Abstract: Wilms tumor 1 (WT1) mutations have recently been identified in approximately 10% of adult acute myeloid leukemia (AML) with normal cytogenetics (CN-AML) and are associated with poor outcome. Using array-based comparative genome hybridization in pediatric CN-AML samples, we detected a WT1 deletion in one sample. The other WT1 allele was mutated. This prompted us to further investigate the role of WT1 aberrations in childhood AML. Mutations were found in 35 of 298 (12%) diagnostic pediatric AML samples. In 19 of 35 (54%) samples, more than one WT1 aberration was found: 15 samples had 2 different mutations, 2 had a homozygous mutation, and 2 had a mutation plus a WT1 deletion. WT1 mutations clustered significantly in the CN-AML subgroup (22%; P < .001) and were associated with FLT3/ITD (43 vs 17%; P < .001). WT1 mutations conferred an independent poor prognostic significance (WT1 mutated vs wild-type patients: 5-year probability of overall survival [pOS] 35% vs 66%, P = .002; probability of event-free survival 22% vs 46%, P < .001; and cumulative incidence of relapse or regression 70% vs 44%, P < .001). Patients with both a WT1 mutation and a FLT3/ITD had a dismal prognosis (5-year pOS 21%). WT1 mutations occur at a significant rate in childhood AML and are a novel independent poor prognostic marker.
Abstract: To investigate the role of genetic and environmental factors in the pathogenesis of type 1 diabetes mellitus (T1D), we carried out a study in Germany aimed at comparing the prevalence and incidence of T1D among children of migrant Italians from high-risk (Sardinia) and low-risk (continental Italy) regions versus German children. Children from Italy were identified by the "Baden-Wuerttemberg (BW) Diabetes Incidence Registry", which registered 4017 newly diagnosed T1D patients, aged 0-14 years, between 1987 and 2003. Data relating to T1D children from Sardinia were elicited from more than 2000 questionnaires. Our findings were: (1) T1D is more frequent among German children than among children of Italian migrants [incidence rate (IR) 14.8/100,000/year, 95% confidence interval (CI) 14.4-15.4 vs. IR 10.8/100,000/year, 95% CI 8.2-13.6); (2) the incidence of T1D among Italian children residing in Germany is similar to that of Italian children in the home country (IR 10.8/100,000/year, 95% CI 8.2-13.6 vs. 8.4/100,000/year, 95% CI 7.9-8.9); (3) the prevalence of T1D among Sardinian children is higher than that among German children (0.11%, 95% CI 0.11-0.12) independent of the place where the Sardinian children are living (Sardinian children in Germany 2.3%, 95% CI 0.5-6.5 vs. Sardinian children in Sardinia 0.30%, 95% CI 0.27-0.32). Conclusion: Children from high- and low-risk areas of Italy have incidence rates of T1D that are closer to those of their native regions than to those of German children, indicating that genetic factors play a predominant role in the pathogenesis of T1D.
Abstract: A standardized, sensitive and universal method for minimal residual disease (MRD) detection in acute myeloid leukemia (AML) is still pending. Although hyperexpression of Wilms' tumor (WT1) gene transcript has been frequently proposed as an MRD marker in AML, wide comparability of the various methods used for evaluating WT1 expression has not been given. We established and standardized a multicenter approach for quantifying WT1 expression by quantitative reverse transcriptase PCR (qRT-PCR), on the basis of a primer/probe set combination at exons 6 and 7. In a series of quality-control rounds, we analyzed 69 childhood AML samples and 47 normal bone marrow (BM) samples from 4 participating centers. Differences in the individual WT1 expressions levels ranged within <0.5 log of the mean in 82% of the cases. In AML samples, the median WT1/1E+04 Abelson (ABL) expression was 3.5E+03 compared with that of 2.3E+01 in healthy BM samples. As 11.5% of childhood AML samples in this cohort harbored WT1 mutations in exon 7, the effect of mutations on WT1 expression has been investigated, showing that mutated cases expressed significantly higher WT1 levels than wild-type cases. Hence, our approach showed high reproducibility and applicability, even in patients with WT1 mutations; therefore, it can be widely used for the quantitation of WT1 expression in future clinical trials.Leukemia advance online publication, 26 March 2009; doi:10.1038/leu.2009.51.
