Johann Greil

Megadose transplantation of purified peripheral blood CD34(+) progenitor cells from HLA-mismatched parental donors in children.

We performed HLA-mismatched stem cell transplantation with megadoses of purified positively selected mobilized peripheral blood CD34+ progenitor cells (PBPC) from related adult donors in 39 children lacking an otherwise suitable donor. The patients received a mean number of 20.7 ± 9.8 × 106/kg purified CD34+ and a mean number of 15.5 ± 20.4 × 103/kg CD3+ T lymphocytes. The first seven patients received short term (<4 weeks) GVHD prophylaxis with cyclosporin A, whereas in all the following 32 patients no GVHD prophylaxis was used. In 38 evaluable patients, five patients experienced primary acute GVHD grade I and one patient grade II. In 32 patients, no signs of primary GVHD were seen and GVHD only occurred after T cell add backs. T cell reconstitution was more rapid if the number of transplanted CD34+ cells exceeded 20 × 106/kg. Of the 39 patients, 15 are alive and well, 13 died due to relapse and 10 transplant-related deaths occurred. We conclude that the HLA barrier can be overcome by transplantation of megadoses of highly purified mismatched CD34+ stem cells. GVHD can be prevented without pharmacological immunosuppression by the efficient T cell depletion associated with the CD34+ positive selection procedure. This approach offers a promising therapeutic option for every child without an otherwise suitable donor. Bone Marrow Transplantation (2001) 27, 777–783.

A Syndrome with Congenital Neutropenia and Mutations in G6PC3

BACKGROUNDnThe main features of severe congenital neutropenia are the onset of severe bacterial infections early in life, a paucity of mature neutrophils, and an increased risk of leukemia. In many patients, the genetic causes of severe congenital neutropenia are unknown.nnnMETHODSnWe performed genomewide genotyping and linkage analysis on two consanguineous pedigrees with a total of five children affected with severe congenital neutropenia. Candidate genes from the linkage interval were sequenced. Functional assays and reconstitution experiments were carried out.nnnRESULTSnAll index patients were susceptible to bacterial infections and had very few mature neutrophils in the bone marrow; structural heart defects, urogenital abnormalities, and venous angiectasia on the trunk and extremities were additional features. Linkage analysis of the two index families yielded a combined multipoint lod score of 5.74 on a linkage interval on chromosome 17q21. Sequencing of G6PC3, the candidate gene encoding glucose-6-phosphatase, catalytic subunit 3, revealed a homozygous missense mutation in exon 6 that abolished the enzymatic activity of glucose-6-phosphatase in all affected children in the two families. The patients neutrophils and fibroblasts had increased susceptibility to apoptosis. The myeloid cells showed evidence of increased endoplasmic reticulum stress and increased activity of glycogen synthase kinase 3beta (GSK-3beta). We identified seven additional, unrelated patients who had severe congenital neutropenia with syndromic features and distinct biallelic mutations in G6PC3.nnnCONCLUSIONSnDefective function of glucose-6-phosphatase, catalytic subunit 3, underlies a severe congenital neutropenia syndrome associated with cardiac and urogenital malformations.

Transplantation of a combination of CD133+ and CD34+ selected progenitor cells from alternative donors.

Positive selected haematopoietic stem cells are increasingly used for allogeneic transplantation with the CD34 antigen employed in most separation techniques. However, the recently described pentaspan molecule CD133 appears to be a marker of more primitive haematopoietic progenitors. Here we report our experience with a new CD133‐based selection method in 10 paediatric patients with matched unrelated (nu2003=u20032) or mismatched‐related donors (nu2003=u20038). These patients received a combination of stem cells (medianu2003=u200329·3u2003×u2003106/kg), selected with either anti‐CD34 or anti‐CD133 coated microbeads. The proportion of CD133+ selected cells was gradually increased from patient to patient from 10% to 100%. Comparison of CD133+ and CD34+ separation procedures revealed similar purity and recovery of target populations but a lower depletion of T cells by CD133+ selection (3·7u2003log vs. 4·1u2003log, Pu2003<u20030·001). Both separation procedures produced >90% CD34+/CD133+ double positive target cells. Engraftment occurred in all patients (sustained primary, nu2003=u20038; after reconditioning, nu2003=u20032). No primary acute graft versus host disease (GvHD)u2003≥u2003grade II or chronic GvHD was observed. The patients showed a rapid platelet recovery (median time to independence from substitutionu2003=u200313·5u2003d), whereas T cell regeneration was variable. Five patients are alive with a median follow‐up of 10u2003months. Our data demonstrates the feasibility of CD133+ selection for transplantation from alternative donors and encourages further trials with total CD133+ separated grafts.

