactivity to TPO, as measured by testing TPO-synergism to adenosine diphosphate in platelet activation or by megakaryocyte colony assays. Flow cytometric analysis revealed absent surface expression of the TPO receptor c-Mpl in 3 of 3 patients. Sequence analysis of the c-mpl gene revealed point mutations in 8 of 8 patients: We found frameshift or nonsense mutations that are predicted to result in a complete loss of c-Mpl function in 5 patients. Heterozygous or homozygous missense mutations predicted to lead to amino acid exchanges in the extracellular domain of the receptor were found in 3 other patients. The type of mutations correlated with the clinical course of the disease. We propose a defective c-Mpl expression due to c-mpl mutations as the cause for thrombocytopenia and progression into pancytopenia seen in patients with CAMT.

Description: Abstract 2372 Thrombocytopenia-absent radii syndrome (TAR) is a rare congenital disorder characterized by bilateral radius aplasia and thrombocytopenia due to virtual absence of bone marrow megakaryocytes (MKs). As other blood lineages are not affected, TAR is considered to be a bone marrow failure syndrome. At birth, platelet counts are often below 50/nL, but ameliorate in many patients during the first two years of life, however, without reaching the lower norm. Platelet reactivity in reponse to thrombopoietin is abrogated, mostly when patients are young while it is restored to normal in older patients. Interestingly, this patterning is not correlated with platelet counts. In 2007 we described a microdeletion on chromosome 1q21 in all patients analyzed as necessary, but not sufficient to cause TAR, as it was also present in unaffected family members. Using next generation sequencing, we recently identified the presence of one of two single nucleotide polymorphisms (SNPs) in non-coding, regulatory regions of the gene RBM8A whose compound inheritance together with the microdeletion causes TAR syndrome (Nature Genetics, 44, 435). All patients inherited one of the two SNPs from one of one parents and the microdeletion from the other. In 25% of cases the microdeletion was acquired de novo. This gene, which is located within the microdeleted region, encodes for Y14, a major subunit of the exon junction complex. In the German/Swiss/Polish cohort of 28 patients, 20 patients had the more frequent SNP in the 5'UTR, whereas 8 patients had a previously undescribed SNP in intron 1. The 5'UTR G-〉A introduces a novel binding site for transcriptional repressor Evi1. This factor is expressed in megakaryocytes and still present in platelets. Using electrophoretic mobility shift assays (EMSA) with lysates from platelets we found a complex exclusively present when a labeled oligonucleotide with the 5'UTR-SNP G-〉C was used and not with the wildtype sequence. This complex could be partially supershifted with an anti-Evi1 antibody, suggesting that an additional factor might bind to this complex. In contrast, the intronic SNP is predicted to abrogate a binding site for Mzf1. We used EMSA with oligonucleotides of both intronic SNP and wildtype sequence and indeed found a complex binding to the wildtype sequence as well as to a consensus sequence for Mzf1, but no or reduced binding to the intronic SNP. This complex could not be supershifted by adding an anti-Mzf1 antibody. However, when nuclear extracts from Meg01 cells were used, we found that one of three subcomplexes showed reduced binding in the presence of the antibody, even when the intronic SNP was used. As both SNPs showed different binding properties we carefully evaluated clinical data and found that platelet counts were more severely reduced in patients with the 5'UTR compared to patients harboring the intronic SNP. Although case numbers for the intronic SNP are low, in a direct comparison of age-matched patients, platelet counts were always higher in TAR patients with the intronic SNP. However, we did not find any obvious differences in skeletal features between the two groups. Further work is ongoing to better understand the causative role of either SNP for impaired megakaryopoiesis and thrombocytopenia in TAR syndrome. