Favorable outcome in children and adolescents with a high proportion of advanced phase disease using single/multiple autologous or matched/mismatched allogeneic stem cell transplantations.

We determined the indication, outcome, and risk factors of single and multiple hematopoietic stem cell transplantation(s) (HSCT) in children and adolescents mostly with advanced disease. Forty-one out of 483 patients (8.5 %; median age 9 years) diagnosed at the University of Leipzig with hematological and oncological diseases required HSCT from 1999 to 2011. Patients had overall survival (OS) of 63 ± 10 and 63 ± 16 %, event-free survival (EFS) of 57 ± 10 and 42 ± 16 %, relapse incidence (RI) of 39 ± 10 and 44 ± 18 % and nonrelapse mortality (NRM) of 4 ± 4 and 13 ± 9 % at 10 years after one or more allogeneic and autologous HSCT, respectively. One patient in CR1 and five with advanced disease received two HSCT. Four of the six patients maintained/achieved CR for a median of 13 months. Three died of progression and one of NRM. Two patients had a third HSCT and one survived in CR +231 days after HSCT. Risk factors for OS and EFS were disease stage at HSCT and EBMT risk score. Center (pediatric or JACIE accredited pediatric/adult) was not a determinant for survival. Pediatric single and multiple HSCT are important curative approaches for high-risk malignant diseases with low NRM. Efforts to reduce high RI remain the major aim.

Consensus of German transplant centers on hematopoietic stem cell transplantation in Fanconi anemia.

Allogeneic hematopoietic stem cell transplantation (HSCT) is currently the only curative therapy for the severe hematopoietic complications associated with Fanconi anemia (FA). In Germany, it is estimated that 10-15 transplants are performed annually for FA. However, because FA is a DNA repair disorder, standard conditioning regimens confer a high risk of excessive regimen-related toxicities and mortality, and reduced intensity regimens are linked with graft failure in some FA patients. Moreover, development of graft-versus-host disease is a major contributing factor for secondary solid tumors. The relative rarity of the disorder limits HSCT experience at any single center. Consensus meetings were convened to develop a national approach for HSCT in FA. This manuscript outlines current experience and knowledge about HSCT in FA and, based on this analysis, general recommendations reached at these meetings.

Outcomes of mismatched and unrelated donor hematopoietic stem cell transplantation in Fanconi anemia conditioned with chemotherapy only.

Fanconi anemia (FA) is a genomic instability syndrome associated with bone marrow failure, myelodysplastic syndrome (MDS), and/or acute myeloid leukemia (AML) requiring hematopoietic stem cell transplantation (HSCT) to restore normal hematopoiesis. Although low-intensity fludarabine-based preparative regimens without radiation confer excellent outcomes in FA HSCTs with HLA-matched sibling donors, outcomes for FA patients with alternative donors are less encouraging, albeit improving. We present our experience with 17 FA patients who completed mismatched related or unrelated donor HSCT using a non-radiation fludarabine-based preparative regimen at Charité University Medicine Berlin. All patients engrafted; however, one patient had unstable chimerism in the setting of multi-viral infections that necessitated a stem cell boost to revert to full donor chimerism. Forty-seven percent of patients developed grade I acute graft-verus-host disease (aGVHD). No grade II-IV aGVHD or chronic graft-versus-host disease of any severity occurred. At a median follow-up of 30 months, 88 % of patients are alive with normal hematopoiesis. Two patients died of infections 4 months post-transplantation. These results demonstrate that short-term outcomes for FA patients with mismatched and unrelated donor HSCTs can be excellent using chemotherapy only conditioning. Viral reactivation, however, was a major treatment-related complication.

Detection of a novel HLA-DQB1 allele, designated DQB1*06:49.

We report a novel allele HLA-DQB1*06:49 with a G→T transversion, most closely resembling HLA-DQB1*06:02:01.

Randomized controlled trial of taurolidine citrate versus heparin as catheter lock solution in paediatric patients with haematological malignancies.

