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Background The prognosis of children with acute lymphoblastic leukemia (ALL) has improved considerably over the past decades. However, to achieve cure in patients with refractory disease or relapse new treatment options are mandatory. Methods In the multicenter-trial CoALL-08-09, an additional treatment element consisting of the rarely used chemotherapeutic agent amsacrine combined with etoposide and methylprednisolone (AEP) (amsacrine 2 x 100 mg/m2, etoposide 2 x 500 mg/m2 and methylprednisolone 4 x 1000 mg/m2) was implemented into the first-line treatment of pediatric patients with a poor treatment response at the end of induction (EOI) measured by minimal residual disease (MRD). These patients were stratified into a high-risk intensified arm (HR-I) including an AEP element at the end of consolidation. Patients with induction failure (IF), i.e. lack of cytomorphological remission EOI, were eligible for hematopoietic stem cell transplantation (HSCT) after remission had been reached later on. These patients received AEP as a part of their MRD-guided bridging-to-transplant treatment. Results A significant improvement in probality of overall survival (pOS) for the CoALL-08-09 HR-I patients was noted compared to MRD-matched patients from the preceding CoALL-07-03 trial in the absence of severe or persistent treatment-related toxicities. Relapse rate and probability of event-free survival (pEFS) did not differ significantly between trials. In patients with IF a stable or improved MRD response after AEP was observed without severe or persistent treatment-related toxicities. Conclusion In conclusion, AEP is well-tolerated as a component of the HR treatment and useful in bridging-to-transplant settings.

Novel treatment strategies are needed to improve cure for all children with acute lymphoblastic leukemia (ALL). To this end, we investigated the therapeutic potential of clofarabine in primary ALL in trial CoALL 08-09 (clinicaltrials gov. identifier: NCT01228331). The primary study objective was the minimal residual disease (MRD)- based comparative assessment of cytotoxic efficacies of clofarabine 5x40 mg/m2 versus high-dose cytarabine (HIDAC) 4x3g/m2, both in combination with PEG-ASP 2,500 IU/m2 as randomized intervention in early consolidation. The secondary objective was an outcome analysis focused on treatment arm dependence and MRD after randomized intervention. In B-cell precursor (BCP)-ALL, eradication of MRD was more profound after clofarabine compared to cytarabine, with 93 versus 79 of 143 randomized patients per arm reaching MRD-negativity (c2 test P=0.03, leftsided P [Fisher’s exact test]=0.04). MRD status of BCP-ALL after randomized intervention maintained its prognostic relevance, with a significant impact on event-free survival (EFS) and relapse rate. However, no difference in outcome regarding EFS and overall survival (OS) between randomized courses was observed (5-year EFS: clofarabine 85.7, SE=4.1 vs. HIDAC 84.8, SE=4.7 [P=0.96]; OS: 95.7, SE=1.9 vs. 92.2, SE=3.2 [P=0.59]), independent of covariates or overall risk strata. Severe toxicities between randomized and subsequent treatment elements were also without significant difference. In conclusion, clofarabine/PEG-ASP is effective and safe, but greater cytotoxic efficacy of clofarabine compared to HIDAC did not translate into improved outcomes indicating a lack of surrogacy of post-intervention MRD at the trial level as opposed to the patient level, which hampers a broader implementation of this regimen in the frontline treatment of ALL.

Diffuse IDH-mutant astrocytoma mostly occurs in adults and carries a favorable prognosis compared to IDH-wildtype malignant gliomas. Acquired mismatch repair deficiency is known to occur in recurrent IDH-mutant gliomas as resistance mechanism towards alkylating chemotherapy. In this multi-institutional study, we report a novel epigenetic group of 32 IDH-mutant gliomas with proven or suspected hereditary mismatch repair deficiency. None of the tumors exhibited a combined 1p/19q deletion. These primary mismatch repair-deficient IDH-mutant astrocytomas (PMMRDIA) were histologically high-grade and were mainly found in children, adolescents and young adults (median age 14 years). Mismatch repair deficiency syndromes (Lynch or Constitutional Mismatch Repair Deficiency Syndrom (CMMRD)) were clinically diagnosed and/or germline mutations in DNA mismatch repair genes (MLH1, MSH6, MSH2) were found in all cases, except one case with a family and personal history of colon cancer and another case with MSH6-deficiency available only as recurrent tumor. Loss of at least one of the mismatch repair proteins was detected via immunohistochemistry in all, but one case analyzed. Tumors displayed a hypermutant genotype and microsatellite instability was present in more than half of the sequenced cases. Integrated somatic mutational and chromosomal copy number analyses showed frequent inactivation of TP53, RB1 and activation of RTK/PI3K/AKT pathways. In contrast to the majority of IDH-mutant gliomas, more than 60% of the samples in our cohort presented with an unmethylated MGMT promoter. While the rate of immuno-histochemical ATRX loss was reduced, variants of unknown significance were more frequently detected possibly indicating a higher frequency of ATRX inactivation by protein malfunction. Compared to reference cohorts of other IDH-mutant gliomas, primary mismatch repair-deficient IDH-mutant astrocytomas have by far the worst clinical outcome with a median survival of only 15 months irrespective of histological or molecular features. The findings reveal a so far unknown entity of IDH-mutant astrocytoma with high prognostic relevance. Diagnosis can be established by aligning with the characteristic DNA methylation profile, by DNA-sequencing-based proof of mismatch repair deficiency or immunohistochemically demonstrating loss-of-mismatch repair proteins.

Reports on pediatric low-grade diffuse glioma WHO-grade II (DG2) suggest an impaired survival rate, but lack conclusive results for genetically defined DG2-entities. We analyzed the natural history, treatment and prognosis of DG2 and investigated which genetically defined sub-entities proved unfavorable for survival. Within the prospectively registered, population-based German/Swiss SIOP-LGG 2004 cohort 100 patients (age 0.8-17.8 years, 4% neurofibromatosis [NF1]) were diagnosed with a DG2. Following biopsy (41%) or variable extent of resection (59%), 65 patients received no adjuvant treatment. Radiologic progression or severe neurologic symptoms prompted chemotherapy (n = 18) or radiotherapy (n = 17). Multiple lines of salvage treatment were necessary for 19/35 patients. Five years event-free survival dropped to 0.44, while 5 years overall survival was 0.90 (median observation time 8.3 years). Extensive genetic profiling of 65/100 DG2 identified Histone3-K27M-mutation in 4, IDH1-mutation in 11, BRAF-V600-mutation in 12, KIAA1549-BRAF-fusions in 6 patients, while the remaining 32 tumor tissues did not show alterations of these genes. Progression to malignant glioma occurred in 12 cases of all genetically defined subgroups within a range of 0.5 to 10.8 years, except for tumors carrying KIAA1549-BRAF-fusions. Histone3-K27M-mutant tumors proved uniformly fatal within 0.6 to 2.4 years. The current LGG treatment strategy seems appropriate for all DG2-entities, with the exemption of Histone3-K27M-mutant tumors that require a HGG-related treatment strategy. Our data confirm the importance to genetically define pediatric low-grade diffuse gliomas for proper treatment decisions and risk assessment.