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1214 Immune thrombocytopenia (ITP) is the most common acquired thrombocytopenia in children. Typically, external triggers as infections or vaccinations cause the rise of antibodies that crossreact with antigens expressed on the platelet surface. These anti-platelet antibodies are mostly directed against glycoprotein complexes GPIIb/IIIa or GPIb/IX/V, resulting in an increased turnover of antibody-decorated platelets which are then sequestered by the reticuloendothelial system. Recently, it has been suggested that thrombocytopenia might also be due to an insufficient platelet production as serum of some patients with ITP can impair the maturation of CD34+ hematopoietic stem cells to bone marrow megakaryocytes (MKs) in vitro or abrogate the formation of proplatelets in an in vitro culture system. The accelerated platelet turnover demands the generation of platelets de novo. Bone marrow smears often reveal normal or slightly increased MKs, although they seem to be smaller and of altered morphology. However, very little is known about the consequences of anti-platelet antibodies on bone marrow MKs in vivo and in situ. Here, we took advantage of a simple animal model of passive ITP by single or multiple intraperitoneal injections of an anti-GPIb antibody into mice. MKs were evaluated by multi-color immunofluorescence histology on whole femur sections in a modified staining procedure that bypasses decalcification. MK numbers on day 3 were doubled in response to a single injection and tripled on day 8 when mice were injected additionally on day 3 and 7. In these mice platelet counts were up to 2000/nL on day 10, indicating the power to produce platelets. MK area per section was transiently upregulated on day 3 in single injected mice and quadrupled after multiple injections on day 8 before shrinking below norm on day 14. Staining with an anti-rat IgG antibody showed that the antibody was present on MKs within the bone marrow several hours to days after injection. The signal was present for 5 days and no antibody was detected on day 7. MKs had an overall normal morphology and showed no signs of apoptosis or DNA blebbing. All MKs analyzed were negative for TdT in a classical TUNEL assay, indicating that there were no single strand breaks. As platelet counts rose markedly while the antibody was still present on the MK surface, we sought to identify whether the pool of MKs is expanded or formed de novo. To address this, mice where fed with nucleotide analogue EdU for up to 12 days and femur sections stained with Click-It-647 reagent to stain for newly incorporated DNA while mice were treated with anti-platelet antibody or isotype control. We found EdU-positive MKs after 12 days in control isotype-injected mice indicating the de novo formation from hematopoietic stem cells. In antibody-injected mice, newly formed MKs were negative or stained weakly for EdU on day 12, suggesting that they arise partially from an existing pool of progenitors. Finally, we analyzed platelet formation in vivo by imaging of the cranial bone marrow of GPIIb-eYFP-heterozygous mice. The depletion antibody was labeled with Atto-590-fluorophore and injected hours before imaging. Vasculature was counterstained by Quantum dots. We found that MKs residing at the bone marrow were decorated with the antibody and released pre- and proplatelets into the vasculature, indicating that platelet biogenesis can occur in the presence of anti-platelet antibodies on MKs. Our data thus provide novel insight into the pathomechanism of platelet production in patients with ITP. Disclosures: No relevant conflicts of interest to declare.

