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Recent studies have suggested that the lipid-lowering agent simvastatin holds great promise as a cancer therapeutic; it inhibits the growth of multiple tumors, including triple-negative breast cancer. Doxorubicin- and simvastatin-induced cytotoxicity has been associated with the modulation of Ca2+signaling, but the underlying mechanisms remain incompletely understood. Here we identify how Ca2+signaling regulates the breast tumor cell response to doxorubicin and simvastatin. These two drugs inhibit cell survival while increasing apoptosis in two human breast cancer cell lines and five primary breast tumor specimens through the modulation of Ca2+signaling. Signal transduction and functional studies revealed that both simvastatin and doxorubicin trigger persistent cytosolic Ca2+release, thereby stimulating the proapoptotic BIM pathway and mitochondrial Ca2+overload, which are responsible for metabolic dysfunction and apoptosis induction. Simvastatin and doxorubicin suppress the prosurvival ERK1/2 pathway in a Ca2+-independent and Ca2+-dependent manner, respectively. In addition, reduction of the Ca2+signal by chelation or pharmacological inhibition significantly prevents drug-mediated anticancer signaling. Unexpectedly, a scratch-wound assay indicated that these two drugs induce rapid cell migration, while inhibiting cell invasion and colony formation in a Ca2+-dependent manner. Further, the in vivo data for MDA-MB-231 xenografts demonstrate that upon chelation of Ca2+, the ability of both drugs to reduce the tumor burden was significantly reduced via caspase-3 deactivation. Our results establish a calcium-based mechanism as crucial for executing the cell death process triggered by simvastatin and doxorubicin, and suggest that combining simvastatin with doxorubicin may be an effective regimen for the treatment of breast cancer.

Despite advances in supportive therapy to prevent complications of sickle cell disease (SCD), access to care is not universal. Hematopoietic cell transplantation is, to date, the only curative therapy for SCD, but its application is limited by availability of a suitable HLA-matched donor and lack of awareness of the benefits of transplant. Included in this study are 1000 recipients of HLA-identical sibling transplants performed between 1986 and 2013 and reported to the European Society for Blood and Marrow Transplantation, Eurocord, and the Center for International Blood and Marrow Transplant Research. The primary endpoint was event-free survival, defined as being alive without graft failure; risk factors were studied using a Cox regression models. The median age at transplantation was 9 years, and the median follow-up was longer than 5 years. Most patients received a myeloablative conditioning regimen (n = 873; 87%); the remainder received reduced-intensity conditioning regimens (n = 125; 13%). Bone marrow was the predominant stem cell source (n = 839; 84%); peripheral blood and cord blood progenitors were used in 73 (7%) and 88 (9%) patients, respectively. The 5-year event-free survival and overall survival were 91.4% (95% confidence interval, 89.6%-93.3%) and 92.9% (95% confidence interval, 91.1%-94.6%), respectively. Event-free survival was lower with increasing age at transplantation (hazard ratio [HR], 1.09; P< .001) and higher for transplantations performed after 2006 (HR, 0.95; P= .013). Twenty-three patients experienced graft failure, and 70 patients (7%) died, with the most common cause of death being infection. The excellent outcome of a cohort transplanted over the course of 3 decades confirms the role of HLA-identical sibling transplantation for children and adults with SCD.

Despite advances in supportive therapy to prevent complications of sickle cell disease (SCD), access to care is not universal. Hematopoietic cell transplantation is, to date, the only curative therapy for SCD, but its application is limited by availability of a suitable HLA-matched donor and lack of awareness of the benefits of transplant. Included in this study are 1000 recipients of HLA-identical sibling transplants performed between 1986 and 2013 and reported to the European Society for Blood and Marrow Transplantation, Eurocord, and the Center for International Blood and Marrow Transplant Research. The primary endpoint was event-free survival, defined as being alive without graft failure; risk factors were studied using a Cox regression models. The median age at transplantation was 9 years, and the median follow-up was longer than 5 years. Most patients received a myeloablative conditioning regimen (n = 873; 87%); the remainder received reduced-intensity conditioning regimens (n = 125; 13%). Bone marrow was the predominant stem cell source (n = 839; 84%); peripheral blood and cord blood progenitors were used in 73 (7%) and 88 (9%) patients, respectively. The 5-year event-free survival and overall survival were 91.4% (95% confidence interval, 89.6%-93.3%) and 92.9% (95% confidence interval, 91.1%-94.6%), respectively. Event-free survival was lower with increasing age at transplantation (hazard ratio [HR], 1.09; P < .001) and higher for transplantations performed after 2006 (HR, 0.95; P = .013). Twenty-three patients experienced graft failure, and 70 patients (7%) died, with the most common cause of death being infection. The excellent outcome of a cohort transplanted over the course of 3 decades confirms the role of HLA-identical sibling transplantation for children and adults with SCD.

