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Abstract: The EORTC Leukemia Group (LG) has a long history of promoting the study of leukemias and related malignancies and reports here on three of their most significant achievements. In acute myelogenous leukemia (AML), the LG and Italian group GIMEMA started their fruitful collaboration in 1986 with the AML-8 trial with 1519 inclusions. In the AML-8A trial, in patients who reached complete remission, without a HLA identical sibling, autograft provided longer disease-free survival than a second course of consolidation, whereas the best outcome was observed in patients with a donor, who had to be allografted. The AML-10 trial set a new standard of treatment for induction/consolidation with replacement of daunorubicin by either idarubicin or mitoxantrone. The AML-12 trial tested the effect of high-dose cytosine-arabinoside during induction (2109 inclusions, data base locked in August 2011 for final analysis). Development of intergroup trials focusing on subgroups of AML bearing specific genetic abnormalities is now mandatory to validate the “targeted approach” of driving molecular events. In high-risk myelodysplastic syndrome (MDS), the phase III trial conducted by the LG in collaboration with the German MDS Study Group showed that the response rate of decitabine versus best supportive care was higher (complete or partial remissions, 19% versus 0%, and hematologic improvement, 15%versus 2%), progression-free survival was significantly prolonged (median 6.6 versus 3 months), cumulative incidence of AML was significantly decreased (22%versus 33% at one year), but the impact on OS was less evident (median 10.1 versus 8.5 months; hazard ratio 0.88). Quality of life had improved significantly in patients in the decitabine arm. The assessment of HDAC inhibitors, such as vorinostat, will probably be tested in the next trial. Also in MDS, relevant genetic lesions involved in the pathogenesis of this disease were identified using single nucleotide polymorphisms array-based genomic profiling and genomic sequencing in 102 patients with MDS. Acquired abnormalities of the TET2 gene were identified in 26% of the cases and in the EZH2 gene in 5-10% of the patients. TET2 mutations were detected in 96% of the bone marrow cells, including CD34+ progenitor cells, suggesting that TET2 mutations could be an early event during disease evolution. In normal bone marrow, TET2 expression was elevated in granulocytes, suggesting a role in myelopoiesis. Conclusion: during the last 25 years the EORTC LG in cooperation with GIMEMA made a considerable contribution to the improvement of treatment results of patients with acute leukemia or MDS. [Copyright &y& Elsevier]

Donor killer cell immunoglobulin-like receptor (KIR) ligand incompatibility in the graft-versus-host direction is associated with decreased relapse incidence and improved disease-free survival after haploidentical and human leucocyte antigen (HLA)-mismatched unrelated, haematopoietic stem cell transplantation. However, review of all published studies of allogeneic HLA-matched or mismatched stem cell transplantation shows that the results on the relationship between donor–recipient KIR(-ligand) (in)compatibility and outcomes are highly variable, ranging from highly beneficial to detrimental. Reasons for these differences may include the methodology to determine KIR(-ligand) incompatibility, the disease distribution and the transplant protocol or donor type. Two retrospective studies on the effects of KIR-ligand incompatibility in unrelated cord blood transplantation (UCBT) for haematological malignancies have resulted in conflicting results. The Eurocord study showed a favourable effect of KIR-ligand mismatching on relapse incidence and leukaemia-free survival, whereas the Minneapolis study showed no effect on these end points and a detrimental effect on incidence of graft-versus-host disease (GvHD). In patients with non-malignant disorders, KIR-ligand (in)compatibility between donor and recipient was not associated with outcomes in a recent Eurocord analysis. Therefore, the role of natural killer (NK) cell alloreactivity in UCBT is far from clear. It is too early to use a donor–recipient KIR(-ligand) algorithm for selection of a cord blood donor. [Copyright &y& Elsevier]