We used hybrid capture-targeted next-generation sequencing of circulating cell-free DNA (ccfDNA) of pediatric Hodgkin lymphoma (PHL) patients to determine pathogenic mechanisms and assess the clinical utility of this method. Hodgkin-Reed/Sternberg (HRS) cell-derived single nucleotide variants, insertions/deletions, translocations and VH-DH-JH rearrangements were detected in pretherapy ccfDNA of 72 of 96 patients. Number of variants per patient ranged from 1 to 21 with allele frequencies from 0.6 to 42%. Nine translocation breakpoints were detected. Genes involved in JAK/STAT, NFkB and PI3K signaling and antigen presentation were most frequently affected. SOCS1 variants, mainly deletions, were found in most circulating tumor (ct) DNAs, and seven of the nine translocation breakpoints involved SOCS1. Analysis of VH-DH-JH rearrangements revealed an origin of PHL HRS cells from partially selected germinal center B cells. Amounts of pretherapy ctDNA were correlated with metabolic tumor volumes. Furthermore, in all ccfDNA samples of 43 patients with early response assessment quantitative qPET 3, indicative of a favorable clinical course, ctDNA was not detectable. In contrast, in five of six patients with qPET 3, indicative of an unfavorable clinical course, ctDNA remained detectable. ccfDNA analysis of PHL is thus a suitable approach to determine pathogenic mechanisms and monitor therapy response.

We conducted a clinical trial and report the long-term outcome of 773 children with acute lymphoblastic leukemia upon risk-adapted therapy accrued in trial CoALL 07-03 (from the Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia). In a 2-step stratification, patients were allocated to receive either low- or high-risk treatment, based on initial white blood cell count, age, and immunophenotype. A second stratification was performed according to the results of in vitro pharmacosensitivity toward prednisolone, vincristine, and asparaginase (PVA score) and in vivo response after induction therapy (minimal residual disease [MRD]). Therapy was reduced for both risk groups in patients with a low PVA score or negative MRD result, and intensified in patients with a high PVA score. Overall outcome improved significantly compared with the predecessor CoALL 06-97 trial, with identical therapy backbone despite treatment reduction in 15.8% of patients (10-year probability of event-free survival, 83.5% vs 73.9%; overall survival, 90.7% vs 83.8%). Outcome for patients in the reduced treatment arms was superior to that of patients in the standard arms, associated with a profound reduction in frequency and severity of infectious complications. Importantly, we observed a lack of correlation between in vitro and in vivo drug response, as well as a lower predictive value of in vitro drug testing, reflecting an intrinsic limitation of this methodology that prevents its use for treatment stratification in future trials. In conclusion, it might be possible to reduce chemotherapy in children with acute lymphoblastic leukemia selected by stringent in vivo measurement of MRD without jeopardizing overall outcome.

Background Since the FDA approval of clofarabine for the treatment of relapsed or refractory acute lymphoblastic leukemia (ALL) at childhood, several studies have been launched which put clofarabine under scrutiny in combination with other cytostatic drugs as second or third line therapy. As a novel treatment-strategy we introduced the combination of clofarabine with pegylated asparaginase (PEG-ASP) in a randomized fashion in comparison to the standard consolidation course with high dose cytarabine (Hidac) combined with PEG-ASP into the frontline management of ALL within the CoALL 08-09 protocol. The primary objective of the study was to compare the MRD based assessment of the cytotoxic efficacy of the randomized courses in pediatric ALL. Patients and methods CoALL 08-09 was an open, interventional, multi-center, prospective, and randomized clinical trial for patients with newly diagnosed ALL. In March 2010 the trial had been opened for enrollment of patients under 18 years with confirmed diagnosis of acute B- or T-cell precursor leukemia. After a stratified phase I/II, in which patients with high-risk features and high post-induction MRD received the combination of clofarabine and PEG-ASP, the randomized trial started in November 2013. All patients with a measurable MRD at the end of induction (EOI), as measured by RQ-PCR according to EURO-MRD guidelines, were eligible for randomization to receive either the combination of clofarabine 5 x 40 mg/m2 or high dose cytarabine (HIDAC) 4 x 3g/m2 bothin combination with PEG-ASP 2.500 IU/m2 at the beginning of the consolidation phase. Until December 2019 303 study patients were randomized allocating 151 patients towards the clofarabine and 152 patients to the HIDAC arm. Patient characteristics and MRD burden prior to this treatment element were highly comparable. Results The reduction of minimal residual disease was significantly more profound after clofarabine compared to cytarabine (p(Cochran-Armitage)=.01 for BCP-ALL with 93 vs 79 patients reaching MRD negativity (Table 1). MRD status of BCP-ALL after the first randomized consolidation block (day 50) was of prognostic relevance with a statistically significant impact on EFS and relapse rate (Fig. 2a). However, no difference in outcome regarding the event-free and overall survival between the randomized arms was observed (5-year EFS: Clofarabine 85.7, SE=4.1 vs Hidac 84.8, SE=4.7 (p=.96) and OS: 95.7, SE=1.9 vs 92.2, SE=3.2(p=.59) Fig. 2b). This finding was recapitulated independent of gender, age, WBC count at diagnosis, ETV6-RUNX 1 translocation, immunophenotype or overall risk strata). Results for T-ALL patients are in the same range but because of small numbers not statistically significant. No differences in the incidence of severe or persistent toxicity btween randomized treatment elements as well as in the subsequent treatment realization were registered. Conclusion Clofarabine combined with PEG-asparaginase is highly effective and well tolerated in the frontline treatment of ALL. Although the prognostic impact of MRD is still discernible in early consolidation, the greater cytotoxic efficacy of clofarabine reflected by a more frequent eradication of MRD, did not translate into an apparent improvement of outcome likely due to a lack of power in the comparative analysis of randomized patients. Disclosures No relevant conflicts of interest to declare.

