Immune recovery in children undergoing allogeneic stem cell transplantation: absolute CD8+ CD3+ count reconstitution is associated with survival.

To evaluate the correlation between kinetics of immune reconstitution and survival, we prospectively evaluated lymphocyte subsets in 32 paediatric patients undergoing allogeneic stem cell transplantation (SCT) for haematological malignancies. Four-colour flow cytometric analysis was performed at short intervals with a median follow-up of 4 years post SCT. A total of 50% of patients reached age-matched 5th percentile of natural killer, cytotoxic T, B and helper T cells 4, 9, 20 and 28 weeks after SCT, respectively, which increased to more than 80% within 1 year after SCT. Transplantation of peripheral blood stem cells (PBSC) seemed to elicit the fastest reconstitution of CD3+, CD4+ CD3+, CD8+ CD3+ and naïve T cells compared to bone marrow (BM) or CD34-selected PBSC, which did not differ. Most importantly, we observed a significantly higher number of survivors among patients whose CD8+ CD3+ absolute counts rose above the 5th percentile of age-matched normal levels during the first year post SCT compared to patients who never reached these levels (19/25 vs 0/7, P<0.001). This was still present in both subgroups, BM- and CD34-selected grafts (P=0.03, 0.02). These results from a small patient sample underline the importance of particular lymphocyte subsets for the outcome of children undergoing SCT. A larger study with detailed subset analysis is underway.

Ex vivo expansion of highly purified NK cells for immunotherapy after haploidentical stem cell transplantation in children.

BACKGROUND: Allogeneic natural killer (NK) cells are known to show medium to high cytotoxic activity against HLA-nonidentical leukemia or tumor cells. For a possible benefit of post transplant treatment with NK cells after haploidentical stem cell transplantation (haplo-SCT) we developed a clinical scale procedure for NK cell processing observing Good Manufacturing Practice (GMP). METHODS: Allogeneic donor NK cells were selected from 15 unstimulated leukaphereses using two rounds of immunomagnetic T cell depletion, followed by an NK cell enrichment step. CD56 (+)CD3 (-) NK cells were stimulated and expanded in vitro according to GMP. Quality control of NK cell purity, residual T cells and cytotoxic activity was done by multi-coloured flow cytometric analyses. RESULTS: Purification led to an absolute number of 234-1 237 x 10 (6) CD56 (+)CD3 (-) NK cells from leukapheresis harvests with a median purity of 95 % and a 4 to 6(1/2) log depletion of T cells. After two weeks stimulation with IL-2 a five-fold expansion of NK cells with a T cell contamination below 0.1 % was reached. Median cell viability was 95 % after purification and 99 % after expansion. The IL-2 stimulated NK cells showed a highly increased lytic activity against the MHC-I deficient K562 cells compared to freshly isolated NK cells and a medium cytotoxicity against patients' leukemic cells. CONCLUSIONS: Clinical scale enrichment and activation of allogeneic donor NK cells is feasible. High dose NK cell application may be a new treatment option for pediatric patients with leukemia or solid tumors in case of minimal residual disease or unbalanced chimerism post haplo-SCT as we could show for the first three patients .

Hourly monitoring of circulating CD34+ cells to optimize timing of autologous apheresis in pediatric patients.

In order to increase the CD34+ cell yield in children undergoing autologous stem cell transplantation, the optimum time of apheresis after G-CSF administration has still to be found. We prospectively studied the mobilization of CD34+ cells and white blood cells in the peripheral blood (PB) of 20 pediatric patients before leukapheresis. The monitoring schedule covered 12 h, with blood samples taken before and at 2, 4, 5, 6, 7, 8, 10 and 12 h after G-CSF administration when 10 CD34+ cells/mul were reached. CD34+ cells were measured by flow cytometric analysis both in the single- and dual-platform setting. Two different patterns of mobilization (POM) emerged: 12 patients showed an increase in CD34+ cells in PB during the first 4 h after G-CSF (POM I), while eight patients had an initial decrease of CD34+ cells. However, all patients together showed a significant increase of CD34+ cells about 10 h after G-CSF administration. Further studies with more patients, using an enhanced monitoring schedule will be required to refine the results.

Autologous transplantation of CD133 selected hematopoietic progenitor cells in a pediatric patient with relapsed leukemia.

A pediatric patient with very early meningeal relapse of his CD34(+) CD133(-) pre-B-ALL was transplanted with 2.5 x 10(6)/kg CD133 selected autologous progenitor cells. Enrichment of CD133(+) cells resulted in a purity of 92.3 +/- 3.5% CD133(+). Hematopoietic engraftment with >1.0 x 10(9)/l neutrophils and >50 x 10(9)/l platelets was reached within 13 and 24 days, respectively. At a follow-up of 11(1/2) months after autologous transplantation, the patient is in complete remission. To our knowledge, the successful transplantation with a CD133 selected graft is the first one to be reported worldwide. CD133 selected cells may serve as an alternative in the case of CD34(+) malignancy.

Novel WT1 mutation, 11p LOH, and t(7;12) (p22;q22) chromosomal translocation identified in a Wilms' tumor case.