Clinical studies have indicated that HLA-DPB1 functions as a classical transplantation antigen in allogeneic stem cell transplantation. Mismatching for HLA-DPB1 was associated with an increased risk of graft-versus-host disease (GVHD), but also a decreased risk of disease relapse. However, specific HLA-DPB1 mismatches were associated with poor clinical outcome. It was suggested that this unfavorable effect was caused by a difference in immunogenicity between HLA-DPB1 alleles. To analyze whether immunogenicity of HLA-DPB1 mismatches could be predicted based on the presence or absence of specific amino acid sequences we developed a model to generate allo-HLA-DPB1 responses in vitro. We tested in total 48 different stimulator/responder combinations by stimulating CD4+ T cells from 5 HLA-DPB1 homozygous individuals with the same antigen-presenting cells transduced with different allo-HLA-DPB1 molecules. HLA-DPB1 molecules used for stimulation comprised 76% to 99% of HLA-DPB1 molecules present in different ethnic populations. We show that all HLA-DPB1 mismatches as defined by allele typing resulted in high-frequency immune responses. Furthermore, we show that crossrecognition of different HLA-DPB1 molecules is a broadly observed phenomenon. We confirm previously described patterns in crossrecognition, and demonstrate that a high degree in similarity between HLA-DPB1 molecules is predictive for crossrecognition, but not for immunogenicity. [ABSTRACT FROM AUTHOR]

Objective: B–lineage acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia in lymphatic blastic phase in adults have poor prognoses despite intensive chemotherapy. Novel targeted treatment modalities emerge, but their evaluation requires relevant in vitro models of lymphoblastic leukemia. Presently available cell lines do not fully represent this heterogeneous disease. Available in vitro culturing protocols do not support long–term proliferation of primary cells. We therefore aimed to develop a culture system that allows long–term proliferation of primary human B–lineage lymphoblastic leukemia. Materials and Methods: Primary lymphoblastic leukemia cells were cultured in a defined serum–free medium, in the absence or presence of human hematopoietic growth factors or serum. Results: In the defined serum–free medium, cells from 12 of 34 cases immediately proliferated in vitro. In the absence of hematopoietic growth factors and serum these cases proliferated for more than 1 year without signs of exhaustion. The culturing system supported different subtypes of lymphoblastic leukemia. Two chronic myeloid leukemia in lymphatic blastic phase, four bcr/abl–positive ALL, one etv6/abl–positive ALL, 2 e2a–pbx1−positive ALL, and one t(9;11)–positive ALL could be long–term expanded, as well as two ALL that displayed nontypical cytogenetics. Not all bcr/abl– or e2a–pbx1−positive ALL proliferated in vitro, demonstrating heterogeneity within these subtypes. The proliferating bcr/abl– and etv6/abl–positive cells displayed sensitivity to imatinib, demonstrating that their proliferation depended on the activity of these oncoproteins. Conclusion: The serum–free culturing system may be a valuable instrument in the study of ALL cell biology, as well as in the evaluation of novel targeted therapeutics. [Copyright &y& Elsevier]

Abstract: Administration of recombinant-human G-CSF (rhG-CSF) is highly efficient in mobilizing hematopoietic stem and progenitor cells (HSC/HPC) from the bone marrow (BM) toward the peripheral blood. This study was designed to investigate whether repeated G-CSF-induced HSC/HPC mobilization in mice could lead to a depletion of the bone marrow HSC/HPC pool with subsequent loss of mobilizing capacity. To test this hypothesis Balb/c mice were treated with a maximum of 12 repeated 5-day cycles of either 10 μg rhG-CSF/day or 0.25 μg rmG-CSF/day. Repeated administration of rhG-CSF lead to strong inhibition of HSC/HPC mobilization toward the peripheral blood and spleen after >4 cycles because of the induction of anti–rhG-CSF antibodies. In contrast, after repeated administration of rmG-CSF, HSC/HPC mobilizing capacity remained intact for up to 12 cycles. The number of CFU-GM per femur did not significantly change for up to 12 cycles. We conclude that repeated administration of G-CSF does not lead to depletion of the bone marrow HSC/HPC pool. [Copyright &y& Elsevier]