Background Until today, adult and pediatric clinical trials investigating single-agent or combinatorial HDAC inhibitors including vorinostat in solid tumors have largely failed to demonstrate efficacy. These results may in part be explained by data from preclinical models showing significant activity only at higher concentrations compared to those achieved with current dosing regimens. In the current pediatric trial, we applied an intra-patient dose escalation design. The purpose of this trial was to determine a safe dose recommendation (SDR) of single-agent vorinostat for intra-patient dose escalation, pharmacokinetic analyses (PK), and activity evaluation in children (3–18 years) with relapsed or therapy-refractory malignancies. Results A phase I intra-patient dose (de)escalation was performed until individual maximum tolerated dose (MTD). The starting dose was 180 mg/m2/day with weekly dose escalations of 50 mg/m2 until DLT/maximum dose. After MTD determination, patients seamlessly continued in phase II with disease assessments every 3 months. PK and plasma cytokine profiles were determined. Fifty of 52 patients received treatment. n = 27/50 (54%) completed the intra-patient (de)escalation and entered phase II. An SDR of 130 mg/m2/day was determined (maximum, 580 mg/m2/day). n = 46/50 (92%) patients experienced treatment-related AEs which were mostly reversible and included thrombocytopenia, fatigue, nausea, diarrhea, anemia, and vomiting. n = 6/50 (12%) had treatment-related SAEs. No treatment-related deaths occurred. Higher dose levels resulted in higher Cmax. Five patients achieved prolonged disease control (> 12 months) and showed a higher Cmax (> 270 ng/mL) and MTDs. Best overall response (combining PR and SD, no CR observed) rate in phase II was 6/27 (22%) with a median PFS and OS of 5.3 and 22.4 months. Low levels of baseline cytokine expression were significantly correlated with favorable outcome. Conclusion An SDR of 130 mg/m2/day for individual dose escalation was determined. Higher drug exposure was associated with responses and long-term disease stabilization with manageable toxicity. Patients with low expression of plasma cytokine levels at baseline were able to tolerate higher doses of vorinostat and benefited from treatment. Baseline cytokine profile is a promising potential predictive biomarker. Trial registration ClinicalTrials.gov, NCT01422499. Registered 24 August 2011

Anthracyclines are integral components of antileukemic treatment. Apart from cardiotoxicity, myelosuppression and infectious complications have been described for doxorubicin (DOX) and daunorubicin (DNR) as predominant side effects, but little is known about their differential toxicities. To address the question whether DNR is associated with a lower rate of infectious complications compared with DOX, 307 children with newly diagnosed acute lymphoblastic leukemia, enrolled in trial CoALL 08-09, were randomized to receive either DOX 30 mg/m

Pediatric tumors of the central nervous system composed of oligoid tumor cells showing diffuse leptomeningeal spread without a primary mass lesion seem to represent a novel tumor entity. The terms “diffuse leptomeningeal glioneural tumor” or—preferably—“disseminated oligodendroglial-like leptomeningeal tumor of childhood” (DOGLT) were proposed. Four patients were identified with clinico-neuropathologic findings compatible with DOGLT and a mean follow-up time of 54 months was determined. Seven different biopsies obtained from the four patients were histologically evaluated. Clinical course, diagnostic measures, histopathologic and radiologic features and treatment suggestions were recorded, on the basis of which diagnostic and therapeutic algorithm was proposed. Patients with DOGLT presented with hydrocephalus as first symptom, requiring neurosurgical therapy. Open arachnoid biopsy was necessary to confirm diagnosis. The oligoid cells in a desmoplastic or focally myxoid matrix showed OLIG2-, MAP2-, S-100 and rare HuC/HuD protein-immunopositivity. IDH1 (R132H)- and CD99-immunohistochemistry was negative in all patients. None of the evaluable biopsies of three patients showed chromosome 1p/19q deletion, neither as isolated nor combined allelic loss. Chemotherapy according to the SIOP-LGG 2004 standard induction and consolidation protocol resulted in complete response and partial response, respectively, in 50 % of the patients. However, after discontinuation of chemotherapy, two patients experienced tumor progression and one of them succumbed to the disease after 19 months. Radiological criteria as well as preliminary treatment results are presented after observation of four clinical cases. Prognosis and long-term clinical courses remain to be observed.

Accumulation of CD3(+) T-cell receptor (TCR)αβ(+)CD4(-)CD8(-) double-negative T cells (DNT) is a hallmark of autoimmune lymphoproliferative syndrome (ALPS). DNT origin and differentiation pathways remain controversial. Here we show that human ALPS DNT have features of terminally differentiated effector memory T cells reexpressing CD45RA(+) (TEMRA), but are CD27(+)CD28(+)KLRG1(-) and do not express the transcription factor T-bet. This unique phenotype was also detected among CD4(+) or CD8(+) ALPS TEMRA cells. T-cell receptor β deep sequencing revealed a significant fraction of shared CDR3 sequences between ALPS DNT and both CD4(+) and CD8(+)TEMRA cells. Moreover, in ALPS patients with a germ line FAS mutation and somatic loss of heterozygosity, in whom biallelic mutant cells can be tracked by absent Fas expression, Fas-negative T cells accumulated not only among DNT, but also among CD4(+) and CD8(+)TEMRA cells. These data indicate that in human Fas deficiency DNT cannot only derive from CD8(+), but also from CD4(+) T cells. Furthermore, defective Fas signaling leads to aberrant transcriptional programs and differentiation of subsets of CD4(+) and CD8(+) T cells. Accumulation of these cells before their double-negative state appears to be an important early event in the pathogenesis of lymphoproliferation in ALPS patients.

The original publication contains the following errors: In the title, the words ‘oligodendroglial cell-like’ should read ‘oligodendroglial-like’. (The correct title is shown in this erratum.) The images in Figs. 3 and 4 were incorrect. The correct figures are shown below. All the figure citations except the citation of Fig. 1 were incorrect. All citations of Fig. 2 actually concern Fig. 5, all citations of Fig. 3 actually concern Fig. 6, all citations of Fig. 4 actually concern Fig. 2, all citations of Fig. 5 actually concern Fig. 3 and all citations of Fig. 6 actually concern Fig. 4.

Familial hemophagocytic lymphohistiocytosis (FHL) is a genetically determined hyperinflammatory syndrome caused by uncontrolled immune response mediated by T-lymphocytes, natural killer (NK) cells, and macrophages. STXBP2 mutations have recently been associated with FHL5. To better characterize the genetic and clinical spectrum of FHL5, we analyzed a cohort of 185 patients with suspected FHL for mutations in STXBP2. We detected biallelic mutations in 37 patients from 28 families of various ethnic origins. Missense mutations and mutations affecting 1 of the exon 15 splice sites were the predominant changes detectable in this cohort. Patients with exon 15 splice-site mutations (n = 13) developed clinical manifestations significantly later than patients with other mutations (median age, 4.1 year vs 2 months) and showed less severe impairment of degranulation and cytotoxic function of NK cells and CTLs. Patients with FHL5 showed several atypical features, including sensorineural hearing deficit, abnormal bleeding, and, most frequently, severe diarrhea that was only present in early-onset disease. In conclusion, we report the largest cohort of patients with FHL5 so far, describe an extended disease spectrum, and demonstrate for the first time a clear genotype-phenotype correlation.

