Andre Willasch

How and when should we monitor chimerism after allogeneic stem cell transplantation

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.

Preemptive immunotherapy in childhood acute myeloid leukemia for patients showing evidence of mixed chimerism after allogeneic stem cell transplantation

Previous studies have shown that children with acute myeloid leukemia (AML) who developed mixed chimerism (MC) were at high risk for relapse after allogeneic stem-cell transplantation (allo-SCT). We investigated the feasibility of intensified preemptive immunotherapy in children receiving allo-SCT for AML. Eighty-four children were registered in our trial from May 2005 to April 2009; of these, 71 fulfilled the inclusion criteria and were treated according to the study protocol. Serial and semiquantitative analyses of posttransplantation chimerism were performed. Defined immunotherapy approaches were considered in MC patients. Continuous complete chimerism (CC) was observed in 51 of 71 patients. MC was detected in 20 patients and was followed by immunotherapy in 13. Six of 13 MC patients returned to CC without toxicity and remained in long-term remission. Overall, the probability of event-free survival (pEFS) was 66% (95% confidence interval [95% CI] = 53%-76%) for all patients and 46% (95% CI = 19%-70%) in MC patients with intervention; however, this number increased to 71% (95% CI = 26%-92%) in 7 of 13 MC patients on immunotherapy who were in remission at the time of transplantation. All MC patients without intervention relapsed. These results suggest that MC is a prognostic factor for impending relapse in childhood AML, and that preemptive immunotherapy may improve the outcome in defined high-risk patients after transplantation.

Standardization of WT1 mRNA quantitation for minimal residual disease monitoring in childhood AML and implications of WT1 gene mutations: a European multicenter study

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.

Continuous rise in incidence of childhood Type 1 diabetes in Germany

Aimsu2003 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.

Children with myelodysplastic syndrome (MDS) and increasing mixed chimaerism after allogeneic stem cell transplantation have a poor outcome which can be improved by pre-emptive immunotherapy

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 (nu2003=u200365; advanced MDSu2003=u200344, refractory cytopeniau2003=u200321) 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.

Diabetes incidence in children of different nationalities: an epidemiological approach to the pathogenesis of 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 2121 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. [Diabetologia (2001) 44 [Suppl 3]: B 21–B 26]

Monitoring of post-transplant remission of childhood malignancies: is there a standard?

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.

Enrichment of cell subpopulations applying automated MACS technique: purity, recovery and applicability for PCR-based chimerism analysis.

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 μl. 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.

Incidence of diabetes mellitus among children of Italian migrants substantiates the role of genetic factors in the pathogenesis of type 1 diabetes

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 0–14xa0years, 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.

Feasibility of IL-15-activated cytokine-induced killer cell infusions after haploidentical stem cell transplantation

Feasibility of IL-15-activated cytokine-induced killer cell infusions after haploidentical stem cell transplantation