Objective: Umbilical cord blood (UCB) is considered as an attractive alternative source of hematopoietic stem cells for allogeneic stem cell transplantations in patients who lack human leukocyte antigen (HLA)-matched donors. However, the low cell dose adversely affects hematopoietic recovery and therefore limits application of UCB transplantation in adults. Transplantation of multiple UCB units could be a strategy to overcome cell dose limitations. Materials and Methods: To investigate the effect of double cord transplantation, nonobese diabetic/severe combined immunodeficient mice were transplanted with human hematopoietic progenitor cells (CD34+) derived from two UCB units with HLA disparity. Human cell engraftment and donor origin was determined by flow cytometry. Results: Double CB transplantation resulted in increased engraftment levels in the bone marrow and peripheral blood in comparison with recipients of a single unit. Because this effect could be due to the higher cell dose (2.105 vs 1.105 cells), double CB transplantation was compared with single units containing equal cell numbers (2.105). In some cases, engraftment levels in recipients of single units containing 2.105 cells were significantly higher than after transplantation of 1.105 cells. These engraftment levels were similar to those observed after double CB transplantation. Chimerism analysis indicated that increased engraftment in recipients of two units was predominantly derived from one unit, whereas in other cases the contribution of the two units was similar. Conclusion: These results indicate that engraftment may be enhanced by addition of a second unrelated CB that might be attributed to a cell dose effect or due to a graft-facilitating effect. [Copyright &y& Elsevier]

Objective: Donor lymphocyte infusion (DLI) after allogeneic stem cell transplantation is largely unsuccessful in the treatment of acute lymphoblastic leukemia (ALL). To allow identification of the causes of this failure, we established an animal model of DLI in human ALL. Methods: NOD/scid mice were inoculated with primary human ALL cells. Cells from five different patients were studied. After engraftment, DLI was performed by infusion of human leukocyte antigen (HLA)-identical donor T cells or HLA-disparate donor T cells. Results: DLI resulted in expansion of activated, leukemia-reactive T cells in all donor–patient combinations. After 40 days of expansion, T cells abruptly declined in numbers and displayed loss of cytotoxicity. At this moment, remissions were observed in three of five donor–patient combinations. In animals engrafted with the two unresponsive ALL, remissions could be achieved when HLA-disparate DLI was given. Conclusion: Our results indicate that the inefficacy of DLI in ALL may be due to the limitation of the proliferative capacity of ALL-reactive T cells and that the antileukemic efficacy during the limited time span of proliferation depends on the antigenic disparity between the donor and the patient. The model can be used to study whether alternative strategies may result in more sustained antileukemic responses after DLI in ALL. [Copyright &y& Elsevier]

Acute lymphoblastic leukaemia (ALL) in adults is a relatively rare neoplasm with a curability rate around 30% at 5 years. This consideration makes it imperative to dissect further the biological mechanisms of disease, in order to selectively implement an hitherto unsatisfactory success rate. The recognition of discrete ALL subtypes (some of which deserve specific therapeutic approaches, like T-lineage ALL (T-ALL) and mature B-lineage ALL (B-ALL)) is possible through an accurate combination of cytomorphology, immunophenotytpe and cytogenetic assays and has been a major result of clinical research studies conducted over the past 20 years. Two–three major prognostic groups are now easily identifiable, with a survival probability ranging from <10 to 20% (Philadelphia-positive ALL) to about 50–60% (low-risk T-ALL and selected patients with B-lineage ALL). These issues are extensively reviewed and form the basis of current knowledge. The second major point relates to the emerging importance of studies that reveal a dysregulated gene activity and its clinical counterpart. It is now clear that prognostication is a complex matter ranging from patient-related issues to cytogenetics to molecular biology, including the evaluation of minimal residual disease (MRD) and possibly gene array tests. On these bases, the role of a correct, highly personalised therapeutic choice will soon become fundamental. Therapeutic progress may be obtainable through a careful integration of chemotherapy, stem cell transplantation, and the new targeted treatments with highly specific metabolic inhibitors and humanised monoclonal antibodies. [Copyright &y& Elsevier]