Abstract: The Wilms tumor antigen, WT1, is expressed at high levels in various types of leukemia and solid tumors, including lung, breast, colon cancer and soft tissue sarcomas. The WT1 protein has been found to be highly immunogenic, and spontaneous humoral and cytotoxic T-cell responses have been detected in patients suffering from leukemia. Furthermore, major histocompatibility complexes class I- and II-restricted WT1 peptide epitopes have been shown to elicit immune responses in patients with WT1-expressing tumors. As a consequence, WT1 has become an attractive target for anticancer immunotherapy. In this study, we investigated the feasibility of generating WT1-specific T cells for adoptive immunotherapy after allogeneic stem cell transplantation. We analyzed the incidence of T cells specific for WT1 peptide epitopes in cancer patients and healthy volunteers. It is noted that we could generate WT1-specific responses in nine of ten healthy volunteer donors and established T-cell clones specific for two WT1-derived peptide epitopes. These in vitro expanded WT1-specific T cells effectively lysed WT1-expressing tumor cell lines, indicating the potential clinical impact of ex vivo expanded donor-derived WT1-specific T cells for adoptive immunotherapy after allogeneic stem cell transplantation.Leukemia advance online publication, 9 April 2009; doi:10.1038/leu.2009.70.
Abstract: PURPOSE: Minimal residual disease (MRD) before allogeneic stem-cell transplantation was shown to predict outcome in children with relapsed acute lymphoblastic leukemia (ALL) in retrospective analysis. To verify this, the Acute Lymphoblastic Leukemia Relapse Berlin-Frankfurt-Münster (ALL-REZ BFM) Study Group conducted a prospective trial. PATIENTS AND METHODS: Between March 1999 and July 2005, 91 children with relapsed ALL treated according to the ALL-REZ BFM 96 or 2002 protocols and receiving stem-cell transplantation in >or= second remission were enrolled. MRD quantification was performed by real-time polymerase chain reaction using T-cell receptor and immunoglobulin gene rearrangements. RESULTS: Probability of event-free survival (pEFS) and cumulative incidence of relapse (CIR) in 45 patients with MRD >or= 10(-4) leukemic cells was 0.27 and 0.57 compared with 0.60 and 0.13 in 46 patients with MRD less than 10(-4) leukemic cells (EFS, P = .004; CIR, P < .001). Intermediate-risk patients (strategic group S1) with MRD >or= 10(-4) leukemic cells (n = 14) had a pEFS of 0.20 and CIR of 0.73, whereas patients with MRD less than 10(-4) leukemic cells (n = 21) had a pEFS of 0.68 and CIR of 0.09 (EFS, P = .020; CIR, P < .001). High-risk patients (S3/4, third complete remission) who received transplantation with an MRD load of less than 10(-4) leukemic cells (n = 25) showed a pEFS and CRI of 0.53 and 0.18, respectively. In contrast, pEFS and CRI were 0.30 and 0.50 in patients who received transplantation with an MRD load of >or= 10(-4) leukemic cells. Multivariate Cox regression analysis revealed MRD as the only independent parameter predictive for EFS (P = .006). CONCLUSION: MRD is an important predictor for post-transplantation outcome. As a result, new strategies with modified stem-cell transplantation procedures will be evaluated in ALL-BFM trials.
