Publications by authors named "Hermann Kreyenberg"

34 Publications

Blinatumomab in pediatric patients with relapsed/refractory B-cell precursor acute lymphoblastic leukemia.

Eur J Haematol 2021 Apr 11;106(4):473-483. Epub 2021 Jan 11.

Department I - General Pediatrics, Hematology/Oncology, Children's Hospital, University Hospital Tübingen, Tübingen, Germany.

Objective: Pediatric patients with relapsed or refractory acute lymphoblastic leukemia have a poor prognosis. We here assess the response rates, adverse events, and long-term follow-up of pediatric patients with relapsed/refractory acute lymphoblastic leukemia receiving blinatumomab.

Methods: Retrospective analysis of a single-center experience with blinatumomab in 38 patients over a period of 10 years.

Results: The median age at onset of therapy was 10 years (1-21 years). Seventy-one percent of patients had undergone at least one hematopoietic stem cell transplantation (HSCT) prior to treatment with blinatumomab. We observed a response to blinatumomab in 13/38 patients (34%). The predominant side effect was febrile reactions, nearly half of the patients developed a cytokine release syndrome. Eight events of neurotoxicity were registered over the 78 cycles (15%). To date, nine patients (24%) are alive and in complete molecular remission. All survivors underwent haploidentical HSCT after treatment with blinatumomab.

Conclusions: Despite heavy pretreatment of most of our patients, severe adverse events were rare and response rates encouraging. Blinatumomab is a valuable bridging salvage therapy for relapsed or refractory patients to a second or even third HSCT.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/ejh.13569DOI Listing
April 2021

ERBB2-CAR-Engineered Cytokine-Induced Killer Cells Exhibit Both CAR-Mediated and Innate Immunity Against High-Risk Rhabdomyosarcoma.

Front Immunol 2020 19;11:581468. Epub 2020 Oct 19.

Division for Stem Cell Transplantation, Immunology, and Intensive Care Medicine, Department for Children and Adolescents, University Hospital Frankfurt, Goethe University Frankfurt, Frankfurt, Germany.

High-risk rhabdomyosarcoma (RMS) occurring in childhood to young adulthood is associated with a poor prognosis; especially children above the age of 10 with advanced stage alveolar RMS still succumb to the disease within a median of 2 years. The advent of chimeric antigen receptor (CAR)-engineered T cells marked significant progress in the treatment of refractory B cell malignancies, but experience for solid tumors has proven challenging. We speculate that this is at least in part due to the poor quality of the patient's own T cells and therefore propose using CAR-modified cytokine-induced killer (CIK) cells as effector cells. CIK cells are a heterogeneous population of polyclonal T cells that acquire phenotypic and cytotoxic properties of natural killer (NK) cells through the cultivation process, becoming so-called T-NK cells. CIK cells can be genetically modified to express CARs. They are minimally alloreactive and can therefore be acquired from haploidentical first-degree relatives. Here, we explored the potential of ERBB2-CAR-modified random-donor CIK cells as a treatment for RMS in xenotolerant mice bearing disseminated high-risk RMS tumors. In otherwise untreated mice, RMS tumors engrafted 13-35 days after intravenous tumor cell injection, as shown by bioluminescence imaging, immunohistochemistry, and polymerase chain reaction for human gDNA, and mice died shortly thereafter (median/range: 62/56-66 days, = 5). Wild-type (WT) CIK cells given at an early stage delayed and eliminated RMS engraftment in 4 of 6 (67%) mice, while ERBB2-CAR CIK cells inhibited initial tumor load in 8 of 8 (100%) mice. WT CIK cells were detectable but not as active as CAR CIK cells at distant tumor sites. CIK cell therapies during advanced RMS delayed but did not inhibit tumor progression compared to untreated controls. ERBB2-CAR CIK cell therapy also supported innate immunity as evidenced by selective accumulation of NK and T-NK cell subpopulations in disseminated RMS tumors, which was not observed for WT CIK cells. Our data underscore the power of heterogenous immune cell populations (T, NK, and T-NK cells) to control solid tumors, which can be further enhanced with CARs, suggesting ERBB2-CAR CIK cells as a potential treatment for high-risk RMS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2020.581468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641627PMC
October 2020

More precisely defining risk peri-HCT in pediatric ALL: pre- vs post-MRD measures, serial positivity, and risk modeling.

Blood Adv 2019 11;3(21):3393-3405

Children's Center for Cancer and Blood Diseases, Children's Hospital Los Angeles, Los Angeles, CA.

Detection of minimal residual disease (MRD) pre- and post-hematopoietic cell transplantation (HCT) for pediatric acute lymphoblastic leukemia (ALL) has been associated with relapse and poor survival. Published studies have had insufficient numbers to: (1) compare the prognostic value of pre-HCT and post-HCT MRD; (2) determine clinical factors post-HCT associated with better outcomes in MRD+ patients; and (3) use MRD and other clinical factors to develop and validate a prognostic model for relapse in pediatric patients with ALL who undergo allogeneic HCT. To address these issues, we assembled an international database including sibling (n = 191), unrelated (n = 259), mismatched (n = 56), and cord blood (n = 110) grafts given after myeloablative conditioning. Although high and very high MRD pre-HCT were significant predictors in univariate analysis, with bivariate analysis using MRD pre-HCT and post-HCT, MRD pre-HCT at any level was less predictive than even low-level MRD post-HCT. Patients with MRD pre-HCT must become MRD low/negative at 1 to 2 months and negative within 3 to 6 months after HCT for successful therapy. Factors associated with improved outcome of patients with detectable MRD post-HCT included acute graft-versus-host disease. We derived a risk score with an MRD cohort from Europe, North America, and Australia using negative predictive characteristics (late disease status, non-total body irradiation regimen, and MRD [high, very high]) defining good, intermediate, and poor risk groups with 2-year cumulative incidences of relapse of 21%, 38%, and 47%, respectively. We validated the score in a second, more contemporaneous cohort and noted 2-year cumulative incidences of relapse of 13%, 26%, and 47% (P < .001) for the defined risk groups.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/bloodadvances.2019000449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6855112PMC
November 2019

ADCC can improve graft vs leukemia effect after T- and B-cell depleted haploidentical stem cell transplantation in pediatric B-lineage ALL.