Donor lymphocyte infusion (DLI) results in complete cytogenetic remission (CCR) of relapsed chronic-phase chronic myeloid leukemia (CML-CP) after allogeneic stem cell transplantation (SCT) in up to 80% of patients. The main complication of DLI is graft-versus-host disease (GVHD). Decreasing the dose of DLI is associated with less GVHD but also with a longer interval between treatment and CCR. We postulated that combining α-interferon (α-IFN) with DLI would enable us to decrease the dose of DLI, thereby limiting GVHD, and at the same time to decrease the interval between DLI and CCR for patients with either a hematologic or cytogenetic relapse. For molecular relapses, we hypothesized that because of a lower tumor load, very low doses of DLI without α-IFN could be an effective treatment. Two groups of CML-CP patients treated with DLI at a very low dose of 0.5 to 1.0 × 107 mononuclear cells per kilogram, containing 2 to 6 × 106 CD3+ T cells per kilogram, were analyzed: 13 patients with a cytogenetic or a hematologic relapse after allogeneic SCT (group A) were treated with additional α-IFN therapy at a dose of 3 × 106 U 5 d/wk, and 8 patients with a molecular relapse were treated without α-IFN (group B). Twelve patients from group A reached a CCR. The median interval between DLI and CCR was 7 weeks (range, 5–18 weeks) for group A. All patients with a CCR reached complete donor chimerism at a median of 10 weeks after DLI (range, 6–121 weeks). Eleven patients reached molecular remission at a median of 15 weeks after DLI (range, 8–34 weeks). In group B, all patients reached a molecular remission at a median of 14 weeks (range, 12–29 weeks). Five patients from group A developed acute GVHD grade II to IV and extensive chronic GVHD. In group B, 1 patient developed acute GVHD grade II to IV and subsequently developed extensive chronic GVHD. With a median follow-up of 62 months, 10 patients in group A are alive and in continuous CCR. One patient had a molecular relapse, for which she successfully received additional DLI; another patient reached molecular remission only after 5 doses of DLI. Two patients from group A died of a gram-negative sepsis, and 1 died of an acute myocardial infection. In group B, all patients are alive and in molecular remission with a median follow-up of 20 months. One patient’s disease progressed but was successfully treated with DLI plus α-IFN. In conclusion, very-low-dose DLI in combination with α-IFN as treatment for cytogenetic or hematologic relapses of CML-CP after allogeneic SCT reduced the interval to obtain a CCR with acceptable GVHD when compared with the literature. Patients with a CCR also reached complete donor chimerism and complete molecular remissions. For patients with a molecular relapse, very-low-dose DLI alone is sufficient to induce molecular remissions in most patients and is associated with limited GVHD. [Copyright &y& Elsevier]

: ObjectiveUnresponsiveness to chemotherapy is a major problem in the treatment of leukemia, which can be caused by unresponsiveness of noncycling cells to cell cycle-dependent cytotoxic agents. Targeted toxins consisting of a targeting and activating cytokine (granulocyte-macrophage colony-stimulating factor [GM-CSF]) and diphtheria toxin (DT) can be used to overcome this kind of resistance of leukemic cells. In this study we manipulated the cell cycle and proliferative status of leukemic cells, explored the effect on sensitivity to DT, and determined the ability of DT388GMCSF fusion proteins to activate and subsequently kill leukemic cells.: Materials and MethodsWe used the GM-CSF–dependent myeloid leukemic cell line AML-193 as a model. GM-CSF or granulocyte colony-stimulating factor (G-CSF) was used to manipulate the cell cycle and proliferative state of AML-193 cells. Cell death was quantified by 51Cr release assays. The results obtained in the AML-193 cell line model were confirmed using primary leukemic blasts.: ResultsSimilar to treatment with chemotherapy and immunotherapy, leukemic cells in resting G0 phase were relatively resistant to DT-induced cell death. Synchronized recruitment of leukemic cells into activated phases of the cell cycle by low concentrations of GM-CSF or G-CSF resulted in significant increased DT sensitivity. DT388GMCSF fusion proteins specifically targeted GM-CSF receptor-expressing cells, resulting in recruitment of leukemic cells from G0 phase of the cell cycle and subsequent kill of these cells.: ConclusionLeukemic cells in G0 phase, which are resistant to conventional chemotherapy, Fas-induced immunotherapy, and DT alone, can be synchronically activated and subsequently killed by DT388GMCSF fusion proteins. [Copyright &y& Elsevier]