Glioblastoma multiforme (GBM) is a lethal brain tumour in adults and children. However, DNA copy number and gene expression signatures indicate differences between adult and paediatric cases. To explore the genetic events underlying this distinction, we sequenced the exomes of 48 paediatric GBM samples. Somatic mutations in the H3.3-ATRX-DAXX chromatin remodelling pathway were identified in 44% of tumours (21/48). Recurrent mutations in H3F3A, which encodes the replication-independent histone 3 variant H3.3, were observed in 31% of tumours, and led to amino acid substitutions at two critical positions within the histone tail (K27M, G34R/G34V) involved in key regulatory post-translational modifications. Mutations in ATRX (α-thalassaemia/mental retardation syndrome X-linked) and DAXX (death-domain associated protein), encoding two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, were identified in 31% of samples overall, and in 100% of tumours harbouring a G34R or G34V H3.3 mutation. Somatic TP53 mutations were identified in 54% of all cases, and in 86% of samples with H3F3A and/or ATRX mutations. Screening of a large cohort of gliomas of various grades and histologies (n = 784) showed H3F3A mutations to be specific to GBM and highly prevalent in children and young adults. Furthermore, the presence of H3F3A/ATRX-DAXX/TP53 mutations was strongly associated with alternative lengthening of telomeres and specific gene expression profiles. This is, to our knowledge, the first report to highlight recurrent mutations in a regulatory histone in humans, and our data suggest that defects of the chromatin architecture underlie paediatric and young adult GBM pathogenesis.

Activation of the MAPK signaling pathway has been shown to be a unifying molecular feature in pilocytic astrocytoma (PA). Genetically, tandem duplications at chromosome 7q34 resulting in KIAA1549-BRAF fusion genes constitute the most common mechanism identified to date. To elucidate alternative mechanisms of aberrant MAPK activation in PA, we screened 125 primary tumors for RAF fusion genes and mutations in KRAS, NRAS, HRAS, PTPN11, BRAF and RAF1. Using microarray-based comparative genomic hybridization (aCGH), we identified in three cases an interstitial deletion of ~2.5 Mb as a novel recurrent mechanism forming BRAF gene fusions with FAM131B, a currently uncharacterized gene on chromosome 7q34. This deletion removes the BRAF N-terminal inhibitory domains, giving a constitutively active BRAF kinase. Functional characterization of the novel FAM131B-BRAF fusion demonstrated constitutive MEK phosphorylation potential and transforming activity in vitro. In addition, our study confirmed previously reported BRAF and RAF1 fusion variants in 72% (90/125) of PA. Mutations in BRAF (8/125), KRAS (2/125) and NF1 (4/125) and the rare RAF1 gene fusions (2/125) were mutually exclusive with BRAF rearrangements, with the exception of two cases in our series that concomitantly harbored more than one hit in the MAPK pathway. In summary, our findings further underline the fundamental role of RAF kinase fusion products as a tumor-specific marker and an ideally suited drug target for PA.

Die hamolytischen Anamien werden in korpuskular-bedingte (Veranderungen der Blutkorperchen selbst) und extrakorpuskularbedingte Formen (von den Blutkorperchen unabhangige Ursachen) eingeteilt. Die Diagnostik und Therapie dieser Erkrankungen von Erythrozytenmembrandefekten bis zu immunhamolytischen Anamien sind in diesem Beitrag zusammengefasst.

An orbital embryonal rhabdomyosarcoma (RMS) is a rare malignancy in children, but clinical findings are typical. We detail the case of an 8-year-old female with orbital RMS and discuss the therapeutic options. Orbital RMS was apparent with painless exophthalmos of the right globe and diplopia. Head MRI showed tumor masses behind and inferior to the globe. Open biopsy led to the histological diagnosis. Metastasis or infiltration of orbital bone was not observed. Chemotherapy was carried out in accordance with the Cooperative Weichteilsarkom Studie (CWS) 2002 protocol. Tumor regression was detected after the first course of chemotherapy; we decided to excise the residual tumor with preservation of the globe. The CWS 2002 protocol was subsequently completed. Radiotherapy was not done. One year after treatment, RMS recurrence was not observed. After interdisciplinary treatment, mutilation was avoided after exenteration of the orbit or radiation treatment to the growing facial skeleton. That was possible due to excision of the residual tumor in a second step, leading to down-staging of the RMS.

Paroxysmal dyskinesias are episodic movement disorders that can be inherited or are sporadic in nature. The pathophysiology underlying these disorders remains largely unknown but may involve disrupted ion homeostasis due to defects in cell-surface channels or nutrient transporters. In this study, we describe a family with paroxysmal exertion-induced dyskinesia (PED) over 3 generations. Their PED was accompanied by epilepsy, mild developmental delay, reduced CSF glucose levels, hemolytic anemia with echinocytosis, and altered erythrocyte ion concentrations. Using a candidate gene approach, we identified a causative deletion of 4 highly conserved amino acids (Q282_S285del) in the pore region of the glucose transporter 1 (GLUT1). Functional studies in Xenopus oocytes and human erythrocytes revealed that this mutation decreased glucose transport and caused a cation leak that alters intracellular concentrations of sodium, potassium, and calcium. We screened 4 additional families, in which PED is combined with epilepsy, developmental delay, or migraine, but not with hemolysis or echinocytosis, and identified 2 additional GLUT1 mutations (A275T, G314S) that decreased glucose transport but did not affect cation permeability. Combining these data with brain imaging studies, we propose that the dyskinesias result from an exertion-induced energy deficit that may cause episodic dysfunction of the basal ganglia, and that the hemolysis with echinocytosis may result from alterations in intracellular electrolytes caused by a cation leak through mutant GLUT1.

HISTORY AND CLINICAL FINDINGS Three members of one family (mother, now aged 44 years; daughter, now aged 23 years, and son, now aged 19 years) have had chronic haemolytic anaemia since their infancy. All three have had several blood transfusions a year because of haemoglobin (Hb) concentrations as low as 5 g/dl due to intermittent increases in haemolysis. Despite an unclear diagnosis all three had splenectomies: their spleens had been enlarged to about 5-7 cm below the rib margin. After splenectomy their haemoglobin concentration rose to 9-12 g/dl and the previously raised reticulocyte count of 150-250/1000 rose further to 400-700/1000. None of the patients had symptoms other than a slight decrease in exercise tolerance. They were admitted to hospital for further evaluation. Physical examination was unremarkable except for slight jaundice and well healed splenectomy scar. TESTS All three patients had normochromic haemolytic anaemia (Hb 10-12 g/dl), bilirubin 2-3 mg/dl, lactate dehydrogenase activity 400-600 U/l). The reticulocyte count was raised to 420-690/1000. In all of them pyruvate kinase (PK) activity was normal or slightly increased if related to Hb concentration. Reticulocytes from healthy persons were isolated by density-gradient centrifugation so that samples could be prepared with different numbers of reticulocytes: PK activity was found to be dependent on the sample's reticulocyte count. In the three patients PK deficiency could, therefore, only be diagnosed by taking into account the raised reticulocyte count. CONCLUSION As reticulocytes have a higher PK activity than erythrocytes the reticulocyte count must be taken into account when assessing the significance of a given value of this enzyme's activity.