Abstract: Enrichment of cell subpopulations is a prerequisite for lineage-specific chimerism analysis (LCA), a frequent approach in follow-up after allo-SCT. An efficient enrichment technique is Magnetic Cell Sorting (MACS) using the AutoMACS separator. However, evaluation of purity, recovery and applicability for PCR-based chimerism analysis of MACS-enriched subpopulations from post-transplant peripheral blood, providing reduced cell numbers and/or unbalanced proportions of subpopulations, is currently unavailable. We performed enrichment of CD3-, CD14-, CD15-, CD19- and CD56-positive subpopulations using 'Whole Blood MicroBeads' and AutoMACS separator in 137 prospectively collected peripheral blood samples from 15 paediatric patients after allo-CD3-/CD19-depleted SCT. Purity was assessed by immune phenotyping. Recovery and applicability for chimerism analysis was evaluated. Excellent purity >90% was achieved in CD14-, CD15-positive cells in 81%, 95% of the isolates and in 86% of CD3 and CD19 isolates, if ACC was >400 cells per mul. Median purity of CD56-positive isolates was 78.9%. Recovery >90% was between 93 (CD56) and 37% (CD15). Conventional and real-time PCR-based chimerism analysis was feasible in virtually all samples. Isolation of cell subpopulations by automated cell enrichment in post-transplant peripheral blood is feasible and fast providing excellent purity and recovery for routine lineage-specific chimerism analysis.Bone Marrow Transplantation advance online publication, 4 May 2009; doi:10.1038/bmt.2009.89.
Abstract: Chimerism analysis has become an important tool for the peri-transplant surveillance of engraftment. It offers the possibility to realize imminent graft rejection and it can serve as an indicator for the recurrence of the underlying malignant or non-malignant disease. In addition to this analysis, the characterization of residual disease (MRD) prior to and in the course of follow-up post transplant has become an important prognostic factor to highlight patients at highest risk for relapse. Consecutive post transplant MRD monitoring, together with chimerism analysis, allows the detection of impending relapse in a substantial group of children transplanted for acute leukemia. Consequently, these investigations have become the basis for treatment intervention, for example, to avoid graft rejection, to maintain engraftment and to treat imminent relapse by pre-emptive immunotherapy.
Abstract: AIMS: To assess the incidence and the trend in incidence of Type 1 diabetes (T1DM) in children and adolescents < 15 years of age in Baden-Württemberg (BW), Germany. METHODS: BW is Germany's third largest federal state. All 31 paediatric departments in BW and one diabetes centre participated in the study. Case registration was done according to the EURODIAB criteria. The degree of ascertainment was 97.2%. RESULTS: From 1987 to 2003, the age- and sex-standardized incidence rate was 14.1/100,000 per year [95% confidence interval (CI) 13.7, 14.6, n = 4017]. The estimated annual increase in incidence was 3.8% (95% CI 1.1, 6.6). Compared with the first years of our registry, the current mean number of new cases of T1DM has doubled (1987-1989, n = 153; 2000-2003, n = 302). Generally, the highest rise in incidence occurred in the youngest age group of 0-4-year-old patients (5.8%; 95% CI 2.5, 9.3), followed by the age groups 5-9 (3.4%; 95% CI 0.8, 6.0) and 10-14 (2.7%; 95% CI 0.3, 5.1). CONCLUSIONS: In Germany, the number of children and adolescents with new-onset T1DM has been rising at a faster pace than expected. A distinct shift to younger age at onset has been observed in Germany.
Abstract: Sequence polymorphisms (SPs) can serve as genetic markers for quantitative polymerase chain reactions (qPCR) for chimerism analysis, providing a significantly higher sensitivity compared to short tandem repeat PCR. In this study, a panel of 29 selected markers was evaluated in 317 patients with leukemia and myelodysplastic syndrome, who received allogeneic stem cell transplantation. In total, 5415 posttransplantation samples were analyzed. Recipient genotype discrimination was possible in 96% with a mean number of 2.5 (1-7) informative markers per recipient/donor pair. Marker specific standard dilution series from volunteers' DNA served as standard for quantification of chimerism. Sensitivity of the method was < or =1 x 10-3 (0.1% of recipient cells) in 83.3% of the assays. By this method, it was possible to very accurately detect autologous signals in the range from 0% to 0.5% (95% confidence interval [CI] +/-0.2), from 0.5% to 1% (95% CI +/-0.4), from 1% to 2% (95% CI +/-0.6) and from 2% to 5% (95% CI +/-1.2). Reproducibility of the quantified autologous signals was independent from the amount of DNA. This is the first report on a SP-based chimerism system allowing for the performance of chimerism analyses for virtually all patients with high sensitivity, excellent reproducibility, and precision of measurement.