About 5-10% of sporadic Wilms' tumors (WT) are associated with mutations in the Wilms' tumor 1 gene (WT1). More than 90% of patients with Denys-Drash syndrome (DDS; characterized by renal nephropathy, gonadal anomaly, and predisposition to WT) show constitutional intragenic WT1 mutations. We describe a novel WT1 stop-mutation in exon 2. This heterozygous germline mutation was detected in a one-year-old girl who was bilaterally affected with Wilms' tumor but without any other clinical manifestations of DDS. The C-to-A transversion is predicted to result in a polypeptide comprising only the first 165 amino acids of the WT1 protein. Loss of heterozygosity (LOH) studies comparing tumor DNA with lymphocyte DNA revealed LOH for the entire short arm of chromosome 11 in tumor tissue. In addition to the chromosome 11 lesions, the tumor showed a seemingly balanced chromosomal translocation t(7;12) (p22;q22) as the only visible cytogenetic aberration.

Treatment results in children and adolescents with loco-regional recurrences of abdominal germ cell tumors (GCTs): a pilot-study with PEI chemotherapy and regional deep hyperthermia (RHT) in comparison to a matched cohort.

In this study treatment results in children and adolescents (n = 32) suffering from loco-regional abdominal relapses of germ cell tumors (GCT) (7 embryonal carcinoma, 17 Yolk sac tumors, 8 immature teratomas) aged from 1;0 to 23;3 years (mean = 10;11 years) were evaluated. In this pilot study 9 patients were treated with cisplatinum (40 mg/m2 on days 1 and 4), etoposide (100 mg/m2 on days 1 to 4), and ifosfamide (2000 mg/m2 on days 1 to 4) (PEI) +/- radiation in combination with regional deep hyperthermia (RHI). In sedation RHT was induced by non-invasive heat applicators (Sigma-40 and Sigma 60, BSD Medical Corporation, Utah, USA). In 7 out of these 9 patients with recurrent GCT a tumor response (5 CR, 2 PR, 1 SD, 1 PD) was found. In addition, in 2 patients a complete tumor resection could be achieved inspite of 2 previous incomplete tumor resections each. Five out of 9 patients are living event-free after an observation period ranging from 8 to 40 months (median = 15 months). Treatment results of this RHT study population were compared with treatment results in patients with recurrent GCT, who received conventional relapse therapy (chemotherapy/ surgery +/- radiation) alone. In this matched cohort 5 out of 23 patients are living event-free after an observation time ranging from 1 to 120 months (median = 8 months). According to Kaplan-Maier life table analysis, patients with relapse therapy combined with RHT have an event-free survival (EFS) of 0.41 +/- 0.33 whereas the matched cohort without RHT have an EFS of 0.16 +/- 0.25. The difference in treatment results of both groups is significant (Wilcoxon/p = 0.03). From the data presented in this study we conclude that children with loco-regional recurrences of extracranial non-testicular GCT have an unfavorable prognosis, unless local tumor control can be achieved. The additional application of RHT in combination with conventional therapy (PEI chemotherapy +/- radiation) can improve local tumor control and EFS in GCT patients with loco-regional recurrences. Therefore, based upon these results in the future MAKEI trial RHT will be applied to GCT patients with poor response to neoadjuvant chemotherapy alone as first line treatment.

[Thrombocyte function in children with Type I diabetes mellitus. Cross-sectional study]. / Thrombozytenfunktion bei Kindern mit Diabetes mellitus Typ I. Querschnittstudie.

Platelet count, spontaneous platelet aggregation, ADP- and collagen-induced platelet aggregation platelet adhesion, platelet volume, shape change, beta-thromboglobulin and von-Willebrand-factor have been investigated in 51 insulin dependent diabetic children without clinical signs of diabetic angiopathy. Compared to an age matched healthy control group diabetic children showed a significant enhancement of spontaneous platelet aggregation, elevated plasma levels of von-Willebrand-factor, increased platelet shape change and adhesion. No alterations could be found in ADP--and collagen--induced platelet aggregation and in beta-thromboglobulin levels. Significant correlations could be found between the total glycosylated haemoglobin concentrations (Hb A1) and spontaneous platelet aggregation, as well as between duration of diabetes Hb A1, and platelet volume. In this study we could demonstrate changes in platelet function in diabetic children without clinical signs of diabetic angiopathy. However these changes could be due to metabolic adjustment and may precede diabetic vasculopathy.

15 parameters of coagulation and fibrinolysis in children with type I diabetes mellitus (onset period).

15 parameters of coagulation and fibrinolysis were investigated in 38 children with type I diabetes mellitus without clinical signs of diabetic angiopathy. Compared to an age matched non diabetic control group spontaneous platelet aggregation was enhanced, plasma levels for factor VIII C, von Willebrand factor, antithrombin III and C-1-inactivator were elevated, alpha-2-macroglobulin was decreased at onset of the disease. During remission (3, 6, 12 months) these changes reverted to normal. Alpha-2-antiplasmin decreased after 12 months. If, during partial remission, diabetic duration was longer than one year an increase of factor VIII C was seen again. In comparison to the controls no significant alterations were found for ristocetin cofactor, fibrinogen, plasminogen and alpha-1-antichymotrypsin. It seems likely that changes in plasmatic coagulation, fibrinolysis and platelet function during the onset period of diabetes mellitus type I are due to metabolic changes and precede diabetic angiopathy.