Mutations of the hematopoietic transcription factor GATA1 (GATA1s) are pathognomonic in newborn with transient leukemia and children with Down syndrome and myeloid leukemia (ML-DS). Both TL and ML-DS can also occur in children with trisomy 21 mosaic.Between 2002 and 2011, 15 newborns and infants were diagnosed with DS mosaic. 9 of them presented with TL and 8 children suffered from ML-DS; 2 of them with a history of TL. In children without stigmata the special morphology and immunophenotype of blasts triggered the screening for GATA1 mutation and trisomy 21 mosaic.All newborns with TL achieved complete remission (CR). Due to clinical symptoms caused by the leukemic blasts, in 3 children low-dose cytarabine was applied. 1 patient died due to cardiac defect. In all patients GATA 1 s was confirmed. 6 children with ML-DS were initially treated according the AML-BFM protocol. After ML-DS was confirmed, therapy was continued with the intensity reduced schedule according to the ML-DS 2006 protocol. All children are still in CR (follow-up 1.8-7 years, median 2.7 yrs). 2 children with unknown trisomy 21 mosaic were diagnosed as acute megakaryoblastic leukemia (AMKL) and treated according the high risk arm of the AML-BFM 2004 including allogeneic stem cell transplantation in one child). GATA1 mutation was identified retrospectively. Both children are alive in CR.GATA1s associated leukemia has to be excluded in all young children with AMKL (

Pediatric tumors of the central nervous system composed of oligoid tumor cells showing diffuse leptomeningeal spread without a primary mass lesion seem to represent a novel tumor entity. The terms "diffuse leptomeningeal glioneural tumor" or-preferably-"disseminated oligodendroglial-like leptomeningeal tumor of childhood" (DOGLT) were proposed. Four patients were identified with clinico-neuropathologic findings compatible with DOGLT and a mean follow-up time of 54 months was determined. Seven different biopsies obtained from the four patients were histologically evaluated. Clinical course, diagnostic measures, histopathologic and radiologic features and treatment suggestions were recorded, on the basis of which diagnostic and therapeutic algorithm was proposed. Patients with DOGLT presented with hydrocephalus as first symptom, requiring neurosurgical therapy. Open arachnoid biopsy was necessary to confirm diagnosis. The oligoid cells in a desmoplastic or focally myxoid matrix showed OLIG2-, MAP2-, S-100 and rare HuC/HuD protein-immunopositivity. IDH1 (R132H)- and CD99-immunohistochemistry was negative in all patients. None of the evaluable biopsies of three patients showed chromosome 1p/19q deletion, neither as isolated nor combined allelic loss. Chemotherapy according to the SIOP-LGG 2004 standard induction and consolidation protocol resulted in complete response and partial response, respectively, in 50 % of the patients. However, after discontinuation of chemotherapy, two patients experienced tumor progression and one of them succumbed to the disease after 19 months. Radiological criteria as well as preliminary treatment results are presented after observation of four clinical cases. Prognosis and long-term clinical courses remain to be observed.

The present study shows that stress signaling plays a role in differentiation of K562, PANC1, HT29 and HL60 tumor cells: (1) Butyrate induced differentiation in K562, PANC1, and HT29 cells can be inhibited by SB203580, a specific inhibitor of p38 stress activated protein kinase. (2) Heat shock and hyperosmolarity increase expression of differentiation markers in K562, HT29, HL60 and in K562, PANC1, and HT29 cells, respectively. (3) Conversely, environmental stress induced differentiation in K562, HT29, and PANC1 cells can be inhibited by SB203580 and quercetin, a compound with heat shock pathway inhibiting activity. (4) Butyrate and environmental stress enhance either additively or synergistically differentiation of K562, HT29, PANC1 or HL60 cells, respectively. Stress signaling pathways might be an interesting pharmacologic target for differentiation therapy of malignant disease.

Fragestellung und Hintergrund. Bei Patienten mit Thalassaemia major fuhrt die Hamosiderose haufig zu Endokrinopathien. Wir untersuchten die Entwicklung von Glukosetoleranzstorungen und den Einfluss therapeutischer Masnahmen wie intensivierter Chelattherapie und Diat. Methode und Patienten. 60 Patienten mit Thalassaemia major im Alter von 4–36 Jahren, die regelmasige subkutane Deferoxamininfusionen sowie Bluttransfusionen erhielten, wurden hinsichtlich moglicher Endokrinopathien untersucht. Zur Untersuchung der Insulinsekretion dienten orale und i.-v.-Glukosebelastungen sowie der i.-v.-Glukagontest. Bei Patienten mit gestorter Glukosetoleranz wurde der Einfluss einer intensivierten (i. v.) Deferoxamintherapie und diatetischer Masnahmen auf Blutglukose- und Seruminsulinkonzentrationen untersucht. Ergebnisse. Glukosetoleranzstorungen zahlen ab dem 2. Lebensjahrzehnt zu den 4 haufigsten Hormonerkrankungen bei Patienten mit Thalassaemia major. Storungen der Glukosetoleranz gehen in einem Fruhstadium mit einer Hyperinsulinamie einher, spater entwickelt sich ein Insulinmangeldiabetes durch Erschopfung der β-Zell-Aktivitat. In der Fruhphase der Erkrankung fuhrten bei einigen Patienten eine Intensivierung der Deferoxamintherapie oder alleinige diatetische Behandlung zu einer Verbesserung der Glukosetoleranz. Schlussfolgerungen. Storungen der Glukosetoleranz lassen sich im Fruhstadium durch Diat und Intensivierung der Chelattherapie bessern. Die Manifestation eines Diabetes mellitus kann damit moglicherweise aufgehalten werden. Im fortgeschrittenen Stadium der Glukosetoleranzstorung liegt ein Insulinmangel vor, weshalb eine Insulinsubstitution notwendig wird.