Abstract: BACKGROUND AND OBJECTIVES: After allogeneic stem cell transplantation treatment failures are mostly caused by graft rejection or graft-versus-host disease (GVHD). T-cell depletion is an appropriate tool to prevent GvHD. However, it might be associated with an increased risk of graft rejection, which can be recognized by serial and quantitative characterization of chimerism. Thus, pre-emptive immunotherapy might be helpful to avoid graft rejection. DESIGN AND METHODS: We present the outcome of 56 transplants performed in 53 children with non-malignant diseases. T-cell depletion was conducted in 27/56 grafts. When increasing mixed chimerism over 30% autologous cells occurred low dose donor lymphocyte transfusions (DLT) were performed. RESULTS: During the course of the follow-up 29 out of 53 patients achieved complete chimerism or low mixed chimerism (0-1%) and 28/29 remained in continuous complete remission. Donor engraftment failed in 2/53 patients who died of serious infection. Increasing mixed chimerism was found in 19 out of 56 transplantations. Fifteen of these 19 patients received additional immunotherapy with DLT. Eleven out of the 15 remained in complete remission. One of the 15 patients developed GvHD grade III that turned out to extensive chronic GvHD. The 3-year overall survival was 100% for patients transplanted from matched related or unrelated donors and 75% for patients transplanted from mismatched donors. INTERPRETATION AND CONCLUSIONS: We demonstrated that children transplanted for non-malignant diseases have an excellent overall survival. T-cell depletion is associated with an increased risk of graft rejection. Pre-emptive immunotherapy with DLT, administered on the basis of increasing mixed chimerism, is feasible and might prevent graft rejection.
Abstract: We recently reported that virtually all children with acute leukaemia and myelodysplastic syndrome (MDS) who develop the phenotype of increasing mixed chimaerism (MC) after allogeneic stem cell transplantation (allo-SCT) will relapse. We therefore performed a prospective, multi-centre study focused on children with MDS (n = 65; advanced MDS = 44, refractory cytopenia = 21) after allo-SCT in order to determine to what extent relapse can be prevented by pre-emptive immunotherapy on the basis of increasing MC. Analyses of chimaerism in 44 patients with advanced MDS revealed 31 cases with complete chimaerism (CC)/low-level MC/transient MC, 11 cases with increasing MC and two cases with decreasing MC. The same analyses in 21 MDS patients with refractory cytopenia revealed 17 cases with CC/low-level MC, one case with increasing MC and three cases with decreasing MC. Pre-emptive immunotherapy performed on each patient that showed increasing MC improved event-free survival from 0%, as seen in prior studies, to 50%. We therefore conclude that pre-emptive immunotherapy is an effective treatment option to prevent impending relapse in children with MDS after allo-SCT.
Abstract: Summary:Chimerism analysis has become an important tool for the peri-transplant surveillance of engraftment. It offers the possibility to realize impending graft rejection and can serve as an indicator for the recurrence of the underlying malignant or nonmalignant disease. Most recently, these investigations have become the basis for treatment intervention, for example, to avoid graft rejection, to maintain engraftment and to treat imminent relapse by pre-emptive immunotherapy. This invited review focuses on the clinical implications of characterization of hematopoietic chimerism in stem cell transplantation.