Description: A large proportion of patients with mutations in the Wiskott Aldrich syndrome (WAS) protein gene exhibit the milder phenotype termed X-linked thrombocytopenia (XLT). Intravenous immunoglobulin and antibiotic prophylaxis until stem cell transplantation (SCT) at an early age is the treatment of choice for classical WAS patients. For patients with XLT the optimal treatment is much less clear. Most of them reach adulthood without significant problems. However, severe complications such as infections, bleeding, autoimmune disorders and malignancies have been observed in these patients, albeit at a lower rate than in classical WAS patients. In order to provide a basis for treatment decisons in XLT patients we intended to define the natural course of disease in XLT patients and assess the probability of severe disease related complications. A retrospective survey at centers treating primary immunodeficiency patients was carried out and data were collected for patients with a documented WASP gene mutation and a WAS disease severity score of 2 or less, implicating thrombocytopenia and mild eczema and minor infections only. Data from a total of 182 patients from 12 countries could be analyzed. Median age at last follow-up was 11.2 years (range 0.4–74.6). Overall probability of survival censored for SCT was favorable in this cohort with 95%, 86% and 78% at 20, 40 and 60 years of age respectively. We did however observe a high rate of severe disease related events such as potentially life threatening infection or bleeding, autoimmune disease or malignancy. Therefore the probability to be alive without a serious event was only 66%, 45% and 29% at 20, 40 and 60 years respectively. Out of all 182 patients 13 (7%) developed severe infectious events, 3 of which were fatal. Severe bleeding episodes occurred in 23/182 (13%), 4 of which were fatal. Autoimmune disease developed in 22/182 (12%) and 9/182 (5%) were diagnosed with malignancy, 6 of whom have died. Overall and event free survival probabilities were not significantly influenced by the type of mutation or the presence of WASProtein. Patients receiving any antibiotic prophylaxis or IVIG had no survival benefit compared to others. Splenectomy, which was performed in 39/182 patients (21%), posed a significant risk to experience a severe infectious event (p

Description: A large proportion of patients with mutations in the Wiskott-Aldrich syndrome (WAS) protein gene exhibit the milder phenotype termed X-linked thrombocytopenia (XLT). Whereas stem cell transplantation at an early age is the treatment of choice for patients with WAS, therapeutic options for patients with XLT are controversial. In a retrospective multicenter study we defined the clinical phenotype of XLT and determined the probability of severe disease-related complications in patients older than 2 years with documented WAS gene mutations and mild-to-moderate eczema or mild, infrequent infections. Enrolled were 173 patients (median age, 11.5 years) from 12 countries spanning 2830 patient-years. Serious bleeding episodes occurred in 13.9%, life-threatening infections in 6.9%, autoimmunity in 12.1%, and malignancy in 5.2% of patients. Overall and event-free survival probabilities were not significantly influenced by the type of mutation or intravenous immunoglobulin or antibiotic prophylaxis. Splenectomy resulted in increased risk of severe infections. This analysis of the clinical outcome and molecular basis of patients with XLT shows excellent long-term survival but also a high probability of severe disease-related complications. These observations will allow better decision making when considering treatment options for individual patients with XLT.

Description: CAMT is a rare disease characterized by thrombocytopenia in infancy due to ineffective megakaryopoiesis. We retrospectively analyzed clinical parameters of 21 patients diagnosed with CAMT, characterized by severe thrombocytopenia at birth, normal bone marrow cellularity and severely reduced numbers of megakaryocytes. 18 children developed postnatal bleeding symptoms with a median platelet count of 21/nl. 2 children suffered from a postnatal cerebral bleeding and intrauterine cerebral bleeding was suspected in 4 more children. We observed differences in the course of disease: 12 children formed a homogeneous group regarding the hematological parameters. Their platelet counts remained on a very low level, bone marrow cellularity decreased during the first year of life and they developed severe aplastic anemia in early infancy (2 to 53 months). 