Bone Marrow Transplant 2019 08;54(Suppl 2):689-693

Department of Pediatric Hematology and Oncology, University Children's Hospital, University of Tuebingen, Tuebingen, Germany.

Posttransplant relapsed B-cell precursor ALL can be cured by 2nd hematopoietic stem cell transplantation (HSCT) in 20% of patients. The major cause of death after second HSCT is leukemic relapse. One reliable predictor for survival after 2nd-HSCT are posttransplant MRD levels. Patients with detectable or increase of MRD are likely to relapse. Patients in complete molecular remission show the best leukemia-free survival and lowest cumulative incidence (CI) of relapse. As patients who undergo second or subsequent HSCT are high-risk patients, we evaluated the prophylactic use of the chimeric Fc-optimized CD19-4G7SDIE-mAb. Posttransplant relapsed CD19 BCP-ALL patients, who underwent a second or subsequent haplo-HSCT from a T- and B-cell depleted graft received posttransplant prophylactic CD19-4G7SDIE-mAb treatment on compassionate use in complete molecular remission, to increase the antileukemic activity of the new reconstituting immune system by recruiting Fc-expressing effector cells. NK cells recovered early and robust. The 3 year overall survival in 15 evaluable patients was 56%, the 3 year event-free survival was 55% and the CI of relapse 38%. Compared to a historical control group, the CI of relapse was markedly lower and consecutively the EFS higher. Posttransplant-targeted therapy may overcome the need for unspecific GvL effect of undesired GvHD, that can cause severe morbidity and mortality. Due to a low adverse event profile the CD19-4G7SDIE-mAb may be suitable for broad administration to consolidate posttransplant MRD negativity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41409-019-0606-1DOI Listing
August 2019

AlloHSCT in paediatric ALL and AML in complete remission: improvement over time impacted by accreditation?

Bone Marrow Transplant 2019 05 26;54(5):737-745. Epub 2018 Sep 26.

Division for Stem Cell Transplantation and Immunology, Department for Children and Adolescents, University Hospital, Goethe University, Frankfurt/Main, Germany.

Allogeneic hematopoietic stem cell transplantation (alloHSCT) has become a well-established treatment option for many patients suffering from malignant and non-malignant diseases. In the past decade, high-resolution HLA-typing, remission surveillance, pre-emptive immune intervention, and standardisation in supportive care measures have substantially improved transplant outcomes. This retrospective study evaluated transplant procedures in 162 paediatric patients with acute lymphoblastic leukaemia (n = 124) or acute myeloid leukaemia (n = 38) who received their first alloHSCT in our institution over an 11-year period. We observed a significant reduction in risk of non-relapse mortality (NRM) over time (HR = 0.34, 95% CI 0.12-0.98; P = 0.05), the 4-year NRM estimate decreased from 20% in 2005-2008 to 7% in 2012-2016 (P = 0.02) and an increase in survival after relapse. There was no significant difference in patients who received a graft from a sibling, haplo, or an unrelated donor with regard to their overall survival (P = 0.45), event-free survival (P = 0.61), and non-relapse mortality (P = 0.19). Our data suggest that a specific transplant infrastructure with a highly experienced team in an accredited transplant centre likely contributes to better transplant outcomes for acute leukaemia patients in complete remission regardless of donor type.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41409-018-0341-zDOI Listing
May 2019

Pre-Emptive Immunotherapy for Clearance of Molecular Disease in Childhood Acute Lymphoblastic Leukemia after Transplantation.

Biol Blood Marrow Transplant 2017 01 11;23(1):87-95. Epub 2016 Oct 11.

University Hospital Frankfurt/Main, Department for Children and Adolescents, Division for Stem Cell Transplantation and Immunology, Goethe-University Frankfurt/Main, Germany.

Monitoring of minimal residual disease (MRD) or chimerism may help guide pre-emptive immunotherapy (IT) with a view to preventing relapse in childhood acute lymphoblastic leukemia (ALL) after transplantation. Patients with ALL who consecutively underwent transplantation in Frankfurt/Main, Germany between January 1, 2005 and July 1, 2014 were included in this retrospective study. Chimerism monitoring was performed in all, and MRD assessment was performed in 58 of 89 patients. IT was guided in 19 of 24 patients with mixed chimerism (MC) and MRD and by MRD only in another 4 patients with complete chimerism (CC). The 3-year probabilities of event-free survival (EFS) were .69 ± .06 for the cohort without IT and .69 ± .10 for IT patients. Incidences of relapse (CIR) and treatment-related mortality (CITRM) were equally distributed between both cohorts (without IT: 3-year CIR, .21 ± .05, 3-year CITRM, .10 ± .04; IT patients: 3-year CIR, .18 ± .09, 3-year CITRM .13 ± .07). Accordingly, 3-year EFS and 3-year CIR were similar in CC and MC patients with IT, whereas MC patients without IT experienced relapse. IT was neither associated with an enhanced immune recovery nor an increased risk for acute graft-versus-host disease. Relapse prevention by IT in patients at risk may lead to the same favorable outcome as found in CC and MRD-negative-patients. This underlines the importance of excellent MRD and chimerism monitoring after transplantation as the basis for IT to improve survival in childhood ALL.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbmt.2016.10.006DOI Listing
January 2017

Mesenchymal stromal cells from pooled mononuclear cells of multiple bone marrow donors as rescue therapy in pediatric severe steroid-refractory graft-versus-host disease: a multicenter survey.

Haematologica 2016 08 12;101(8):985-94. Epub 2016 May 12.