BACKGROUND: A catheter lock solution containing 1.35% taurolidine and 4% citrate could potentially disrupt bacterial surface adherence and consecutive biofilm production due to the anti-adherence properties of taurolidine and the anticlotting and chelator activities of both compounds. AIM: To compare the impact on microbial catheter colonization and infectious complications of heparin and taurolidine citrate as central venous catheter (CVC) lock solutions in paediatric patients with haematological malignancies. METHODS: Seventy-one patients aged 1.4-18 years were randomized to two treatment groups using either heparin (N = 36) or taurolidine citrate (N = 35). Infectious complications and clinical side-effects were prospectively monitored and microbial colonization of catheters was assessed at the time of removal. FINDINGS: There were two bloodstream infections in the taurolidine citrate group versus nine in the heparin group (0.3 vs 1.3 infections per 1000 catheter-days; P = 0.03). Fever of unknown origin and catheter occlusions were observed with a similar frequency in both groups. Microbial colonization was found in 25.4% catheters. The time of no-lock use, but not the type of lock solution or time of observation, was a significant predictor of catheter colonization (P = 0.004). Colonization was not observed in CVCs used immediately with taurolidine citrate lock. Seven patients in the taurolidine citrate group (20%) experienced side-effects (nausea, vomiting, abnormal taste sensations). CONCLUSION: The use of taurolidine citrate lock solution was associated with a significant reduction in bloodstream infection in immunocompromised paediatric patients. Taurolidine citrate may prevent colonization of CVCs if used from the time of insertion, but not after a period of no-lock catheter use.

A novel HLA-DQB1*03:02 variant designated DQB1*03:02:05.

The new DQB1*03:02:05 differs from DQB1*03:02:01 by two nucleotide exchanges in exon 2.

Hyperbilirubinemia and rapid fatal hepatic failure in severe combined immunodeficiency caused by adenosine deaminase deficiency (ADA-SCID).

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.

Delayed onset of (severe) combined immunodeficiency (S)CID (T-B+NK+): complete IL-7 receptor deficiency in a 22 months old girl.

BACKGROUND: Usually IL-7 receptor deficiency presents as (T-B+NK+) (Severe) Combined Immunodeficiency (SCID) within the first six months of life. All published IL-7R-deficient patients so far have been diagnosed and received stem cell transplantation within the first year of life. PATIENT AND METHODS: We present a female patient born to non-consanguineous German parents with delayed manifestation. She presented with superinfected dermatitis at 6 months of life and developed a first pneumonia at age 9 months. On admission to our department at 22 months the patient presented with severe T cell lymphopenia. PNEUMOCYSTIS JIROVECI pneumonia was diagnosed from broncho-alveolar lavage fluid. RESULTS: Sequencing of IL7RA in the patient revealed compound heterozygous mutations. FACS analysis showed no expression of IL-7 receptor alpha-chain on the patient's lympho- and monocytes. The patient successfully received haematopoietic stem cell transplantation from a 9/10 matched unrelated donor at age 24 months. CONCLUSION: [corrected] Despite almost absent T cell functions clinical symptoms occurred late compared to previously published patients. Thus even in patients with moderate clinical symptoms and delayed onset a (T-B+NK+) (Severe) Combined Immunodeficiency ((S)CID)) due to missing IL-7 receptor signalling must be considered.

Allogeneic stem cell transplantation for pediatric and adolescent patients with CML: results from the prospective trial CML-paed I.

PURPOSE: Stem cell transplantation (SCT) can definitely cure chronic myeloid leukemia (CML), a rare disease in childhood. We prospectively evaluated the results of early SCT in pediatric CML after standardized pretreatment with hydroxyurea+/-interferon. PATIENTS AND METHODS: Between 1995 and 2004, 200 children (median age: 12.4 years) were enrolled and stratified: given the availability of an HLA-matched related donor (MRD), SCT was scheduled within 6 months and otherwise from an unrelated donor (UD) within 12 months following diagnosis. RESULTS: 176 patients underwent SCT; from MRD within median 4 months and from UD within median 11 months after diagnosis. At SCT, 158 patients were in chronic phase (CP1 or CP2), 9 patients were in accelerated phase and 9 patients were in blast crisis (BC). The conditioning regimen - total body irradiation or busulfan - exerted no different impact on overall survival (OS). Probability of OS at 5 years was 87+/-11% if grafted from a sibling (n=41), 52+/-9% from matched UD (MUD, n=71), and 45+/-16% from mismatched donors (MMD, n=55), respectively. A trend for better OS in CP1 was observed if SCT was performed within 6 months (n=49; 74+/-9%), compared to 7-12 months (n=52; 62+/-15%), and >12 months (n=43; 62+/-17%) after diagnosis, respectively (p=0.157). Probability of relapse at 5 years was 20+/-12%. Transplant-related mortality and graft-versus-host disease mainly contributed to the inferior outcome in UD and HLA-mismatched SCT. CONCLUSION: These data from the first prospective trial on CML restricted to children and adolescents might be considered for decision making when balancing the risks of SCT against the increasing use of imatinib as upfront treatment for CML.