Abstract: BACKGROUND: Since 1987, patients with newly diagnosed diabetes mellitus type 1 under 15 yr of age have been registered in Baden-Wuerttemberg (BW), Germany. AIM: Our aim was to describe the frequency and the clinical presentation of diabetic ketoacidosis (DKA) at onset of type 1 diabetes mellitus in children. METHODS: All 31 pediatric departments in BW and one diabetes center participated in this study. Hospital records of 2121 children below 15 yr of age were examined retrospectively. DKA was defined as glucose > 250 mg/dL, pH < 7.30 or bicarbonate < 15 mmol/L and ketonuria. Statistical analysis was done after logarithmic transformation. RESULTS: 26.3% (n = 558) of all patients presented with DKA. The mean age of these patients was 7.9 yr. The frequency of DKA is higher in girls than in boys (28.9 vs. 23.8%; p = 0.0079). Those aged 0-4 yr suffered most frequently (p < 0.0001) from ketoacidosis (36.0%). The percentage of DKA in newly diagnosed cases was constant over 10 yr. 23.3% of all patients with DKA presented with an altered level of consciousness; 10.9% of these had clinical signs of coma. No deaths occurred. The proportion of ketoacidosis does not increase concurrently with the number of diabetes manifestations in winter. CONCLUSION: The proportion of DKA in children with newly diagnosed diabetes mellitus is significant. In particular, children < 5 yr and girls face an increased risk. DKA may be the result of a particularly aggressive subtype of diabetes.
Abstract: OBJECTIVE: On the basis of 2121 case observations between 1987 and 1997, we describe the clinical and laboratory characteristics of diabetes mellitus type 1 at its onset. Our objective is to analyze whether clinical presentation follows a uniform pattern or whether there is evidence for different subtypes. RESEARCH DESIGN AND METHODS: Thirty-one pediatric hospitals and one diabetes center in Baden-Wuerttemberg (BW), Germany, participated in this study. The hospital records of 2121 children below 15 yr of age were examined retrospectively. Statistical analysis was done after logarithmic transformation into a normal distribution. RESULTS: The average duration of symptoms was found to be 15.2 d (95% CI (Confidence Intervals) = 14.3-16.1) ranging between 2.0 and 180 d (95% central range). The most frequent symptoms were polyuria and polydipsia; 7.2% presented with altered level of consciousness. The mean blood glucose value was 407.9 mg/dL (95% CI = 400.0-416.0), corresponding to 23.3 mmol/L (95% CI = 22.8-23.8). The median pH value was 7.35 (95% CI = 7.34-7.36), and the median base excess was -5 mmol/L (95% CI =-5 to -4). The younger patients had a shorter duration of symptoms and suffered most frequently from ketoacidosis. CONCLUSIONS: Although the symptoms of diabetes at its onset follow a uniform pattern, the clinical presentation and duration of symptoms indicate that there may be various forms of type 1 diabetes.
Abstract: AIMS/HYPOTHESIS: Incidence studies of children with Type I (insulin-dependent) diabetes mellitus and different ethnic backgrounds are known to provide important insights into the pathogenesis of the disease. For this reason, we compared the incidence rate in Baden-Württemberg, Germany, of children who were not of German descent with that of German children as well as with the reported incidence rates pertaining to the countries of origin of the children who were not of German descent. METHODS: Our study was based on the Baden-Württemberg incidence register, part of the EURODIAB TIGER network, which includes 2,121 children aged 0-14 years, diagnosed as having Type I diabetes between 1987 and 1997. The study covered a population at risk of 1.8 million children, which represents 13.3% of the total number of children in Germany. RESULTS: The total incidence rate was found to be 12.5 per 100,000 per year (95 %-CI 12.0-13.0); for German children alone it was calculated as 13.5 (95%-CI 12.9-14.1) and for children who were not of German descent it was significantly lower at 6.9 per 100,000 per year (95%-CI 5.8-8.0). The percentage of children who were not of German descent with Type I diabetes (8.3 %) is smaller than that among the general population (15.2%). Children from former Yugoslavia, Italy and Greece had incidence rates closer to their countries of origin than to the incidence rate of German children. CONCLUSION/INTERPRETATION: Our findings indicate that genetic factors play a predominant role in the pathogenesis of Type I diabetes. However, the influence of certain aspects of life-style, which remain constant even after immigration, cannot be excluded.