University Hospital Frankfurt, Department for Children and Adolescents, Division for Stem Cell Transplantation and Immunology, Frankfurt am Main, Germany

To circumvent donor-to-donor heterogeneity which may lead to inconsistent results after treatment of acute graft-versus-host disease with mesenchymal stromal cells generated from single donors we developed a novel approach by generating these cells from pooled bone marrow mononuclear cells of 8 healthy "3(rd)-party" donors. Generated cells were frozen in 209 vials and designated as mesenchymal stromal cell bank. These vials served as a source for generation of clinical grade mesenchymal stromal cell end-products, which exhibited typical mesenchymal stromal cell phenotype, trilineage differentiation potential and at later passages expressed replicative senescence-related markers (p21 and p16). Genetic analysis demonstrated their genomic stability (normal karyotype and a diploid pattern). Importantly, clinical end-products exerted a significantly higher allosuppressive potential than the mean allosuppressive potential of mesenchymal stromal cells generated from the same donors individually. Administration of 81 mesenchymal stromal cell end-products to 26 patients with severe steroid-resistant acute graft-versus-host disease in 7 stem cell transplant centers who were refractory to many lines of treatment, induced a 77% overall response at the primary end point (day 28). Remarkably, although the cohort of patients was highly challenging (96% grade III/IV and only 4% grade II graft-versus-host disease), after treatment with mesenchymal stromal cell end-products the overall survival rate at two years follow up was 71±11% for the entire patient cohort, compared to 51.4±9.0% in graft-versus-host disease clinical studies, in which mesenchymal stromal cells were derived from single donors. Mesenchymal stromal cell end-products may, therefore, provide a novel therapeutic tool for the effective treatment of severe acute graft-versus-host disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2015.140368DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967578PMC
August 2016

Absence of Maternal Microchimerism in Regional Lymph Nodes of Children With Biliary Atresia.

J Pediatr Gastroenterol Nutr 2016 06;62(6):804-7

*Pediatric Surgery, Klinikum Brandenburg, Medizinische Hochschule Theodor Fontane, Brandenburg †Department of Human Genetics, Hannover Medical School (MHH), Hannover ‡Division for Stem Cell Transplantation and Immunology, Department for Children and Adolescents, University Hospital, Goethe University, Frankfurt/Main, Germany.

Objective: Maternal lymphocytes have been cited as a potential cause of infantile biliary atresia (BA). When hepatoportoenterostomy is performed, locoregional lymphadenopathy is frequently encountered.

Methods: We screened enlarged nodes from 6 consecutive nonsyndromatic BA patients (age: 68 days ± 18.9 days) for maternal elements using DNA fingerprinting with short tandem repeat analysis and quantitative real-time polymerase chain reaction for allelic (single nucleotide) sequence polymorphisms.

Results: Although being partly positive in infants' peripheral blood, no maternal microchimerism could be demonstrated in any of the lymph nodes.

Conclusion: This result challenges the hypothesis that maternal cells play a role in hilar lymphadenopathy of children with BA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/MPG.0000000000001093DOI Listing
June 2016

Detection of relapse after hematopoietic stem cell transplantation in childhood by monitoring of WT1 expression and chimerism.

J Cancer Res Clin Oncol 2015 Jul 24;141(7):1283-90. Epub 2015 Jan 24.

Department of Pediatrics, Jena University Hospital, Kochstr. 2, 07745, Jena, Germany.

Purpose: The measurement of minimal residual disease after hematopoietic stem cell transplantation is crucial for the prevention of hematological relapses. But many methods are limited to certain groups of patients. We present a comparison of two markers which are universally applicable: WT1 expression and chimerism status.

Methods: We analyzed 1,848 peripheral blood and bone marrow samples of 148 children, adolescents and young adults with malignant hematological diseases. Median follow-up time was 739 days after transplantation.

Results: All patients suffering from hematological relapse showed high WT1 levels. Approximately half (51%) of the 37 relapses could have been detected early through an increase in WT1 expression. WT1 kinetics revealed to be more specific than single elevated WT1 levels (p < 0.05) and chimerism analysis (p < 0.05). Combined with chimerism analysis, 74% of relapses were detectable in advance. Elevated WT1 expression levels after transplantation reached the highest sensitivity (64.9%) as a single marker, although differences were not significant. When the dynamics of both markers as well as any elevated WT1 expression were taken into account, a sensitivity of 81.5% and a specificity of 83.7% were obtained.

Conclusions: Hence, we conclude that WT1 is a useful marker for monitoring of minimal residual disease after transplantation, if specific targets are not available. WT1 expression and chimerism status should be mutually evaluated to decide about immunotherapeutic interventions aimed at preventing morphological relapse.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00432-015-1919-0DOI Listing
July 2015

Monitoring of minimal residual disease after allogeneic stem-cell transplantation in relapsed childhood acute lymphoblastic leukemia allows for the identification of impending relapse: results of the ALL-BFM-SCT 2003 trial.

J Clin Oncol 2015 Apr 20;33(11):1275-84. Epub 2015 Jan 20.

Peter Bader, Hermann Kreyenberg, Emilia Salzmann-Manrique, Andre M. Willasch, and Thomas E. Klingebiel, University Hospital for Children and Adolescents, Frankfurt/Main; Arend von Stackelberg and Cornelia Eckert, Children's Hospital Charité, Berlin, Berlin; Roland Meisel, Heinrich-Heine-University, Düsseldorf; Daniel Stachel, University Hospital Erlangen, Erlangen; Martin Schrappe, Julia Alten, Andre Schrauder, Christian-Albrechts-University and Medical Center Schleswig-Holstein, Kiel; Ansgar Schulz, University Medical Center, Ulm; Peter Lang, University Children's Hospital, Tuebingen; Ingo Müller, University Medical Center Hamburg-Eppendorf, Hamburg; Michael H. Albert, University Children's Hospital, Munich, Germany; and Ulrike Poetschger and Christina Peters, St Anna Children's Hospital, Vienna, Austria.

