Identification and selective depletion of alloreactive T-cells for adoptive immunotherapy.

BACKGROUND: T-cell-depleted allografts may exhibit delayed T-cell recovery, severe infections, and relapse after haploidentical hematopoietic stem cell transplantation (HSCT). Required donor lymphocyte infusions containing nonalloreactive cells may transfer immune function without causing graft-versus-host disease. METHODS: We developed an ex vivo approach for the immunomagnetic depletion of alloreactive CD25+, CD69+, and HLA-DR+ T-cells. To achieve highest rates of alloantigen expression, we cocultured peripheral blood mononuclear cells (PBMNCs) with PBMNCs (A/B*), dendritic cells (A/B* DCs), or cytokine-pretreated PBMNCs (A/B* cyt cells). Functional analyses were performed after depletion. RESULTS: After coculture with PBMNCs (A/B* cells), 29% of T-cells became CD25+, CD69+, and HLA-DR+. In modified mixed lymphocyte reactions (MLR) (A/B* cyt cells and A/B* DCs), 35% and 37% of T-cells became CD25+, CD69+, and HLA-DR+. Alloactivation was confirmed by interferon gamma release and proliferation. Immuno-magnetic depletion produced <1% alloactivated cells. Furthermore, this depletion strategy was allospecific and hardly impaired the immune function of the retained cells. DISCUSSION: The efficiency of immunomagnetic depletion depended on the stimulatory capacity of stimulator cells and was improved by using cytokine-pretreated PBMNCs for alloactivation. Overall, this approach might be a promising strategy for restoration of the immune system, particularly after haploidentical HSCT.

High dose chemotherapy and autologous hematopoietic cell transplantation for Wilms tumor: a study of the European Society for Blood and Marrow Transplantation.

Survival for subgroups of patients with Wilms tumor (WT), such as those who suffer from relapse, is disappointing. Some patients' treatment plans include high-dose chemotherapy (HDT) with autologous hematopoietic cell transplantation (aHCT), although proof for its benefit is lacking. To increase the level of evidence regarding children with WT receiving aHCT as consolidation of first or second remission (after first relapse), we extracted relevant data from the European Blood and Marrow Transplantation Registry concerning 69 patients. Different HDT regimens were administered, mostly either melphalan-containing (n = 34) or thiotepa-containing (n = 14). For the whole population, 5-year overall survival (OS) and event-free survival (EFS) probabilities were 0.67 (±0.06) and 0.63 (±0.06), respectively (median observation time 7.8 years); for children transplanted in first remission, OS and EFS were 0.69 (±0.09) and 0.72 (±0.08). In univariate analysis, male gender and relapse in multiple sites were associated with lower OS probabilities. The use of a given pretransplant regimen (i.e. melphalan alone versus regimens with multiple drugs) did not seem to influence EFS/OS probability after aHCT, but significantly influenced platelet engraftment (more delayed with thiotepa). We here provide further data to improve the basis for future evidence-based clinical decision-making when using HDT and aHCT in relapsed/refractory WT.

Continuous rise in incidence of childhood Type 1 diabetes in Germany.

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.

Treatment of relapse after allogeneic stem cell transplantation in children and adolescents with ALL: the Frankfurt experience.

Therapy for post-transplant relapse of paediatric ALL is limited. Standardised curative approaches are not available. We hereby describe our local procedure in this life-threatening situation. A total of 101 ALL patients received their first allogeneic stem cell transplantation (SCT) in our institution. After relapse, our primary therapeutic goal was to cure the patient with high-dose chemotherapy or specific immunotherapy (HDCHT/SIT) followed by a second SCT from a haploidentical donor (transplant approach). If this was not feasible, low-dose chemotherapy and donor lymphocyte infusions (LDCHT+DLI) were offered (non-transplant approach). A total of 23 patients suffered a post-transplant relapse. Eight patients received HDCHT/SIT, followed by haploidentical SCT in 7/8. Ten received LDCHT+DLI. The eight patients treated with a second transplant and the ten treated with the non-transplant approach had a 4-year overall survival of 56% and 40%, respectively (P=0.232). Prerequisites for successful treatment of post-transplant relapse by either a second transplant or experimental non-transplant approaches are good clinical condition and the capacity to achieve haematological remission by the induction treatment element.

State-of-the-art fertility preservation in children and adolescents undergoing haematopoietic stem cell transplantation: a report on the expert meeting of the Paediatric Diseases Working Party (PDWP) of the European Society for Blood and Marrow Transplantation (EBMT) in Baden, Austria, 29-30 September 2015.