Quantitative proton magnetic resonance spectroscopy of children with adrenoleukodystrophy before and after hematopoietic stem cell transplantation.

About 35-40 % of boys with X-linked adrenoleukodystrophy (ALD) develop a rapidly progressive cerebral form which leads to severe neurologic disability and death within 3-5 years after onset of clinical symptoms. Because previous proton magnetic resonance spectroscopy (MRS) studies of ALD identified metabolite patterns characteristic of demyelination, gliosis, and neuroaxonal loss, this work tested the hypothesis that MRS--apart from indicating disease progression--provides criteria for the outcome after hematopoietic stem cell transplantation (HSCT) which has been promising at an early stage of the active disease. Follow-up quantitative proton MRS was performed in frontal and occipital white matter of ALD patients (n = 12) before and up to 5 years after HSCT. The observed metabolite alterations were retrospectively correlated with the clinical outcome representing either a stable condition (n = 5), a further deterioration (n = 5), or death (n = 2). While disease progression of patients before HSCT was mainly characterized by a further increase of elevated choline-containing compounds (Cho) as an indicator of active demyelination, a positive outcome after HSCT was correlated with high N-acetylaspartate (tNAA) levels in affected white matter before HSCT yielding positive and negative predictive values for tNAA of 80 %. Although to be confirmed in a larger cohort of patients, the present findings suggest the preservation of neuroaxonal integrity as a prerequisite for an arrested course. Conversely, the combination of increased Cho with markedly reduced tNAA before HSCT apparently reflects a degree of tissue degeneration which precludes a successful therapeutic intervention.

Second German consensus on immunogenetic donor search for allotransplantation of hematopoietic stem cells.

The present paper summarizes the results of the second German consensus meeting on immunogenetic donor search for allotransplantation of hematopoietic stem cells held in Essen in November 1999 under the auspices of the German Society for Immunogenetics (DGI) and the German Working Party for Blood and Marrow Transplantation (DAG-KBT). Immunogeneticists and transplant physicians from all over the country agreed to update the national standards for: (1) search strategy including the role of unrelated and extended family donor search after unsuccessful core family donor search, (2) histocompatibility loci to be typed, (3) histocompatibility typing techniques to be used (HLA serology vs DNA-based HLA typing, cellular tests, serum cross-match), and (4) acceptable HLA mismatches in the context of a defined underlying disease, donor type, and conditioning regimen.

Platelet glycoprotein complex Ia/IIa antibodies cause neonatal alloimmune thrombocytopenia but do not inhibit megakaryopoiesis and platelet recovery after allogeneic cord blood stem cell transplantation.

A sibling cord blood (CB) transplantation was performed in a boy with Wiskott-Aldrich syndrome. The CB (31 x 10(6) CD34(+) cells) derived from a newborn sister with neonatal alloimmune thrombocytopenia (NAIT) with 40,000 platelets/microl, caused by a maternal anti-HPA-5b and HLA-A2 antibody. Maternal serum did not inhibit clonogenicity after in vitro testing of megakaryopoiesis. Accordingly, this CB was accepted for sibling transplantation. The transplantation showed a good course with fast and sustained hematopoietic reconstitution (granulocytes >500/microl on day +16, platelets >50,000/microl on day +30). This case demonstrates a successful CB transplantation from a donor suffering from NAIT.

Association of complementation group and mutation type with clinical outcome in fanconi anemia. European Fanconi Anemia Research Group.

Fanconi anemia (FA) is a clinically and genetically heterogeneous disorder. Clinical care is complicated by variable age at onset and severity of hematologic symptoms. Recent advances in the molecular biology of FA have allowed us to investigate the relationship between FA genotype and the nature and severity of the clinical phenotype. Two hundred forty-five patients from all 7 known complementation groups (FA-A to FA-G) were studied. Mutations were detected in one of the cloned FANC genes in 169 patients; in the remainder the complementation group was assigned by cell fusion or Western blotting. A range of qualitative and quantitative clinical parameters was compared for each complementation group and for different classes of mutation. Significant phenotypic differences were found. FA-G patients had more severe cytopenia and a higher incidence of leukemia. Somatic abnormalities were less prevalent in FA-C, but more common in the rare groups FA-D, FA-E, and FA-F. In FA-A, patients homozygous for null mutations had an earlier onset of anemia and a higher incidence of leukemia than those with mutations producing an altered protein. In FA-C, there was a later age of onset of aplastic anemia and fewer somatic abnormalities in patients with the 322delG mutation, but there were more somatic abnormalities in patients with IVS4 + 4A --> T. This study indicates that FA patients with mutations in the FANCG gene and patients homozygous for null mutations in FANCA are high-risk groups with a poor hematologic outcome and should be considered as candidates both for frequent monitoring and early therapeutic intervention. (Blood. 2000;96:4064-4070)