Purpose: To elucidate the impact of minimal residual disease (MRD) after allogeneic transplantation, the Acute Lymphoblastic Leukemia Berlin-Frankfurt-Münster Stem Cell Transplantation Group (ALL-BFM-SCT) conducted a prospective clinical trial.

Patients And Methods: In the ALL-BFM-SCT 2003 trial, MRD was assessed in the bone marrow at days +30, +60, +90, +180, and +365 after transplantation in 113 patients with relapsed disease. Standardized quantification of MRD was performed according to the guidelines of the Euro-MRD Group.

Results: All patients showed a 3-year probability of event-free survival (pEFS) of 55%. The cumulative incidence rates of relapse and treatment-related mortality were 32% and 12%, respectively. The pEFS was 60% for patients who received their transplantations in second complete remission, 50% for patients in ≥ third complete remission, and 0% for patients not in remission (P = .015). At all time points, the level of MRD was inversely correlated with event-free survival (EFS; P < .004) and positively correlated with the cumulative incidence of relapse (P < .01). A multivariable Cox model was fitted for each time point, which showed that MRD ≥ 10(-4) leukemic cells was consistently correlated with inferior EFS (P < .003). The accuracy of MRD measurements in predicting relapse was investigated with time-dependent receiver operating curves at days +30, +60, +90, and +180. From day +60 onward, the discriminatory power of MRD detection to predict the probability of relapse after 1, 3, 6, and 9 months was more than 96%, more than 87%, more than 71%, and more than 61%, respectively.

Conclusion: MRD after transplantation was a reliable marker for predicting impending relapses and could thus serve as the basis for pre-emptive therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1200/JCO.2014.58.4631DOI Listing
April 2015

SMAC Mimetic BV6 Enables Sensitization of Resistant Tumor Cells but also Affects Cytokine-Induced Killer (CIK) Cells: A Potential Challenge for Combination Therapy.

Front Pediatr 2014 18;2:75. Epub 2014 Jul 18.

Division for Stem Cell Transplantation and Immunology, Department for Children and Adolescents Medicine, University Hospital Frankfurt, Goethe University Frankfurt am Main , Frankfurt , Germany.

Allogeneic hematopoietic stem cell transplantation (HSCT) is an established treatment option for high-risk hematological malignancies, and may also be offered to patients with solid malignancies refractory to conventional therapies. In case of patients' relapse, refractory tumor cells may then be targeted by cellular therapy-based combination strategies. Here, we investigated the potential of small molecule IAP (SMAC mimetic) BV6 in increasing cytokine-induced killer (CIK) cell-mediated cytotoxicity against different tumor targets. Four-hour pre-incubation with 2.5 μMol BV6 moderately enhanced CIK cell-mediated lysis of hematological (H9, THP-1, and Tanoue) and solid malignancies (RH1, RH30, and TE671). However, BV6 also increased apoptosis of non-malignant cells like peripheral blood mononuclear cells and most notably had an inhibitory effect on immune cells potentially limiting their cytotoxic potential. Hence, cytotoxicity increased in a dose-dependent manner when BV6 was removed before CIK cells were added to tumor targets. However, cytotoxic potential was not further increasable by extending BV6 pre-incubation period of target cells from 4 to 12 h. Molecular studies revealed that BV6 sensitization of target cells involved activation of caspases. Here, we provide evidence that SMAC mimetic may sensitize targets cells for CIK cell-induced cell death. However, BV6 also increased apoptosis of non-malignant cells like CIK cells and peripheral mononuclear cells. These findings may therefore be important for cell- and small molecule IAP-based combination therapies of resistant cancers after allogeneic HSCT.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fped.2014.00075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4103003PMC
August 2014

Monitoring of hematopoietic chimerism after transplantation for pediatric myelodysplastic syndrome: real-time or conventional short tandem repeat PCR in peripheral blood or bone marrow?

Biol Blood Marrow Transplant 2014 Dec 31;20(12):1918-25. Epub 2014 Jul 31.

Division for Stem Cell Transplantation and Immunology, Department for Children and Adolescents, University Hospital, Goethe University, Frankfurt/Main, Germany.

Quantitative real-time PCR (qPCR) has been proposed as a highly sensitive method for monitoring hematopoietic chimerism and may serve as a surrogate marker for the detection of minimal residual disease minimal residual disease in myelodysplastic syndrome (MDS), until specific methods of detection become available. Because a systematic comparison of the clinical utility of qPCR with the gold standard short tandem repeat (STR)-PCR has not been reported, we retrospectively measured chimerism by qPCR in 54 children transplanted for MDS in a previous study. Results obtained by STR-PCR in the initial study served as comparison. Because the detection limit of qPCR was sufficiently low to detect an autologous background, we defined the sample as mixed chimera if the proportion of recipient-derived cells exceeded .5%. The true positive rates were 100% versus 80% (qPCR versus STR-PCR, not significant), and mixed chimerism in most cases was detected earlier by qPCR than by STR-PCR (median, 31 days) when chimerism was quantified concurrently in peripheral blood and bone marrow. Both methods revealed a substantial rate of false positives (22.7% versus 13.6%, not significant), indicating the importance of serial testing of chimerism to monitor its progression. Finally, we propose criteria for monitoring chimerism in pediatric MDS with regard to the subtypes, specimens, PCR method, and timing of sampling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbmt.2014.07.030DOI Listing
December 2014

Pediatric posttransplant relapsed/refractory B-precursor acute lymphoblastic leukemia shows durable remission by therapy with the T-cell engaging bispecific antibody blinatumomab.

Haematologica 2014 Jul 11;99(7):1212-9. Epub 2014 Apr 11.

University Children's Hospital, Department of Pediatric Hematology and Oncology, University of Tuebingen.

