Search

The article focuses on a study conducted on haploidentical stem cell transplantation for the treatment of leukemia, which can be done from parent to children. Topics discussed include transplant modality and graft manipulation being determining factors for stem cell donation, fetal microchimerism (FM) due to bidirectional cell transfer during third trimester of pregnancy, and determination of killer immunoglobulin-like receptors present on maternal natural killer cells.

Hematopoietic stem cell transplantation is becoming an increasingly important approach to treatment of different malignant and non-malignant disorders. There is thus growing demand for diagnostic assays permitting the surveillance of donor/recipient chimerism posttransplant. Current techniques are heterogeneous, rendering uniform evaluation and comparison of diagnostic results between centers difficult. Leading laboratories from 10 European countries have therefore performed a collaborative study supported by a European grant, the EuroChimerism Concerted Action, with the aim to develop a standardized diagnostic methodology for the detection and monitoring of chimerism in patients undergoing allogeneic stem cell transplantation. Following extensive analysis of a large set of microsatellite/short tandem repeat (STR) loci, the EuroChimerism (EUC) panel comprising 13 STR markers was established with the aim to optimally meet the specific requirements of quantitative chimerism analysis. Based on highly stringent selection criteria, the EUC panel provides multiple informative markers in any transplant setting. The standardized STR-PCR tests permit detection of donor- or recipient-derived cells at a sensitivity ranging between 0.8 and 1.6%. Moreover, the EUC assay facilitates accurate and reproducible quantification of donor and recipient hematopoietic cells. Wide use of the European-harmonized protocol for chimerism analysis presented will provide a basis for optimal diagnostic support and timely treatment decisions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented regarding the administration of CD19/CD3-bispecific antibody blinatumomab to observe complete remission in pediatric patients with post-transplant relapsed acute lymphoblastic leukemia (ALL).

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (2009) 23, 1472–1479; doi:10.1038/leu.2009.51; published online 26 March 2009 [ABSTRACT FROM AUTHOR]

A letter to the editor is presented discussing alterations of phenotypic or genotypic markers due to clonal development between disease episodes interfere with the detection of minimal residual disease (MRD) in acute leukemia.

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.Bone Marrow Transplantation (2005) 35, 107-119. doi:10.1038/sj.bmt.1704715 Published online 25 October 2004 [ABSTRACT FROM AUTHOR]

Summary:Children with leukemias and increasing mixed chimerism (increasing MC) after allogeneic stem cell transplantation have the highest risk to relapse. Early immunological intervention was found to be effective in these cases. To substantiate this on a defined group of pediatric acute myelogenous leukemia (AML) patients, we performed serial analysis of post transplant chimerism and pre-emptive immunotherapy in patients with increasing MC. In total, 81 children were monitored, 62 patients revealed complete chimerism (CC), low-level MC or decreasing MC. Increasing MC was detected in 19 cases. Despite early immunological intervention relapse was still significantly more frequent in patients with increasing MC (9/19) than in patients with CC, low-level or decreasing MC (8/62, P<0.005). The probability of 3-year event-free survival (EFS) was 52% for all patients (n=81), 59% for patients with CC, low-level MC, 60% for patients with decreasing MC (n=62), and 28% for patients with increasing MC (n=19, P<0.005). Patients with increasing MC who received early immunological intervention showed a significantly enhanced probability for event-free survival (pEFS 36%, n=15) compared to patients with increasing MC without intervention (pEFS 0%, n=4, P<0.05). These results prove that pediatric AML patients with increasing MC are at highest risk for relapse and that early immunological intervention can prevent relapse in these patients.Bone Marrow Transplantation (2004) 33, 815-821. doi:10.1038/sj.bmt.1704444 Published online 1 March 2004 [ABSTRACT FROM AUTHOR]

Summary:Allogeneic stem cell transplantation (allo-SCT) is a well-established treatment modality for children with severe aplastic anemia (SAA). Treatment failures are rare and mostly caused by graft rejection. Increasing mixed chimerism represents a stage at the very beginning of graft rejection, where immunological intervention might be an effective prophylactic approach. To substantiate this, we: (1) monitored peripheral blood cells from children with SAA after allo-SCT and performed pre-emptive immunotherapy in patients with increasing MC. In all, 23/34 courses of 32 children with SAA after allo-SCT showed a complete chimerism (CC) throughout and 10/34 developed different types of mixed chimerism (MC). Altogether, 4/10 with MC spontaneously developed decreasing MC, 2/10 courses persisted with low proportions of autologous cells below 30% (stable-MC), 4/10 developed increasing MC and one patient showed an autologous recovery. All patients with CC, decreasing MC or stable MC remained in continuous complete remission (CCR). In all, 2/4 patients with increasing MC developed graft rejection. Based on these observations, 2/4 new patients with increasing MC received low-dose DLIs prophylactically, and remained in CCR without any GVHD. These results substantiate that low-dose DLI in children with SAA and increasing MC can prevent graft rejection with a calculable risk to induce severe GVHD.Bone Marrow Transplantation (2004) 33, 219-223. doi:10.1038/sj.bmt.1704337 Published online 1 December 2003 [ABSTRACT FROM AUTHOR]

Leukemia (2003) 17, 237–240. doi:10.1038/sj.leu.2402761 [ABSTRACT FROM AUTHOR]

We have retrospectively investigated the relationship between the level of minimal residual disease (MRD) detected in bone marrow taken prior to conditioning therapy and outcome following stem cell transplantation for high risk childhood ALL. Forty-one patients, in whom both a molecular marker of MRD and sufficient archival material was available, were included in the study. All were in remission at BMT: eight in CR1, 32 in CR2 and five in greater than CR2. MRD was measured by PCR amplification of antigen receptor gene rearrangements and clone-specific oligoprobing, the median sensitivity of detection being one leukaemic cell in 10000 normals. Results were classified as high-level positive (if a clonal band was evident after electrophoresis), low-level positive (if MRD was detected only after oligoprobing) and negative. MRD was detected at high levels in 17 patients, at low levels in 10 patients and 14 patients were MRD negative at the time of transplant. The 5year event-free survival for these groups was 23%, 48% and 78%, respectively (P = 0.022). Limited multivariate analysis confirmed the significance of MRD (P = 0.0095) vs CR status, donor type, sex, immunophenotype and acute GvHD. This study confirms the strong relationship between MRD level and outcome following allogeneic transplantation. In contrast to a previous study we observed that a minority of children with high-level pre-BMT MRD can enter long lasting remission. The possible role for acute GVHD coupled with a graft-versus-leukaemia effect in the clearance of high level MRD in patients with ALL is discussed. [ABSTRACT FROM AUTHOR]

Discusses the results of three major reports on the importance of minimal residual disease level prior to hematopoietic stem cell transplantation (HSCT) as a prognostic marker in pediatric acute lymphoblastic leukemia patients. Patient categories according to the minimal residual disease level prior to HSCT; Results of statistical analysis; Factors considered for the multivariate analysis.

Reports on the case of a male patient diagnosed with lymphoid blast crisis chronic myeloid leukemia. Results of the treatment with imatinib mesylate; Analysis of cerebrospinal fluid; Efficacy of imatinib mesylate treatment to the patient.

Bone Marrow Transplantation (2002) 30, S28–S31. doi:10.1038/sj.bmt.1703751 [ABSTRACT FROM AUTHOR]