Adenovirus infection and treatment with cidofovir in children after liver transplantation

Abstract:u2002 In a retrospective study, serum samples from 21 pediatric liver transplant recipients were analysed by quantitative real‐time PCR for ADV infection up to 24u2003wk after Tx. ADV DNA was detected in serum of eight children after Tx, one of whom developed life‐threatening fulminant hepatitis and sepsis. None of these children were symptomatic at the time of first detection of ADV DNA in serum after Tx. Seven children with positive ADV PCR had low adenoviral loads, showed no increase in viral load and remained clinically asymptomatic in the follow‐up period of 24u2003wk. After 10u2003wk under immunosuppression one child presented clinically with adenoviral sepsis and severe necrotizing hepatitis. This patient revealed a dramatic increase of ADV from baseline titers up to 1.3u2003×u2003109u2003copies/mL serum within 10u2003wk after Tx. ADV was also detected in a liver biopsy of this child at 1.2u2003×u2003104u2003copies/cell and typed by sequence analysis as human ADV species C, type 6, a rarely detected ADV type and first described in a liver transplant patient. Immunosuppression was reduced in this patient immediately and the antiviral drug cidofovir administered intravenously followed by viral suppression and clinical improvement of the child.

Successful treatment of primary refractory acute myeloid leukemia with megadose stem cell transplantation, bone marrow boost and reduced intensity conditioning avoiding chronic graft vs. host disease and severe late toxicity

Abstract:u2002 We report on a 9‐yr‐old boy suffering from primary refractory AML. Remission was not achieved after two courses of induction therapy, leading to prolonged aplasia for more than 3u2003months and severe infection. Therefore, the boy was treated with a reduced intensity conditioning regimen consisting of fludarabine, cyclophosphamide and OKT3. Megadose transplantation of highly enriched CD34+ peripheral stem cells from his HLA‐identical brother, followed on day +11 by a boost of unmanipulated bone marrow, was performed. Regeneration of donor hematopoiesis was rapid and led to resolution of infection. No additional donor lymphocyte infusions were necessary. More than 4 1/2u2003yr after the transplant the boy remains in complete continuous remission with no evidence for chronic GvHD or other late effects. Therefore, we conclude that reduced intensity conditioning in combination with allogeneic megadose stem cell transplantation and bone marrow boost may have helped to achieve cure from primary refractory AML as well as a good quality of life for this boy.

Telomeres and telomerase in paediatric patients with T-cell acute lymphoblastic leukaemia (T-ALL).

Telomeres and telomerase in paediatric patients with T-cell acute lymphoblastic leukaemia (T-ALL)

Transplantation of CD34+ enriched allografts in children with nonmalignant diseases: does graft manipulation necessarily result in high incidence of graft failure?

Transplantation of CD34+ enriched allografts in children with nonmalignant diseases: does graft manipulation necessarily result in high incidence of graft failure?

Bearbeitung und Transplantation hämatopoetischer Stammzellen

ZusammenfassungHintergrund Neue Methoden zur Anreicherung und Depletion definierter Zellpopulationen haben es möglich gemacht, hämatopoetische Stammzellen in hoher Reinheit von gesunden Spendern zu gewinnen. Patienten und Methoden 116 Patienten mit Leukämien, Lymphomen und nichtmalignen Erkrankungen wurden mit hochreinen Stammzellen von HLA-identischen Fremdspendern oder nichtidentischen (haploidenten) Familienspendern transplantiert.Ergebnisse Akute und chronische GvHD konnten massiv reduziert werden, bei gleichzeitig stabilem Engraftment. Patienten mit ALL in Remission, die ansonsten nicht hätten transplantiert werden können, hatten nach Stammzellspende ihrer Eltern ein ereignisfreies 5-Jahres-Überleben von 46%. Für Kinder mit nichtmalignen Erkrankungen konnte ebenfalls ein hoher Nutzen gezeigt werden.Schlussfolgerung Durch moderne Stammzellbearbeitungsmethoden können den individuellen Erfordernissen entsprechende Transplantate gewonnen und HLA-Barrieren überwunden werden. Somit ist prinzipiell für jedes Kind ein Spender verfügbar. Im Fall eines fehlenden HLA-identen Spenders sprechen diese Ergebnisse für die Entscheidung zugunsten alternativer Spender in Gestalt der Eltern.AbstractBackground New graft manipulation procedures make it possible to obtain highly purified hematopoietic stem cells from healthy donors.Patients and Methods A total of 116 children with leukemias, lymphomas, and nonmalignant diseases received purified (97%) peripheral stem cells from matched unrelated donors or mismatched related (haploidentical) donors.Results Acute and chronic GVHD were vastly minimized and a high overall rate of engraftment was achieved. Event-free survival at 5 years was 46% after haploidentical transplantation for children with ALL in remission who otherwise lacked a matched donor. Moreover, excellent results were observed in patients with nonmalignant diseases.ConclusionsGraft manipulation results in well-characterized transplants with minimal risk of GVHD and makes a donor available for each patient by recruiting the parents. Thus, alternative donors should be strongly considered in all patients who need a stem cell transplantation but lack an identical donor.