: ObjectiveAllogeneic stem cell transplantation (alloSCT) following reduced-intensity conditioning offers a relatively nontoxic regimen while preserving rapid and sustained engraftment. Acute and chronic graft-vs-host disease (GVHD) is, however, a significant cause of severe morbidity. To reduce the incidence of GVHD, we treated a group of high-risk patients with a reduced-intensity conditioning regimen followed by in vitro T-cell–depleted alloSCT using Campath 1-H incubation.: Patients and methodsEighteen patients were treated with fludarabine (6×30 mg/m2), busulphan (2×3.2 mg/kg), and ATG (4×10 mg/kg) followed by the infusion of high-dose T-cell–depleted peripheral stem cells from sibling donors. No posttransplant GVHD prophylaxis was administered. At 6 months after alloSCT, low-dose donor lymphocyte infusion (DLI) was administered.: ResultsAll patients had sustained engraftment of donor cells with a median of 95% donor cells at 3 months after alloSCT. Minimal acute and no chronic GVHD was observed after alloSCT. A high incidence of cytomegalovirus (CMV) reactivation but no CMV disease was observed. Eleven patients received DLI at a median of 6.5 months after alloSCT. Acute GVHD grade II–III developed in 6 patients. All patients showed improvement of donor chimerism after DLI. With a median follow-up of 211 days, 11 patients are alive. Particular in patients with chronic lymphocytic leukemia and acute myeloid leukemia, a significant graft-vs-tumor effect was observed.: ConclusionsIn vitro T-cell–depleted alloSCT following reduced-intensity conditioning leads to durable donor engraftment without GVHD. The high levels of donor chimerism allow the subsequent use of cellular immunotherapy to treat residual disease. [Copyright &y& Elsevier]

Minor histocompatibility antigens (mHags) play a major role in graft rejection, the induction of detrimental graft-vs-host disease (GVHD), and the development of the beneficial graft-vs-leukemia (GVL) effect after allogeneic stem cell transplantation (SCT). mHags can be defined as amino acid polymorphisms in cellular proteins that can lead to differential presentation of antigenic peptides in HLA molecules and therefore to differential recognition by T cells. The tissue distribution of the mHags and the HLA molecules by which they can be presented play a significant role in the clinical outcome of T-cell responses against these antigens. In part, differential recognition by T cells of mHags specifically expressed in hematopoietic cells, including the malignant cells from the recipient may result in GVL reactivity without concurrent GVHD. Furthermore, T-cell responses against proteins solely expressed in hematopoietic cell lineages from which the malignancy is derived may be appropriate mediators of GVL reactivity without GVHD induction. Characterization of clinical immune responses in patients treated for relapsed leukemia after allogeneic SCT with donor lymphocyte infusion in the absence of GVHD may lead to the characterization of new mHags that can be exploited to generate tumor-specific immune responses. By in vitro generation of T-cell responses against defined mHags, the efficacy and specificity of cellular immunotherapy against hematologic malignancies in the context of allogeneic transplantation may be improved. [Copyright &y& Elsevier]

AraC resistance in vitro is explained by inactivation of dCK, while resistance to DNR is described by overexpression of multidrug efflux pumps like Pgp or MRP. Thusfar, no correlation between resistance mechanisms in vitro and in patients with AML has been documented. We generated AraC and DNR double resistant cell lines to investigate resistance mechanisms of both agents. In these cell lines involvement of dCK was extensively investigated and Pgp expression and activity was determined. Our data implicate that similar resistance mechanisms like inactivation of dCK coincided by alternatively spliced dCK forms and overexpression of Pgp are induced in single—as well as in double resistant leukemic cell lines. [Copyright &y& Elsevier]