Nowadays, allogeneic haematopoietic stem cell transplantation (allo-HSCT) is a well-established treatment procedure and often the only cure for many patients with malignant and non-malignant diseases. Decrease in short-term complications has substantially contributed to increased survival. Therefore long-term sequelae are reaching the focus of patient care. One of the most important risks of stem cell transplant survivors is infertility. As well as in the field of allo-HSCT also the field of reproductive medicine has achieved substantial advances to offer potential options for fertility preservation in both boys and girls. Access to these procedures as well as their financing differs significantly throughout Europe. As all European children and adolescents should have the same possibility, the Paediatric Diseases Working Party of the European Society for Blood and Marrow Transplantation organised an expert meeting in September 2015. This manuscript describes the recommendations for the diagnosis and pre-emptive procedures that should be offered to all children and adolescents in Europe who have to undergo an allo-HSCT.

Fertility preservation issues in pediatric hematopoietic stem cell transplantation: practical approaches from the consensus of the Pediatric Diseases Working Party of the EBMT and the International BFM Study Group.

Fertility preservation is an urgent challenge in the transplant setting. A panel of transplanters and fertility specialists within the Pediatric Diseases Working Party of the European Society for Blood and Marrow Transplantation (EBMT) and the International BFM Study Group provides specific guidelines. Patients and families should be informed of possible gender- and age-specific cryopreservation strategies that should be tailored according to the underlying disease, clinical condition and previous exposure to chemotherapy. Semen collection should be routinely offered to all postpubertal boys at the diagnosis of any disease requiring therapy that could potentially impair fertility. Testicular tissue collection might be offered to postpubertal boys; nevertheless, its use has been unsuccessful to date. Oocyte collection after hormonal hyperstimulation should be offered to postpubertal girls facing gonadotoxic therapies that could be delayed for the 2 weeks required for the procedure. Ovarian tissue collection could be offered to pre-/post-pubertal girls. Pregnancies have been reported after postpubertal ovarian tissue reimplantation; however, to date, no pregnancy has been reported after the reimplantation of prepubertal ovarian tissue or in vitro maturation of pre-/post-pubertal ovarian tissue. Possible future advances in reproductive medicine could change this scenario. Health authorities should prioritize fertility preservation projects in pediatric transplantation to improve patient care and quality of life.

Maternal CD4+ microchimerism in HIV-exposed newborns after spontaneous vaginal delivery or caesarean section.

BACKGROUND: Maternal CD4+ cell microchimerism may be greater after caesarean section compared to spontaneous vaginal delivery and could cause mother-to-child transmission (MTCT) in HIV-exposed newborns. AIMS: To evaluate maternal CD4+ cell microchimerism in HIV-exposed newborns after spontaneous vaginal delivery or caesarean section. STUDY DESIGN AND SUBJECTS: In this prospective single-centre study, neonates whose mothers were infected with HIV and had normal MTCT risk according to the German Austrian Guidelines were considered for study enrolment. Maternal CD4+ cell microchimerism in the newborns' umbilical cord blood was measured and compared by mode of delivery. RESULTS: Thirty-seven HIV-infected mothers and their 39 newborns were included in the study. None of the 17 (0.0%) newborns delivered vaginally had quantifiable maternal CD4+ cells (95% confidence interval (CI): 0.00-0.00) in their circulation at birth compared with four of 16 (25.0%) newborns delivered via planned caesarean section, who showed 0.01-0.66% maternal cells (95% CI: -0.06-0.16; P=0.02) in their circulation. The intention to treat analysis, which included six additional newborns delivered by unplanned caesarean section, showed quantifiable maternal CD4+ cells in one (0.05%; 95% CI: -0.02-0.04) of 23 (4.3%) newborn at birth compared to four of 16 (25.0%) born via planned caesarean section (95% CI: -0.06-0.16; P=0.04). There was no MTCT in any of the newborns. CONCLUSION: In this small cohort, spontaneous vaginal delivery in HIV-infected women with normal MTCT risk was associated with lower maternal CD4+ cell transfer to newborns compared to planned caesarean section.

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.

Incidence of type 1 diabetes in childhood before and after the reunification of Germany--an analysis of epidemiological data, 1960-2006.