Primary myelosarcomas are rare manifestations of acute myeloid leukaemia (AML) that precede bone marrow involvement. Out of 744 children observed during the AML-Berlin-Frankfurt-Munster (BFM) studies 87 and 93, 34 children presented with extramedullar myelosarcomas and no blasts (n = 21; 2.8%), or a low blast count (n = 13; 1.7%) in the bone marrow. Owing to the initially mild and variable symptoms, in some children (n = 12) diagnostic procedures were delayed and treatment intensity was reduced. At 0.65 +/- 0.13, the cumulative incidence of relapse was significantly higher than in other AML patients (0.28 +/- 0.02). The 5-year event-free survival was 0.19 +/- 0.08 (compared with 0.48 +/- 0.02 in AML-BFM studies 87/93; P(log rank) < 0.03). Overall, 18 out of 34 patients died from disease (estimated 5 year survival 0.44 +/- 0.09 compared with 0.55 +/- 0.02 in the AML-BFM-studies 87/93; P(log rank) = 0.35, n.s.). An early diagnostic workup is needed in children with unusual skin lesions or tumours, considering myelosarcoma as a primary manifestation of AML. Intensive AML-specific chemotherapy is recommended soon after diagnosis.

Rhabdoid tumors of the central nervous system are rare malignancies with a still almost uniformly fatal outcome. There is still no proven curative therapy available. We report our experience with nine patients with central nervous system rhabdoid tumors. Gross complete surgical removal of the tumor was achieved in six patients. Seven patients received intensive chemotherapy. Four of these were treated in addition with both neuroaxis radiotherapy and a local boost directed to the tumor region, while two patients received local radiotherapy only. The therapy was reasonably well tolerated in most cases. Despite the aggressive therapy, eight of the nine patients died from progressive tumor disease, and one patient died from hemorrhagic brain stem lesions of unknown etiology. The mean survival time was 10 months after diagnosis. Conventional treatment, although aggressive, cannot change the fatal prognosis of central nervous system rhabdoid tumors. As these neoplasms are so rare, a coordinated register would probably be a good idea, offering a means of learning more about the tumor's biology and possible strategies of treatment.

Butyrate induces cytodifferentiation in many tumor cells of different origin, suggesting that an as yet unidentified common mechanism inherent to malignant cells is the target of butyrate action. This study determined the role of different mitogen-activated protein (MAP) kinase signal transduction pathways in butyrate-induced erythroid differentiation of K562 human leukemia cells. Using a panel of anti-ERK, JNK, and p38 phosphospecific antibodies, the study showed that phosphorylation of ERK and JNK is decreased following treatment of cells with butyrate, whereas phosphorylation of p38 is increased. In contrast, a K562 subline defective in butyrate-mediated induction of erythroid differentiation did not reveal these changes in phosphorylation patterns. Inhibition of ERK activity by UO126 induces erythroid differentiation and acts synergistically with butyrate on hemoglobin synthesis and inhibition of cell proliferation, whereas inhibition of p38 activity by SB203580 completely abolished induction of hemoglobin expression by butyrate. Taken together, our data suggest a model in which butyrate induces erythroid differentiation of K562 cells by inhibition of ERK and activation of p38 signal transduction pathways.

BACKGROUND: In chronically transfused patients, conventional blood group typing may be impossible because of mixed-field agglutination. STUDY DESIGN AND METHODS: In 27 patients with congenital anemia and lifelong transfusion history, genotyping for D, RHD, and RHCE was performed with polymerase chain reactions. These results were compared with the blood group typing results documented in the medical record. RESULTS: Two of 27 cases had been typed D-negative by serologic tests and D-positive by genotyping. In 20 patients, the CDE formula had been determined serologically according to the medical record; 4 of these patients were Cc by serologic tests and C/C by genotyping. One patient typed ee by serologic tests, and genotyping revealed heterozygosity (E/e). CONCLUSION: In patients with a lifelong transfusion history, serologic blood group determination may be impossible, and pretransfusion test results are not always available or reliable. In whites, Rh-matched transfusions are possible with genotyping. The genetic background of the RH genes has to be elucidated in other ethnic groups, such as in black patients with sickle cell disease, before genotyping can be applied without restriction.

Isolated myelosarcomas are rare first manifestations of acute myeloid leukemia (AML), preceding bone marrow involvement by weeks to months. Seventeen of 654 children observed during the studies AML-BFM 87 and 93 were diagnosed as extramedullar myelosarcomas (2.6%). The predominantly myelomonocytic or monoblastic tumor cells (M4 or M5 according to FAB classification) mainly infiltrated skin (n = 8). Additional tumors were located in mucosa (n = 2), central nervous system (n = 2), orbita (n = 2), bone (n = 1), glandulae parotis (n = 1) and lymph nodes. Due to the initial mild and variable symptoms in some children the diagnostic measurements were delayed and treatment was inadequate. This might be responsible for the high rate of relapse (79%) and the poor outcome. Ten of 17 patients died from disease (estimated survival 0.27 +/- 0.13 compared to AML-BFM 87/93 0.51 +/- 0.03). Suspect skin lesions or tumors should be considered as isolated myelosarcoma of a primary manifestation of AML. An intensive AML-specific chemotherapy is recommended.

Hintergrund: Das isolierte Myelosarkom der Haut im Kindesalter ist eine seltene Manifestation der akuten myeloischen Leukamie (AML), die dem Nachweis der Leukamie im Knochenmark Wochen bis Monate vorausgehen kann. Studiendesign und Ergebnisse: In den AML-BFM 87/93-Studien zur Behandlung der akuten myeloischen Leukamie im Kindesalter wurden bei 8 Patienten (1,2%) im Alter von 3 Monaten bis 161/2 Jahren isolierte Myelosarkome der Haut diagnostiziert. Histologisch uberwogen monoblastare und myelomonozytare Leukamiezellen (FAB M5, n=5; FAB M4, n=2; FAB M0, n=1). Wahrend 2 Sauglinge, bei der Diagnose 3 und 6 Monate alt, nach lediglich lokaler chirurgischer Tumorexstirpation bzw. einer alleinigen oralen zytostatischen Therapie in anhaltender Vollremission verblieben, war dies bei den alteren Kindern (31/2–161/2 Jahre) nur bei den beiden gemas AML-BFM-Studienprotokoll behandelten Patienten der Fall. Die verzogerte Diagnostik und die milde initiale Symptomatik fuhrten bei den ubrigen 4 Patienten zu einer reduzierten Initialbehandlung. Erst nach dem ersten Rezidiv erfolgte die intensive systemische Therapie (AML-BFM 87/93). Alle 4 Patienten erlitten ein 2. Rezidiv. 3 der Patienten verstarben 21/2–31/2 Jahre nach der Diagnose, 1 Patient befindet sich bei einer Beobachtungszeit von 61/2 Jahren in Vollremission. Schlusfolgerung: Bei ausergewohnlichen Hautinfiltrationen sollte das seltene Myelosarkom in die differentialdiagnostische Uberlegungen einbezogen werden. Nur bei jungen Sauglingen mit solitarem Tumor ohne Knochenmarkbefall scheint eine abwartende Haltung gerechtfertigt zu sein. Fur alle anderen Kinder wird eine fruhzeitige intensive zytostatische Therapie empfohlen.