7 children also presented with physical anomalies like strabismus (2), nystagmus (2), motor and mental retardation (2), growth retardation (2) and cardiac defects (2). In 5 of 7 patients the parents were cousins of first degree. Sequence analysis of the c-mpl gene in 5 children revealed nonsense mutations with a complete loss of the thrombopoietin receptor. This group we classified as CAMT Type I. In contrast, 6 children formed a more heterogeneous group with delayed bone marrow failure. Their platelet counts at birth were slightly increased compared to those of type I patients (median 35/nl). In all children the number of platelets rose during early infancy and achieved a median maximum of 132/nl. At a median age of 4 9/12 years (range 3 to 6 10/12years) 4 children developed aplastic anemia. In one girl bone marrow morphology revealed refractory anemia with excess blasts at the age of 7 1/12 years. She received two bone marrow transplantations (BMT) and finally died from acute myeloid leukemia. Another girl feels well at the age of 14 years without signs of pancytopenia. One girl presented with growth retardation and a second with a small apical ventricular septal defect. Sequence analysis in 3 children revealed different forms of amino acid exchanges in the extracellular domain of c-Mpl. This might correspond to a residual function of c-Mpl. This group we classified as CAMT Type II. Altogether 18 children received BMT. 3 patients with type II CAMT required a second BMT due to primary graft failure, secondary graft failure and relapse of MDS. BMT with a matched unrelated donor (MUD) was performed in 5 patients, all with a fatal outcome. 8 children died at a mean age of 4 2/12 years: 2 due to bleeding complications and 6 following BMT. We conclude, that c-Mpl deficiency is the main reason for CAMT and can be associated with described physical anomalies. As exemplified the prognosis for patients is poor. Clinical differences can be seen between a total lack and a residual function of the c-Mpl receptor. Besides CAMT due to c-Mpl deficiency the incidence of congenital forms of ineffective megakaryopoiesis was described for other diseases with no defects in the c-mpl gene. (e.g. CAMT with radio-ulnar synostosis, Hoyeraal-Hreidarsson-Syndrome). This heterogeneous group of diseases with normal c-Mpl function we classified as CAMT Type III. Further clinical studies have to be performed to understand the relationship between genotype and clinical phenotype in terms of bone marrow failure, leukemia development and overall survival to better predict the clinical outcome.

Description: Background HD is a B-lineage lymphoma characterized, depending on subtype, by a prominent inflammatory infiltrate and fibrosis. Clinically, inflammatory symptoms like fever, weight loss and night sweats (B-symptoms) and increased blood biomarkers of inflammation, including ESR and CRP, are characteristic of more advanced disease. The clinical trial EURONET-PHL-C2 (Second International Inter-Group Study for Classical Hodgkin's Lymphoma in Children and Adolescents) is a randomized, prospective trial that compares chemo- and radiotherapy treatment concepts of different intensities in patients with intermediate and advanced HD. Patients are stratified by risk into 3 therapy groups (TL-1 to TL-3). An ESR〉 30mm/h had been a risk factor for relapse in previous studies and leads to upstaging from the lowest (TL-1, Ann Arbor stage I and IIa without additional risk factors) to the intermediate risk group TL-2 in the current study. This addon pilot study tested urine proteomic patterns from pediatric patients with HD at diagnosis and compared them to the patterns of normal children. The questions were: Is there a HD-specific pattern, a pattern that identifies high risk or a pattern that correlates with inflammatory markers? Patients and methods Capillary electrophoresis coupled to mass spectrometry (CE-MS) was used to compare the peptide profiles in the mass range of 0.8 to 20 kDa of urine samples (N=34) from 16 children with pediatric Hodgkin lymphoma (PHL) as case and 32 age-matched children with no evidence of a disease (N=28) or with urinary tract infection (N=4) as control groups. Marker selection was based on a two-step strategy. First, a group-wise comparison of rank sum differences was performed on a set of 2418 annotated peptides with distribution frequencies above 30% in at least one of the groups with subsequent adjustment for multiple testing by the method of Bonferroni. In the second step marker candidates were further restricted to those demonstrating a significant positive or negative Spearman rho correlation coefficient (≥0.34 or ≤-0.34) to the Ann-Arbor classification criteria. From the resulting peptides a multivariate peptide marker classifier was established by support vector machine modeling and applied to an independent confirmation set of PHL (N=16, 31 urine samples) and control (N=18, 18 urine samples) patients to determine classification accuracy in receiver operating characteristics (ROC) analysis. Peptides included in the PHL classifier were resolved in their amino acid sequence by tandem mass spectrometry to identify the proteins from which the peptide markers are derived. Results The established multivariate peptide marker model consisting of 40 naturally occurring urinary peptides enabled absolute differentiation between PHL patients and children without signs of disease or urinary tract infection in independent validation as revealed by an area under the ROC curve value of 1.0 (95% confidence interval: 0.93 to 1.00, p