We report on posttransplant relapsed pediatric patients with B-precursor acute lymphoblastic leukemia with no further standard of care therapy who were treated with the T-cell engaging CD19/CD3-bispecific single-chain antibody construct blinatumomab on a compassionate use basis. Blast load was assessed prior to, during and after blinatumomab cycle using flow cytometry to detect minimal residual disease, quantitative polymerase chain reaction for rearrangements of the immunoglobulin or T-cell receptor genes, and bcr/abl mutation detection in one patient with Philadelphia chromosome-positive acute lymphoblastic leukemia. Blinatumomab was administered as a 4-week continuous intravenous infusion at a dosage of 5 or 15 μg/m(2)/day. Nine patients received a total of 18 cycles. Four patients achieved complete remission after the first cycle of treatment; 2 patients showed a complete remission from the second cycle after previous reduction of blast load by chemotherapy. Three patients did not respond, of whom one patient proceeded to a second cycle without additional chemotherapy and again did not respond. Four patients were successfully retransplanted in molecular remission from haploidentical donors. After a median follow up of 398 days, the probability of hematologic event-free survival is 30%. Major toxicities were grade 3 seizures in one patient and grade 3 cytokine release syndrome in 2 patients. Blinatumomab can induce molecular remission in pediatric patients with posttransplant relapsed B-precursor acute lymphoblastic leukemia and facilitate subsequent allogeneic hematopoietic stem cell transplantation from haploidentical donor with subsequent long-term leukemia-free survival.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2013.100073DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4077083PMC
July 2014

Immunomagnetic selection or irradiation eliminates alloreactive cells but also reduces anti-tumor potential of cytokine-induced killer cells: implications for unmanipulated cytokine-induced killer cell infusion.

Cytotherapy 2014 Jun 28;16(6):835-44. Epub 2014 Feb 28.

University Hospital Frankfurt/Main, Department for Children and Adolescents Medicine, Division of Stem Cell Transplantation and Immunology, Goethe-University Frankfurt/Main, Frankfurt/Main, Germany.

Background Aims: Cytokine-induced killer (CIK) cells may offer a novel therapeutic approach for patients with malignancies relapsing after allogeneic stem cell transplantation. Although CIK cells display negligible alloreactivity and cause minimal graft versus-host-disease (GVHD), high CIK cell doses required during relapse may pose a risk for severe GVHD, specifically in the mismatched or haploidentical transplantation setting. Manipulation of CIK cells may reduce risk for GVHD without affecting the anti-tumor potential.

Methods: In this pre-clinical study, we provide a detailed functional comparison of conventional and irradiated, CD56-enriched or T-cell receptor α/β-depleted CIK cells.

Results: In vitro analysis showed retained anti-leukemic and anti-tumor potential after CIK cell manipulation. Even being sequentially infused into immunodeficient mice grafted with malignant cells, cytotoxic effects were fewest after irradiation but were improved by CD56 enrichment and were best with conventional CIK cells. Hence, considering the proliferative capacity of inoculated malignancies and effector cells, a single dose of conventional CIK cells resulted in prolonged disease-free survival and elimination of rhabdomyosarcoma cells, whereas sequential infusions were needed to achieve comparable results in leukemia-bearing mice. However, this mouse model has limitations: highly effective conventional CIK cells demonstrated both limited xenogenic GVHD and low alloreactive potential in vitro.

Conclusions: Our study revealed that conventional CIK cells demonstrate no significant alloreactive potential but provide the strongest anti-tumor efficacy compared with manipulated CIK cells. Conventional CIK cells may therefore be tested in high numbers and short-term intervals in patients with impending relapse even after mismatched transplantation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jcyt.2014.01.003DOI Listing
June 2014

Cytotoxic Capacity of IL-15-Stimulated Cytokine-Induced Killer Cells Against Human Acute Myeloid Leukemia and Rhabdomyosarcoma in Humanized Preclinical Mouse Models.

Front Oncol 2012 9;2:32. Epub 2012 Apr 9.

Department of Pediatric Hematology, Oncology and Hemostaseology, University Children's Hospital of Frankfurt/Main, Goethe-University Frankfurt/Main Frankfurt/Main, Germany.

Allogeneic stem cell transplantation (allo-SCT) has become an important treatment modality for patients with high-risk acute myeloid leukemia (AML) and is also under investigation for soft tissue sarcomas. The therapeutic success is still limited by minimal residual disease (MRD) status ultimately leading to patients' relapse. Adoptive donor lymphocyte infusions based on MRD status using IL-15-expanded cytokine-induced killer (CIK) cells may prevent relapse without causing graft-versus-host-disease (GvHD). To generate preclinical data we developed mouse models to study anti-leukemic- and anti-tumor-potential of CIK cells in vivo. Immunodeficient mice (NOD/SCID/IL-2Rγc(-), NSG) were injected intravenously with human leukemic cell lines THP-1, SH-2 and with human rhabdomyosarcoma (RMS) cell lines RH41 and RH30 at minimal doses required for leukemia or tumor engraftment. Mice transplanted with THP-1 or RH41 cells were randomly assigned for analysis of CIK cell treatment. Organs of mice were analyzed by flow cytometry as well as quantitative polymerase chain reaction for engraftment of malignant cells and CIK cells. Potential of CIK cells to induce GvHD was determined by histological analysis. Tissues of the highest degree of THP-1 cell expansion included bone marrow followed by liver, lung, spleen, peripheral blood (PB), and brain. RH30 and RH41 engraftment mainly took place in liver and lung, but was also detectable in spleen and PB. In spite of delayed CIK cell expansion compared with malignant cells, CIK cells injected at equal amounts were sufficient for significant reduction of RH41 cells, whereas against fast-expanding THP-1 cells 250 times more CIK than THP-1 cells were needed to achieve comparable results. Our preclinical in vivo mouse models showed a reliable 100% engraftment of malignant cells which is essential for analysis of anti-cancer therapy. Furthermore our data demonstrated that IL-15-activated CIK cells have potent cytotoxic capacity against AML and RMS cells without causing GvHD.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fonc.2012.00032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3356002PMC
August 2012

IL-2 stimulated but not unstimulated NK cells induce selective disappearance of peripheral blood cells: concomitant results to a phase I/II study.