Reactivation of fetal hemoglobin synthesis in adulthood can be seen in hematological disorders affecting the erythropoietic system. The objective of the present study was to evaluate the incidence and prognostic significance of increased hemoglobin F in patients with myelodysplastic syndrome. Hemoglobin F concentrations and Ggamma/Ggamma + A gamma-globin chain ratios were determined in 26 patients with primary myelodysplastic syndrome. Median age of the patients was 65 years; all FAB subtypes were included. Increased hemoglobin F concentration of up to 20% of total hemoglobin (normal: below 2%) was seen in 16 patients; ten patients had normal values. There was a significant relation between hemoglobin F concentration and the course of disease, e.g., 12 of the 16 patients with elevated hemoglobin F survived at least 1 year after the examination, in contrast to only three of the ten patients with normal hemoglobin F (p < 0.025). All of six patients with hemoglobin F above 5% survived at least 1 year. There was no significant difference in the hemoglobin F concentration between patients with and without cytogenetic anomalies. The Ggamma/Ggamma + A gamma-globin chain ratio was slightly elevated in all patients, with a weak correlation to the degree of hemoglobin F elevation. The values were not of additional prognostic significance. The data of the present study suggest that the hemoglobin F concentration may be a prognostic parameter in myelodysplastic syndrome; increased hemoglobin F concentration may indicate a better prognosis.

Pharmacological induction of hemoglobin F expression may be a promising approach for the treatment of beta-thalassemia and sickle cell disease. Valproic acid, a drug frequently used for the treatment of seizure disorders, has been shown to enhance fetal hemoglobin synthesis in erythroid cells. However, this effect is only modest and requires relative high concentrations. Therefore, the drug appears not to be applicable for the treatment of beta-globin chain disorders. Here, we describe the identification of novel valproic acid derivatives with potent hemoglobin F inducing activities at concentrations that presumably can be obtained in vivo.

Biochemical and molecular genetic studies were performed on the enzyme variants of two patients compound heterozygous for glucose phosphate isomerase (GPI) deficiency, both suffering from severe haemolytic anaemia. The enzymes of case 1 (GPI 'Zwickau') and case 2 (GPI 'Nordhorn' [25]), revealed reduced GPI activity and remarkable thermolability. Glucose-6-phosphate (Gluc-6-P) concentration was elevated 2.3 times in case 1 and 3.8 times in case 2. Sequencing the patients' GPI genes showed four different point mutations, two of them involving highly conserved amino acids. The c1039 C--T substitution, found in the gene of GPI 'Zwickau', has been described recently [30] and causes an Arg 347--Cys substitution close to the putative catalytic site. The second mutation in this case is a novel c1538 G--A substitution causing a Trp--stop mutation at position 513 apparently resulting in premature RNA degradation thus resulting either in a complete lack of protein or a protein which does not show GPI activity. In the gene of GPI 'Nordhorn' a c1028 A--G mutation was discovered, also previously described [1, 9] causing a Gln 343--Trp substitution. The second mutation was a novel splice site mutation at the border of intron 15 to exon 16: IVS15-(-2) A--C which leads to an aberrant splicing of exon 16, thus resulting either in a truncated and most likely inactive enzyme or in no protein at all.Biochemical and molecular genetic studies performed with the enzyme variants GPI 'Zwickau' and GPI 'Nordhorn' showed that in both cases the simultaneous occurrence of a single amino acid substitution affecting the active site, together with a nonsense mutation leading to the loss of major parts of the enzyme probably explains the severe clinical course of the disease.

Malignant rhabdoid tumors (MRT) are characterized by a typical light microscopic morphology with uniformly round tumor cells, vacuolated cytoplasm with occasional round, hyaline intracytoplasmic, periodic acid-Schiff-positive inclusions, vesicular nuclei with prominent nucleoli and positive immunoreactivity for vimentin. The histogenesis of MRT is controversial. Five cases of primary central nervous system (CNS) rhabdoid tumors in children are presented. Immunohistochemical, light and electron microscopic features are compared with primary CNS malignant rhabdoid tumors reported in the literature. Expression of various neurofilaments in our cases of primary CNS rhabdoid tumors was prominent and we therefore favor a neural differentiation of extrarenal intracerebral rhabdoid tumors.

Glycolysis is the most important source of energy in red cells and working muscles. Inherited defects of glycolysis can cause hemolytic anemia, neurologic abnormalities, and myopathy with exercise ...

Acute myeloid leukemia (AML) is a heterogenous disease according to morphology, immunophenotype, and genetics. The retained capacity of differentiation is the basis for the phenotypic classification of the bulk population of leukemic blasts and the identification of distinct subpopulations. Within the hierarchy of hematopoietic development and differentiation it is still unknown at which stage the malignant transformation occurs. It was our aim to analyze the potential involvement of cells with the immunophenotype of pluripotent stem cells in the leukemic process by the use of cytogenetic and cell sorting techniques. Cytogenetic analyses of bone marrow aspirates were performed in 13 patients with AML (11 de novo and 2 secondary) and showed karyotype abnormalities in 10 cases [2q+, +4, 6p, t(6:9), 7, +8 in 1 patient each and inv(16) in 4 patients each]. Aliquots of the samples were fractionated by fluorescence-activated cell sorting of CD34+ cells. Two subpopulations, CD34+/CD38-(early hematopoietic stem cells) and CD34+/CD38+ (more mature progenitor cells), were screened for karyotype aberations as a marker for leukemic cells. Clonal abnormalities and evaluable metaphases were found in 8 highly purified CD34+/CD38-populations and in 9 of the CD34+/CD38-specimens, respectively. In the majority of cases (CD34+/CD38-, 6 of 8 informative samples; CD34+/CD38+, 5 of 9 informative samples), the highly purified CD34+ specimens also contained cytogenetically normal cells. Secondary, progression-associated chromosomal changes (+8, 12) were identified in the CD34+/CD38-cells of 2 patients. We conclude that clonal karyotypic abnormalities are frequently found in the stem cell-like (CD34+/CD38-) and more mature (CD34+/CD38+) populations of patients with AML, irrespective of the phenotype of the bulk population of leukemic blasts and of the primary or secondary character of the leukemia. Our data suggest that, in AML, malignant transformation as well as disease progression may occur at the level of CD34+/CD38-cells with multilineage potential.