OBJECTIVE: To examine the impact of rapidly changing environmental factors on the incidence of type 1 diabetes mellitus (T1D). METHOD: We compared the frequency of T1D in children before and after the reunification of Germany by means of the registries of the German Democratic Republic (GDR, 1960-1989) and of Baden-Wuerttemberg (BW, 1987-2006). The number of cases of diabetes onset in East Germany after the reunification was predicted by a mathematical model. The observed incidence rate in the Eastern part of Germany after the reunification was taken from the literature 1. RESULTS: In Germany, the incidence rate of T1D in children aged 0-14 was 7.2/100 000/year (95%-CI 6.9-7.5, GDR, 1980-1987), and 10.4/100 000/year (95%-CI 9.5-11.4, BW, 1987-1994). For the whole observation period (1960-2006), the observed incidence rates y could be described by the square of a linear function [GDR: y=(1.86 + 0.040 * (year - 1960))²; r²=0.85; BW: y=(3.03 + 0.085 * (year - 1987))², r²=0.89]. The mean rise in incidence before the reunification was less than half the mean rise after the reunification (mean slope: BW 0.085, 95%-CI 0.080-0.090 vs. GDR 0.040, 95% CI 0.036-0.044). The observed incidence for East Germany after 1989 was higher than the prediction on the basis of the GDR -registry (GDR 12.3/100 000/year vs. Saxony 15.7/100 000/year, 95%-CI 14.2-17.3, n=412; 1999-2003). CONCLUSION: We conclude that the basis for the disease progress is a genetic predisposition. Environmental factors may modify changes in incidence of type 1 diabetes but do not determine the overall risk.

Real-time PCR quantification of major Wilms' tumor gene 1 (WT1) isoforms in acute myeloid leukemia, their characteristic expression patterns and possible functional consequences.

Wilms' tumor gene 1 (WT1) functions including some contradictory effects may be explained by the presence and interactions of its isoforms, however, their evaluation has been so far complicated by several technical problems. We designed unique quantitative PCR systems for direct quantification of the major WT1 isoforms A[EX5-/KTS-], B[+/-], C[-/+] and D[+/+] and verified their sensitivity, specificity and reproducibility in extensive testing. With this method we evaluated WT1 total and isoform expression in 23 normal bone marrow (BM) samples, 73 childhood acute myeloid leukemia (AML), 20 childhood myelodysplastic syndrome (MDS), 9 childhood severe aplastic anemia (SAA), 30 adult AML and 29 adult MDS patients. WT1 isoform patterns showed differences among these samples and clustered them into groups representing the specific diagnoses (P<0.0001). Isoform profiles were independent of total WT1 expression and possess certain common features-overexpression of isoform D and EX5[+] variants. The KTS[+]/KTS[-] ratio was less variable than the EX5[+]/EX5[-] ratio and differed between children and adults (P<0.001); the EX5[+]/EX5[-] ratio varied between diagnoses (AML vs MDS, P<0.001). These findings bring new insights into WT1 isoform function and suggest that the ratio of WT1 isoforms, particularly EX5 variants, is probably crucial for the process of malignant transformation.

Standardization of DNA isolation from low cell numbers for chimerism analysis by PCR of short tandem repeats.

Analysis of short tandem repeats (STR) by PCR analysis is routinely used in chimerism diagnostics to monitor donor engraftment and to diagnose relapse. Some applications require chimerism analysis of low cell numbers, but no standardized protocol is available for DNA isolation from 1000 to 30,000 cells. The EU-supported EuroChimerism Consortium (project QLRT-2001-01485) selected four different protocols for 'small-scale' DNA isolation, which were tested by six laboratories for their ability to recover reproducible amounts of good quality DNA, suited for PCR-based STR analysis. The protocols included two direct lysis methods with and without detergents and proteinase K, and two commercial column-based kits. The direct lysis method using detergents and proteinase K showed the highest DNA recovery and the best performance in the multiplex PowerPlex16 STR assay. DNA isolated with this method also showed the highest sensitivity in chimerism analysis using singleplex PCR reactions of EuroChimerism STR markers. Sensitivity was reached ranging from 1 to 20% of recipient cells in a donor background. In conclusion, the direct lysis method using detergents and proteinase K is a standardized DNA isolation method well suited for chimerism studies on low cell numbers.

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 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.

WT1 peptide-specific T cells generated from peripheral blood of healthy donors: possible implications for adoptive immunotherapy after allogeneic stem cell transplantation.

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.

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.

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.

Diabetes incidence in children of different nationalities: an epidemiological approach to the pathogenesis of diabetes.

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.

Varying clinical presentations at onset of type 1 diabetes mellitus in children--epidemiological evidence for different subtypes of the disease?

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.