Spectrum of mutations in the Fanconi anaemia group G gene, FANCG/XRCC9.

FANCG was the third Faconi anaemia gene identified and proved to be identical to the previously cloned XRCC9 gene. We present the pathogenic mutations and sequence variants we have so far identified in a panel of FA-G patients. Mutation screening was performed by PCR, single strand conformational polymorphism analysis and protein truncation tests. Altogether 18 mutations have been determined in 20 families - 97% of all expected mutant alleles. All mutation types have been found, with the exception of large deletions, the large majority is predicted to lead to shortened proteins. One stop codon mutation, E105X, has been found in several German patients and this founder mutation accounts for 44% of the mutant FANCG alleles in German FA-G patients. Comparison of clinical phenotypes shows that patients homozygous for this mutation have an earlier onset of the haematological disorder than most other FA-G patients. The mouse Fancg sequence was established in order to evaluate missense mutations. A putative missense mutation, L71P, in a possible leucine zipper motif may affect FANCG binding of FANCA and seems to be associated with a milder clinical phenotype.

Transplant activities in Germany in 1998--a survey facilitated by the National Registry for Hemopoietic Stem Cell Transplantation.

To improve the infrastructure of hemopoietic stem-cell transplantations in our country, the German Registry for Hemopoietic Stem-Cell Transplantations (DRST) was established in 1998. The present paper summarizes the current status of the DRST and gives a survey of transplant activities in Germany in 1998 in terms of transplant units, transplant types, transplant frequencies and underlying diseases.

Immunomagnetic selection of CD34+ cells: factors influencing component purity and yield.

BACKGROUND: In immunomagnetic selection of CD34+ cells from HPC transplants, not all factors that affect yield and purity of CD34+ cells are known. METHODS: Forty-three consecutive procedures of immunomagnetic selection of CD34+ cells from peripheral blood HPCs and bone marrow harvests (autologous harvests, n = 27; allogeneic harvests; n=16) were performed by use of a cell selection system (Isolex 300i, Baxter Immunotherapy). The composition of the starting component and the subsets of CD34+ cells were analyzed for correlation with the yield and purity of the final component. RESULTS: The mean purity of the final components was 84.3 percent (range, 27-99%), and the mean yield was 51.4 percent (range, 9.4-80. 4%). Partial regression analysis showed that, among the factors correlating with purity and/or yield, the RBC volume in the starting fraction had the highest predictive impact on the purity and yield of CD34+ cells, even after the exclusion of procedures using bone marrow harvests as an HPC source (beta coefficient, -0.704; p = 0. 001). CONCLUSION: The use of the Isolex 300i system allows efficient recovery of CD34+ cells in routine selection procedures. The volume of RBCs in the starting component should be minimized to ensure a high yield and purity of the final component.

Improved treatment results in childhood B-cell neoplasms with tailored intensification of therapy: A report of the Berlin-Frankfurt-Münster Group Trial NHL-BFM 90.