Description: Abstract 1561 Thrombocytopenia-absent radii syndrome (TARS) is a rare congenital disorder defined by low platelet count and bilaterial aplasia of radii. Additionally, patients have perinatal eosinophilia and leukocytosis and are often anemic during the first years of life. At this time, a moderate, but enigmatic increase in platelet counts has been described, but patients remain thrombocytopenic and eventually continue to suffer from severe episodes of bleeding. Megakaryocytes, the immediate precursor cells of platelets, are scarce in the bone marrow and precursor cells fail to produce megakaryocytic colonies in response to thrombopoietin (TPO). Recently, we demonstrated that all TARS patients harbor a microdeletion on chromosome 1q21 which spans 120–200 kb, comprising 12–18 annotated genes. The deletion is also present in some unaffected parents (carriers) indicating that it is essential but not sufficient for generating the TARS phenotype. We analyzed 158 platelet counts of 33 patients over time and found that platelets increase within 2 years of life in most of our patients, but even in adult patients counts do not reach the lower norm. Thus, we performed an extended analysis of TPO signal transduction in platelets from 20 TARS patients. Overall, Jak2 kinase - despite being expressed in comparable amount - does not become phosphorylated in response to TPO when patients were below age 20, confirming our previous results also performed on young patients. Intriguingly, in platelets isolated from patients over age 20, Jak2 did become phosphorylated. As TPO activates several distinct pathways, we looked for the consequences of this bipartite TPO-responsiveness, including the activation of the alternate januskinase Tyk2, the STAT, the MAPK/ERK, and the Akt pathways. As expected, when Jak2 was not phosphorylated, Tyk2 and all downstream pathways were inactive. In contrast, in the presence of phosphorylated Jak2 (pJak2), all downstream pathways were activated, emphasizing the key role of Jak2 for TPO responsiveness. Platelets from either 20 healthy children or 11 carriers showed normal TPO signaling, excluding that the effect was due to a general age-dependence or a mere consequence of the microdeletion. Densitometric analyses confirmed our overall visual results. Expression levels of the TPO-receptor c-Mpl was not altered in 2 young and 2 adult patients compared to carriers, healthy children and adult controls, arguing against a compensatory upregulation in older patients. Furthermore, we sequenced all coding regions of Jak2 mRNA derived from patient-derived lymphoblastic cell lines (LCL) of one young and one adult patient and could not find any mutations. As bone marrow biopsies are typically not performed, changes in bone marrow cellularity or composition are not directly accessible. Recently, the immature platelet fraction (IPF) has been considered a surrogate marker for megakaryopoiesis. Interestingly, while there was no correlation between platelet count and IPF in 16 patients with TARS, we found a negative correlation between IPF with age. In 9 pediatric patients IPF was elevated (4.6%) compared to the median of 100 pediatric controls (2.7%), while in 7 adult TARS patients the mean IPF was 2.4%. These data provide circumstantial evidence that changes in megakaryopoiesis might drive the change in platelet biogenesis and TPO signaling. Plasma levels of stromal derived factor 1, a chemokine that contributes to restore platelet production in the absence of functional TPO signaling, were within the normal range in 6 patients with TARS. Real-time analysis of mRNA expression in LCL of genes within the microdeleted region indicates comparable expression in 2 unaffected parents with 2 controls, while 3 patients and 2 carriers showed the expected reduced expression. This includes the expression of PIAS3, a negative regulator of the Jak-STAT pathway. PIAS3 protein level, however, was normal in platelet lysates of TARS patients, making a key function for thrombocytopenia in TARS unlikely. Taken together, our data show an unexpected age-dependent change in TPO-signaling in platelets of TARS patients. As this change occurs much later than the amelioration of platelet counts, we suggest that an unknown factor influences platelet biogenesis during childhood. Disclosures: No relevant conflicts of interest to declare.