Solid tumors of the nervous system are the most common childhood cancers after leukemias. Although brain tumors are the leading cause of cancer-related mortality in children, there are not enough adequate model systems to study their biology. We therefore started a pediatric preclinical testing program in Heidelberg to generate orthotopic patient-derived xenograft (PDX) models for a large variety of pediatric brain tumors. Freshly dissected primary material from multiple centers is being sent to us immediately after surgical resections. One part of the tumor is being reserved for pathological and molecular analysis and the other part is being dissociated into a single cell suspension and injected into the brain of immunodeficient mice. After successful engraftment and passaging, extensive molecular characterization of the PDX tumor and the matching primary tumor are being performed. Thus far, we have injected 95 tumors: 36 low-grade gliomas (LGG), 23 medulloblastomas (MB), 13 ependymomas (EPN), 7 high-grade gliomas (HGG), 6 atypical teratoid rhaboid tumors (AT/RT), 3 meningeal tumors (MT), 3 embryonal tumors with multilayered rosettes (ETMR), 2 gliomatosis cerebri (GC), 1 dysembryoplastic neuroepithelial tumor (DNT) and 1 primitive neuroectodermal tumor (PNET). No engraftment was observed for any of the low-grade tumors (LGG, MT, DNT). For high-grade tumors we established initial engraftments of MB (5/23, 22%), EPN (5/13, 38%), HGG (2/7, 29%), AT/RT (2/6, 33%), ETMR (1/3, 33%) and PNET (1/1, 100%). 11 out of 16 (69%) established PDX models were already passaged at least twice in mice and can be used for preclinical experiments. We conclude that it is possible to generate preclinical models for most malignant pediatric brain tumor entities, but not for low-grade tumors using our current protocol. However, even for the malignant entities there seems to be a selection for only the most aggressive subtypes that successfully engraft. Therefore, due to the low engraftment rate of some tumor types, the rarity of pediatric brain tumors and the multitude of different subtypes, international collaborations are absolutely necessary in this field in order to generate and characterize a broad repertoire of PDX models for all pediatric brain tumor subtypes for preclinical testing. Citation Format: Norman L. Mack, Sebastian Brabetz, Florian Selt, Xanthopolous Christina, David Sumerauer, Heidi Bächli, Arnulf Pekrun, Martin U. Schuhmann, Stefan M. Pfister, Olaf Witt, Till Milde, Marcel Kool. Establishment of orthotopic patient-derived xenograft models of pediatric brain tumors – the Heidelberg experience. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A25.

SummaryGlioblastoma (GBM) is a brain tumor that carries a dismal prognosis and displays considerable heterogeneity. We have recently identified recurrent H3F3A mutations affecting two critical amino acids (K27 and G34) of histone H3.3 in one-third of pediatric GBM. Here, we show that each H3F3A mutation defines an epigenetic subgroup of GBM with a distinct global methylation pattern, and that they are mutually exclusive with IDH1 mutations, which characterize a third mutation-defined subgroup. Three further epigenetic subgroups were enriched for hallmark genetic events of adult GBM and/or established transcriptomic signatures. We also demonstrate that the two H3F3A mutations give rise to GBMs in separate anatomic compartments, with differential regulation of transcription factors OLIG1, OLIG2, and FOXG1, possibly reflecting different cellular origins.

The biochemical properties of erythrocyte pyruvate kinase (PK) together with mutations found in the coding sequence of the R-PK gene in five patients with severe hemolytic anemia due to PK deficiency are described. The enzyme variants were designated PK ‘Mosul’ (homozygote), PK ‘Bukarest1,2’, PK ‘Hamburg1’, PK ‘Koln1’, and PK ‘Essen’ (compound heterozygote). PK ‘Mosul’ showed normal positive cooperative substrate binding, PK ‘Bukarest1,2’ exhibited noncooperative behavior, and PK ‘Hamburg1’ and PK ‘Koln1’ displayed mixed cooperativity, whereas PK ‘Essen’ was negative cooperative. PK ‘Mosul’ was found to be homozygous for the mutation 1151 ACG to ATG, resulting in an amino acid substitution 384 Thr to Met. In one allele of PK ‘Bukarest1,2’ a single nucleotide substitution GAG-TAG was found at nucleotide 721, causing a change of 241 Glu to a chain termination codon (PK ‘Bukarest1’). Additionally, in the second allele of this patient a point mutation at position 1594 (CGG-TGG) occurs, changing 532 Arg to Trp (PK ‘Bukarest2’). Direct sequencing showed the heterozygosity of the patient's mother (PK ‘Bukarest1’/normal) at position 721 and of the patient's father (PK ‘Bukarest2’ /normal) at position 1594. A point mutation at position 1529 (CGA-CAA), causing an amino acid substitution 510 Arg-Gln, was identified in PK ‘Hamburg1’ and PK ‘Koln1’. The second mutation in these variants was not detected. In PK ‘Essen’ no mutation in the coding sequence was found at all. Screening for the mutation at position 1529 in further compound heterozygote patients and in normal subjects of Western European origin showed that this exchange is a common mutation responsible for PK deficiency in this population.

Adenosin deaminase (ADA) deficiency is the cause for Severe Combined Immunodeficiency (SCID) in about 15% of patients with SCID, often presenting as T (-)B (-)NK (-)SCID. Treatment options for ADA-SCID are enzyme replacement, bone marrow transplantation or gene therapy. We here describe the first patient with ADA-SCID and fatal hepatic failure despite bone marrow transplantation from a 10/10 HLA identical related donor. As patients with ADA-SCID may be at yet underestimated increased risk for rapid hepatic failure we speculate whether hepatitis in ADA-SCID should lead to the immediate treatment with enzyme replacement by pegylated ADA.

Hereditary spherocytosis is characterized by a reduced spectrin content of the erythrocytes. However, the underlying primary defect remains unclear in the majority of cases. Genetic studies have revealed a linkage to the gene for ankyrin in some families. By means of ELISA we measured the ankyrin, spectrin, and band-3 contents in erythrocytes of 45 patients with typical spherocytosis. They were classified as having mild or moderate spherocytosis, according to clinical severity. Sixteen patients with mild spherocytosis showed slight reductions of ankyrin and spectrin contents. In contrast, 29 patients with moderate spherocytosis exhibited a clear reduction of both ankyrin and spectrin to about 60% of normal. Band 3 and lipid phosphorus, as measures for membrane surface area, were only slightly reduced to 85%. Our results, together with the molecular genetic data indicating the linkage between spherocytosis and the gene for ankyrin, suggest an ankyrin defect or deficiency as the primary lesion in most cases of spherocytosis.