PLoS One 2011 9;6(11):e27351. Epub 2011 Nov 9.

Pediatric Hematology and Oncology, Laboratory for Stem Cell Transplantation and Immunotherapy, Johann Wolfgang Goethe-University Hospital, Frankfurt, Germany.

In an ongoing clinical phase I/II study, 16 pediatric patients suffering from high risk leukemia/tumors received highly purified donor natural killer (NK) cell immunotherapy (NK-DLI) at day (+3) +40 and +100 post haploidentical stem cell transplantation. However, literature about the influence of NK-DLI on recipient's immune system is scarce. Here we present concomitant results of a noninvasive in vivo monitoring approach of recipient's peripheral blood (PB) cells after transfer of either unstimulated (NK-DLI(unstim)) or IL-2 (1000 U/ml, 9-14 days) activated NK cells (NK-DLI(IL-2 stim)) along with their ex vivo secreted cytokine/chemokines. We performed phenotypical and functional characterizations of the NK-DLIs, detailed flow cytometric analyses of various PB cells and comprehensive cytokine/chemokine arrays before and after NK-DLI. Patients of both groups were comparable with regard to remission status, immune reconstitution, donor chimerism, KIR mismatching, stem cell and NK-DLI dose. Only after NK-DLI(IL-2 stim) was a rapid, almost complete loss of CD56(bright)CD16(dim/-) immune regulatory and CD56(dim)CD16(+) cytotoxic NK cells, monocytes, dendritic cells and eosinophils from PB circulation seen 10 min after infusion, while neutrophils significantly increased. The reduction of NK cells was due to both, a decrease in patients' own CD69(-) NCR(low)CD62L(+) NK cells as well as to a diminishing of the transferred cells from the NK-DLI(IL-2 stim) with the CD56(bright)CD16(+/-)CD69(+)NCR(high)CD62L(-) phenotype. All cell counts recovered within the next 24 h. Transfer of NK-DLI(IL-2 stim) translated into significantly increased levels of various cytokines/chemokines (i.e. IFN-γ, IL-6, MIP-1β) in patients' PB. Those remained stable for at least 1 h, presumably leading to endothelial activation, leukocyte adhesion and/or extravasation. In contrast, NK-DLI(unstim) did not cause any of the observed effects. In conclusion, we assume that the adoptive transfer of NK-DLI(IL-2 stim) under the influence of ex vivo and in vivo secreted cytokines/chemokines may promote NK cell trafficking and therefore might enhance efficacy of immunotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0027351PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3212563PMC
April 2012

The cytotoxic potential of interleukin-15-stimulated cytokine-induced killer cells against leukemia cells.

Cytotherapy 2012 Jan 6;14(1):91-103. Epub 2011 Oct 6.

University Children's Hospital of Frankfurt/Main and Department of Pediatric Hematology, Oncology and Hemostaseology, Goethe-University Frankfurt/Main, Frankfurt/Main, Germany.

Background Aims: Cytokine-induced killer (CIK) cells may serve as an alternative approach to adoptive donor lymphocyte infusions (DLI) for patients with acute leukemia relapsing after haplo-identical hematopoietic stem cell transplantation (HSCT). We investigated the feasibility of enhancing CIK cell-mediated cytotoxicity by interleukin (IL)-15 against acute myeloid and lymphoblastic leukemia/lymphoma cells.

Methods: CIK cells were activated using IL-2 (CIK(IL-2)) or IL-15 (CIK(IL-15)) and phenotypically analyzed by fluorescence-activated cell sorting (FACS). Cytotoxic potential was measured by europium release assay.

Results: CIK(IL-2) cells showed potent cytotoxicity against the T-lymphoma cell line H9, T-cell acute lymphoblastic leukemia (T-ALL) cell line MOLT-4 and subtype M4 acute myeloid leukemia (AML) cell line THP-1, but low cytotoxicity against the precursor B (pB)-cell ALL cell line Tanoue. IL-15 stimulation resulted in a significant enhancement of CIK cell-mediated cytotoxicity against acute lymphoblastic leukemia/lymphoma cell lines as well as against primary acute myeloid and defined lymphoblastic leukemia cells. However, the alloreactive potential of CIK(IL-15) cells remained low. Further analysis of CIK(IL-15) cells demonstrated that the NKG2D receptor is apparently involved in the recognition of target cells whereas killer-cell immunoglobulin-like receptor (KIR)-HLA mismatches contributed to a lesser extent to the CIK(IL-15) cell-mediated cytotoxicity. In this context, CD3 (+) CD8 (+) CD25 (+) CD56(-) CIK(IL-15) cell subpopulations were more effective in the lysis of AML cells, in contrast with CD56 (+) CIK(IL-15) cells, which showed the highest cytotoxic potential against ALL cells.

Conclusions: This study provides the first evidence that CIK(IL-15) cells may offer a therapeutic option for patients with refractory or relapsed leukemia following haplo-identical HSCT.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3109/14653249.2011.613931DOI Listing
January 2012

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

Blood 2011 Nov 26;118(20):5681-8. Epub 2011 Sep 26.

Department of Pediatric Hematology, Oncology and Hemostaseology, Goethe-University, University Children's Hospital, Theodor-Stern-Kai 7, Frankfurt/Main, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2011-04-348805DOI Listing
November 2011

Examining the origin of posttransplant lymphoproliferative disorder in a patient after a second allogeneic hematopoeitic stem cell transplantation for relapsed BCR-ABL positive acute lymphoblastic leukemia.

J Pediatr Hematol Oncol 2011 Jan;33(1):50-4

Department of Pediatric Hematology and Oncology, Justus-Liebig University, Giessen, Germany.

Background: Posttransplant lymphoproliferative disease (PTLD) is a serious complication because of the reactivation of the Epstein-Barr virus (EBV). Although after solid organ transplantation (SOT) EBV is typically of recipient origin, after hematopoietic stem cell transplantation (HSCT) donor-derived B cells are usually the source of the EBV-induced lymphoproliferation.