In study NHL-BFM 90, we investigated whether the serum lactate dehydrogenase (LDH) concentration and early response are useful markers for stratification of therapy for childhood B-cell neoplasms in addition to stage, if the outcome of patients with abdominal stage III and LDH >/=500 U/L can be improved by high-dose (HD) methotrexate (MTX) at 5 g/m(2) instead of intermediate-dose (ID) MTX at 500 mg/m(2) in the preceding study 86; whether 2 therapy courses are enough for patients with complete resection; and whether combined systemic and intraventricular chemotherapy is efficacious for central nervous system-positive (CNS(+)) patients. After a cytoreductive prephase, treatment was stratified into 3 risk groups: patients in R1 (completely resected) received 2 5-day courses (ID-MTX, dexamethasone, oxazaphorins, etoposide, cytarabine, doxorubicin, and intrathecal therapy), patients in R2 (extra-abdominal primary only or abdominal tumor and LDH <500 U/L) received 4 courses containing HD-MTX, and patients in R3 (abdominal primary and LDH >/=500 U/L or bone marrow/CNS/multilocal bone disease) received 6 courses. Incomplete responders after 2 courses received an intensification containing HD-cytarabine/etoposide. Patients with no or necrotic tumor thereafter received 3 more courses; 6 patients with viable tumor received autologous bone marrow transplantation. From April 1990 through March 1995, 413 evaluable patients were enrolled (R1, 17%; R2, 40%; and R3, 43%). The 6-year event-free survival (pEFS) was 89% +/- 2% for all and 100%, 96% +/-2%, and 78% +/- 3% in R1, R2, and R3, respectively. The pEFS of patients with abdominal stage III and LDH >/=500 U/L was 81% +/- 4% as compared with 43% +/- 10% in study 86. Of 26 CNS(+) patients, 5 died early, but only 3 relapsed.

Heterogeneous spectrum of mutations in the Fanconi anaemia group A gene.

Fanconi anaemia (FA) is a genetically heterogeneous autosomal recessive disorder associated with chromosomal fragility, bone-marrow failure, congenital abnormalities and cancer. The gene for complementation group A (FAA), which accounts for 60-65% of all cases, has been cloned, and is composed of an open reading frame of 4.3 kb, which is distributed among 43 exons. We have investigated the molecular pathology of FA by screening the FAA gene for mutations in a panel of 90 patients identified by the European FA research group, EUFAR. A highly heterogeneous spectrum of mutations was identified, with 31 different mutations being detected in 34 patients. The mutations were scattered throughout the gene, and most are likely to result in the absence of the FAA protein. A surprisingly high frequency of intragenic deletions was detected, which removed between 1 and 30 exons from the gene. Most microdeletions and insertions occurred at homopolymeric tracts or direct repeats within the coding sequence. These features have not been observed in the other FA gene which has been cloned to date (FAC) and may be indicative of a higher mutation rate in FAA. This would explain why FA group A is much more common than the other complementation groups. The heterogeneity of the mutation spectrum and the frequency of intragenic deletions present a considerable challenge for the molecular diagnosis of FA. A scan of the entire coding sequence of the FAA gene may be required to detect the causative mutations, and scanning protocols will have to include methods which will detect the deletions in compound heterozygotes.

Relapse and clonal disease in children with aplastic anemia (AA) after immunosuppressive therapy (IST): the SAA 94 experience. German/Austrian Pediatric Aplastic Anemia Working Group.

Since the introduction of combined immunosuppressive therapy (IST) into management of aplastic anemia (AA) in childhood response and probability of survival improved. In contrast to bone marrow transplantation (BMT), however, patients after IST are not considered cured as high rates of relapse and development of clonal disease demonstrate. From 11/93 to 9/97 114 children (65 m, 49 f; median age 9.5 y.) from 37 centers in Germany and Austria were registered in the SAA 94 study. 86 patients lacking a matched sibling donor received IST. Most of the patients suffered from very severe (VSAA: PMN < 200/microliter) or severe AA (SAA: PMN < 500/microliter). All patients were treated with combined IST consisting of ALG and Cyclosporin A (CSA). VSAA and SAA patients were additionally treated with G-CSF. Therapy response was evaluated at day 112, after 6, 12 and 18 months. 8/86 patients died, the probability of survival being 87% after 4 years. At d 112 61% of evaluable patients became independent of transfusions (IST response: CR + PR), 13% with normal blood counts (CR). After 6 months 33% showed CR. At 12 and 18 months response improved to 74% resp. 80%, 39% resp. 55% CR. The best response was achieved in the subgroup of VSAA with 90% (PR + CR) and 65% CR after 18 months. 4 patients developed AML 3-19 months after the beginning of IST. In 2/4 pts. an aberrant clone (-7; 5q-) could be detected retrospectively in BM at diagnosis of AA. 3 nonresponders developed chromosomal aberrations (+19; -7, +12; +8) after 4, 12 and 16 months without morphological signs of AML or MDS. Overall 11 relapses occurred at a median time of 12 months (range 5-27 months) after the beginning of IST. 2 of them relapsed under CSA therapy, 2 under tapering of CSA and 7 after cessation of CSA. 7 patients responded again to CSA monotherapy. Overall response rate is 77% with a probability of event free survival (EFS) of 54% after 4 years regarding all complications mentioned as events.