: ObjectiveIn comparison with stem cell transplantation using bone marrow or cytokine-mobilized peripheral blood, cord blood transplantation is characterized by delayed engraftment, in particular platelet recovery. The differences in the kinetics of engraftment may be related to quantitative differences in the numbers of stem cells and megakaryocyte progenitor cells and/or to qualitative differences between megakaryocyte progenitor cells in these grafts. We compared the hematopoietic composition of these grafts and determined the distribution of mature and immature megakaryocyte progenitor cells in cord blood and mobilized peripheral blood and their in vitro kinetic behavior.: MethodsMegakaryocyte progenitor cell subpopulations from cord blood (CB) and mobilized peripheral blood (PBSC) were expanded in vitro in the presence of mpl-ligand. The developmental differences during expansion of megakaryocyte progenitors were analyzed by flow cytometry and progenitor assays.: ResultsWe found that the immature (CD34+/CD41−) subpopulation from CB contains more than 98% of all megakaryocyte progenitor cells, responsible for 99% of all megakaryocytic cells cultured during 2 weeks. The CB CD34+/CD41+ subpopulation shows no contribution to megakaryocytic cell formation. In contrast, in PBSC the mature (CD34+/CD41+) subpopulation contains 7% of all megakaryocyte progenitor cells. Moreover, CD34+ cells from CB and PBSC also showed distinct phenotypic differences during maturation in vitro. PBSC megakaryocyte progenitor cells transiently express both CD34 and CD41 during maturation in vitro, whereas CB progenitor cells transiently lack expression of both markers before differention into (CD34−/CD41+) megakaryocytic cells.: ConclusionThe in vitro data indicate the presence of different developmental stages of megakaryocyte progenitor cells in CB as compared to PBSC. These differences in composition and maturation between CB and PBSC may be related to the different kinetics of engraftment following transplantation of these stem cell sources. [Copyright &y& Elsevier]

: ObjectiveMesenchymal stem cells (MSC) have been implicated as playing an important role in hematopoietic stem cell engraftment. We identified and characterized a new population of MSC derived from human fetal lung. In cotransplantation experiments, we examined the homing of MSC as well as the effect on engraftment of human umbilical cord blood (UCB)-derived CD34+ cells in NOD/SCID mice.: Materials and MethodsCulture-expanded fetal lung-derived CD34+ cells were characterized by immune phenotyping and cultured under conditions promoting differentiation to osteoblasts or adipocytes. Irradiated (3.5 Gy) NOD/SCID mice (n = 51) were transplanted intravenously with 0.03 to 1.0 × 106 UCB CD34+ cells in the presence or absence of 1 × 106 culture-expanded fetal lung-derived MSC, irradiated CD34− cells, B cells, or with cultured MSC only.: ResultsCulture-expanded fetal lung CD34+ cells were identified as MSC based on phenotype (CD105+, SH3+, SH4+, CD160+) and their multilineage potential. Cotransplantation of low doses of UCB CD34+ cells and MSC resulted in a three-fold to four-fold increase in bone marrow engraftment after 6 weeks, whereas no such effect was observed after cotransplantation of irradiated CD34− or B cells. Homing experiments indicated the presence of MSC in the lung, but not in the bone marrow, of NOD/SCID mice.: ConclusionsWe identified a population of MSC derived from human fetal lung. Upon cotransplantation, MSC, but not irradiated CD34− or B cells, promote engraftment of UCB CD34+ cells in bone marrow, spleen, and blood by mechanisms that may not require homing of MSC to the bone marrow. [Copyright &y& Elsevier]

Human leukocyte antigen (HLA) class I expression at the allelic level was analyzed in 397 acute myeloid leukemia (AML) and 186 acute lymphoid leukemia (ALL) using a complement-dependent cytotoxicity assay. Impaired recognition possibly due to HLA downregulation was observed in 2% of the patients with AML and ALL in complete remission, and in 8%–15% in the groups with blasts. In 15 instances of diminished cytotoxicity, leukemic cells and control PHA blasts from the same patients were further analyzed using flow cytometry. In 4/6 ALL and 4/9 AML patients HLA downregulation or complete loss (2 patients) of cell surface expression could be confirmed. No genomic abnormalities were observed. In addition, 12 AML and 13 ALL patients were tested during relapse using flow cytometry. In 1/12 AML patients and 1/13 ALL patients allelic downregulation of cell surface expression was found. In two patients tested, downregulation or loss of cell surface expression of HLA class I antigens corresponded with impaired T cell mediated lysis by HLA restricted cytotoxic T lymphocyte.Treatment of the cells with α- or γ-interferon could restore HLA class I expression and T-cell recognition. In conclusion, downregulation of cell surface expression of HLA class I expression at the allelic level in AML and ALL is infrequent but functionally relevant. HLA downregulation was reversible and T-cell recognition could be restored by α- or γ-interferon. [Copyright &y& Elsevier]