Observation: We report a unique presentation of an EBV-associated PTLD in a 13-year-old boy who underwent 2 subsequent HSCTs from 2 different-sex donors for BCR-ABL-positive acute lymphoblastic leukemia (ALL) and relapses of leukemia, respectively. The PTLD tissue was analyzed for the origin of both B cells and T cells of the lesion. Chimerism analysis done with short tandem repeat systems (STR) showed no autologous signals; approximately 50% of the cells were from the first and second donors, respectively. XY-fluorescence in situ hybridization showed a B-cell PTLD originating from the first donor surrounded by T cells from the second donor.

Conclusion: This case shows that EBV-infected B cells from a first donor can be the source of a PTLD after a second myeloablative HSCT from a different donor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/MPH.0b013e3181ed336fDOI Listing
January 2011

Efficient lysis of rhabdomyosarcoma cells by cytokine-induced killer cells: implications for adoptive immunotherapy after allogeneic stem cell transplantation.

Haematologica 2010 Sep 7;95(9):1579-86. Epub 2010 Apr 7.

University Children's Hospital III, University Children's Hospital III, Department of Hematology/Oncology Department of Hematology/Oncology, Theodor-Stern-Kai 7.

Background: Rhabdomyosarcoma is the most common soft tissue sarcoma in childhood and has a poor prognosis. Here we assessed the capability of ex vivo expanded cytokine-induced killer cells to lyse both alveolar and embryonic rhabdomyosarcoma cell lines and investigated the mechanisms involved.

Design And Methods: Peripheral blood mononuclear cells from six healthy donors were used to generate and expand cytokine-induced killer cells. The phenotype and composition of these cells were determined by multiparameter flow cytometry, while their cytotoxic effect against rhabdomyosarcoma cells was evaluated by a europium release assay.

Results: Cytokine-induced killer cells efficiently lysed cells from both rhabdomyosarcoma cell lines. Antibody-mediated masking of either NKG2D molecule on cytokine-induced killer cells or its ligands on rhabdomyosarcoma cells (major histocompatibility antigen related chain A and B and UL16 binding protein 2) diminished this effect by 50%, suggesting a major role for the NKG2D molecule in rhabdomyosarcoma cell killing. No effect was observed after blocking CD11a, CD3 or TCRalphabeta molecules on cytokine-induced killer cells or CD1d on rhabdomyosar-coma cells. Remarkably, cytokine-induced killer cells used tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to activate caspase-3, as the main caspase responsible for the execution of apoptosis. Accordingly, blocking TRAIL receptors on embryonic rhabdomyosarcoma cell lines significantly reduced the anti-tumor effect of cytokine-induced killer cells. About 50% of T cells within the cytokine-induced killer population had an effector memory phenotype, 20% had a naïve phenotype and approximately 30% of the cells had a central memory phenotype. In addition, cytokine-induced killer cells expressed low levels of activation-induced markers CD69 and CD137 and demonstrated a low alloreactive potential.

Conclusions: Our data suggest that cytokine-induced killer cells may be used as a novel adoptive immunotherapy for the treatment of patients with rhabdomyosarcoma after allogeneic stem cell transplantation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2009.019885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2930961PMC
September 2010

CD271 antigen defines a subset of multipotent stromal cells with immunosuppressive and lymphohematopoietic engraftment-promoting properties.

Haematologica 2010 Apr 23;95(4):651-9. Epub 2010 Feb 23.

Department of Hematology/Oncology, University Children's Hospital III, 60590 Frankfurt am Main, Germany.

Unlabelled: Background In vitro proliferative and differentiation potential of mesenchymal stromal cells generated from CD271(+) bone marrow mononuclear cells (CD271-mesenchymal stromal cells) has been demonstrated in several earlier and recent reports. In the present study we focused, in addition to proliferative and differentiation potential, on in vitro and in vivo immunosuppressive and lymphohematopoietic engraftment-promoting potential of these mesenchymal stromal cells compared to bone marrow-derived mesenchymal stromal cells generated by plastic adherence (plastic adherence-mesenchymal stromal cells).

Design And Methods: We set up a series of experimental protocols in order to determine the phenotype of CD271-mesenchymal stromal cells, and their clonogenic, proliferative, differentiation and immunosuppressive potential. The potential of CD271-mesenchymal stromal cells to improve the engraftment of CD133(+) hematopoietic stem cells at co-transplantation was evaluated in immunodeficient NOD/SCID-IL2Rgamma(null) mice.

Results: In vitro studies demonstrated that CD271-mesenchymal stromal cells differentiate along adipogenic, osteogenic and chondrogenic lineages (trilineage potential), produce significantly higher levels of cytokines than plastic adherence-mesenchymal stromal cells, and significantly inhibit the proliferation of allogeneic T-lymphocytes in mixed lymphocyte reaction assays. Elevated levels of prostaglandin E(2), but not nitric monoxide, mediated the majority of this immunosuppressive effect. In vivo studies showed that CD271-mesenchymal stromal cells promoted significantly greater lymphoid engraftment than did plastic adherence-mesenchymal stromal cells when co-transplanted with CD133(+) hematopoietic stem cells at a ratio of 8:1 in immunodeficient NOD/SCID-IL2Rgamma(null) mice. They induced a 10.4-fold increase in the number of T cells, a 2.5-fold increase in the number of NK cells, and a 3.6-fold increase in the number of B cells, indicating a major qualitative difference between these two mesenchymal stromal cell populations. Conclusions Our results indicate that CD271 antigen provides a versatile marker for prospective isolation and expansion of multipotent mesenchymal stromal cells with immunosuppressive and lymphohematopoietic engraftment-promoting properties. The co-transplantation of such cells together with hematopoietic stem cells in patients with hematologic malignancies may prove valuable in the prevention of impaired/delayed T-cell recovery and graft-versus-host disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2009.015065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2857196PMC
April 2010

Prognostic value of minimal residual disease quantification before allogeneic stem-cell transplantation in relapsed childhood acute lymphoblastic leukemia: the ALL-REZ BFM Study Group.

J Clin Oncol 2009 Jan 8;27(3):377-84. Epub 2008 Dec 8.

Children's Hospital of the JW Goethe University, Frankfurt, Main, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1200/JCO.2008.17.6065DOI Listing
January 2009

Sequence polymorphism systems for quantitative real-time polymerase chain reaction to characterize hematopoietic chimerism-high informativity and sensitivity as well as excellent reproducibility and precision of measurement.

Lab Hematol 2007 ;13(3):73-84

J. W. Goethe University Children's Hospital of Frankfurt, Frankfurt, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1532/LH96.07004DOI Listing
January 2008

Pitfalls in detection of contaminating neuroblastoma cells by tyrosine hydroxylase RT-PCR due to catecholamine-producing hematopoietic cells.

Anticancer Res 2006 May-Jun;26(3A):2075-80

Department of Hematology and Oncology, Children's University Hospital, D-72076 Tübingen, Germany.

Background: RT-PCR analysis of compounds of catecholamine metabolism (in particular tyrosine hydroxylase, TH) is widely used for the detection of contaminating neuroblastoma cells in hematopoietic stem cell preparations. Due to reports in the literature showing that hematopoietic cells are also able to produce catecholamines, we investigated whether TH-RT-PCR is really suitable for this purpose.

Materials And Methods: Besides neuroblastoma cells, mononuclear blood cells, apheresis preparations and hematopoietic stem cells were used for single and nested RT-PCR. In addition to TH, the expressions of dopamine-beta-hydroxylase and noradrenaline transporter were analyzed.

Results: Using single RT-PCR, a clear discrimination between neuroblastoma and hematopoietic cells was possible. However, by using nested RT-PCR, the "neuroblastoma markers" were also detected in a significant percentage of non-mobilized mononuclear blood cells, in mononuclear blood cells of healthy donors mobilized with G-CSF, and in highly purified CD34+ and CD133+ stem cells from healthy mobilized donors.

Conclusion: Our results raise the question of whether the RT-PCR analysis of compounds of catecholamine metabolism is suitable and selective enough to detect the contamination of hematopoietic stem cells by a low number of neuroblastoma cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
August 2006

Outcome of allogeneic stem cell transplantation in children with non-malignant diseases.

Haematologica 2006 Jun;91(6):788-94

University Children's Hospital, Frankfurt, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
June 2006

Chimaerism analyses and subsequent immunological intervention after stem cell transplantation in patients with juvenile myelomonocytic leukaemia.

Br J Haematol 2005 May;129(4):542-9

Department of Paediatrics and Adolescent Medicine, University of Freiburg, Mathildenstrasse 1, 79106 Freiburg, Germany.

Chimaerism analysis was performed by polymerase chain reaction amplification of short-tandem repeat markers in 30 children following haematopoietic stem cell transplantation for juvenile myelomonocytic leukaemia (JMML). Fourteen patients always had complete chimaerism (CC); one of them relapsed after the discontinuation of the study and 13 continued in complete remission (CR). Mixed chimaerism (MC) was noted in 16 patients. Of those 12 patients demonstrated increasing MC (i-MC); 10 relapsed and two achieved CC following discontinuation of immunosuppressive therapy (IST). Four other patients demonstrating only transient MC are alive in CR. MC with up to 20% of autologous cells could be successfully eradicated without induction of severe graft-versus-host disease when IST was reduced or discontinued directly after the first demonstration of MC. At the same time, MC with up to 10% of autologous cells could disappear without intervention. The interval between MC and relapse ranged from 0-320 d (median 38 d). Donor leucocyte infusion was given to six patients with i-MC, but only one patient responded. Peripheral blood seems as valuable as bone marrow for chimaerism studies. In conclusion, serial quantitative chimaerism studies can identify patients with i-MC who are at high risk for relapse of JMML. Immediate withdrawal of IST is advised in these patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1365-2141.2005.05489.xDOI Listing
May 2005

Concurrent detection of minimal residual disease (MRD) in childhood acute lymphoblastic leukaemia by flow cytometry and real-time PCR.

Br J Haematol 2005 Mar;128(6):774-82

Department of Pediatric Haematology and Oncology, University Children's Hospital, D-72076 Tübingen, Germany.

Minimal (i.e. submicroscopic) residual disease (MRD) predicts outcome in childhood acute lymphoblastic leukaemia (ALL). To be used clinically, MRD assays must be reliable and accurate. Two well-established techniques, flow cytometry (FC) and polymerase chain reaction (PCR), can detect leukaemic cells with a sensitivity of 0.01% (10(-4)). We analysed diagnostic samples of 45 ALL-patients (37 B-lineage ALL, eight T-lineage ALL) by four-colour FC and real-time PCR. Leukaemia-associated immunophenotypes, at a sensitivity of MRD detection by FC at the 0.01% level, were identified in 41 cases (91%); antigen-receptor gene rearrangements suitable for MRD detection with a sensitivity of 0.01% or better by PCR were identified in 38 cases (84%). The combined use of FC and PCR allowed MRD monitoring in all 45 patients. In 105 follow-up samples, MRD estimates by both methods were highly concordant, with a deviation factor of <5 by Bland-Altman analysis. Importantly, the concordance between FC and PCR was also observed in regenerating bone marrow samples containing high proportions of CD19(+) cells, and in samples studied 24 h after collection. We conclude that both MRD assays yield generally concordant results. Their combined use should enable MRD monitoring in virtually all patients and prevent false-negative results due to clonal evolution or phenotypic shifts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1365-2141.2005.05401.xDOI Listing
March 2005

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.

Br J Haematol 2005 Mar;128(5):649-58

Department of Hematology/Oncology, University Children's Hospital, D-60590 Frankfurt, Germany.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1365-2141.2004.05354.xDOI Listing
March 2005