According to biologic features, there is a substantial “gray zone” between nodular lymphocyte predominant Hodgkin lymphomas (NLPHLs) (Poppema lymphomas) and T-cellhistiocyte-rich B-cell lymphomas (THRBCLs). Transformation from an NLPHL to a THRBCL can occur and is associated with a worsening of the prognosis. Here is described a case of a 16-year-old boy who presented with an NLPHL with features of THRBCL. Clinical evolution was complicated by 2 relapses leading to autologous and then to allogeneic bone marrow transplantation.

Allogeneic hematopoietic stem-cell transplantation (HSCT) is the only curative treatment for sickle cell disease (SCD); nevertheless, its use has been limited by the risk of transplantation-related mortality (TRM). Between November 1988 and December 2004, 87 consecutive patients with severe SCD ranging from 2 to 22 years of age received transplants in France. Cerebral vasculopathy was the principal indication for transplantation (55 patients). All the patients received grafts from a sibling donor after a myeloablative conditioning regimen (CR). The only change in the CR during the study period was the introduction of antithymocyte globulin (ATG) in March 1992. The rejection rate was 22.6% before the use of ATG but 3% thereafter. With a median follow-up of 6 years (range, 2.0 to 17.9 years), the overall and event-free survival (EFS) rates were 93.1% and 86.1%, respectively. Graft versus host disease (GVHD) was the main cause of TRM. Importantly, cord blood transplant recipients did not develop GVHD. No new ischemic lesions were detected after engraftment, and cerebral velocities were significantly reduced. The outcome improved significantly with time: the EFS rate among the 44 patients receiving transplants after January 2000 was 95.3%. These results indicate that HLA-identical sibling HSCT after myeloablative conditioning with ATG should be considered as a standard of care for SCD children who are at high risk for stroke.

Allogeneic hematopoietic stem-cell transplantation (HSCT) is the only curative treatment for sickle cell disease (SCD); nevertheless, its use has been limited by the risk of transplantation-related mortality (TRM). Between November 1988 and December 2004, 87 consecutive patients with severe SCD ranging from 2 to 22 years of age received transplants in France. Cerebral vasculopathy was the principal indication for transplantation (55 patients). All the patients received grafts from a sibling donor after a myeloablative conditioning regimen (CR). The only change in the CR during the study period was the introduction of antithymocyte globulin (ATG) in March 1992. The rejection rate was 22.6% before the use of ATG but 3% thereafter. With a median follow-up of 6 years (range, 2.0 to 17.9 years), the overall and event-free survival (EFS) rates were 93.1% and 86.1%, respectively. Graft versus host disease (GVHD) was the main cause of TRM. Importantly, cord blood transplant recipients did not develop GVHD. No new ischemic lesions were detected after engraftment, and cerebral velocities were significantly reduced. The outcome improved significantly with time: the EFS rate among the 44 patients receiving transplants after January 2000 was 95.3%. These results indicate that HLA-identical sibling HSCT after myeloablative conditioning with ATG should be considered as a standard of care for SCD children who are at high risk for stroke.

The urokinase plasminogen activator (uPA) is implicated in both cancer cell invasion and angiogenesis. It can interact with a specific receptor (uPAR) via the epidermal growth factor (EGF)-like domain in the urokinase amino-terminal fragment (ATF) in a species-specific manner. Our previous studies showed that adenovirusmediated delivery of murine ATF (AdmATF) suppressed human tumor growth in mouse models, by inhibiting murine angiogenesis. However, we cannot exclude its putative inhibitory action on human cancer cell invasion through a uPAR-independent pathway. To further investigate the mechanisms of ATF, we constructed another adenovirus, AdhmATF, expressing humanized murine ATF (hmATF). hmATF binds to human uPAR but not to murine uPAR. We compared the antagonist effect of both AdmATF and AdhmATF on human and murine cancer cells. In vitro, the supernatant from AdhmATF-infected cells repressed 79% of membrane-associated uPA activity on human MDA-MB-231 cells, whereas that from AdmATF-infected cells repressed 35% of membrane-associated uPA activity. On murine LLC cells, the supernatant from AdhmATF-infected cells inhibited 29% of cell surface uPA activity, whereas that from AdmATF-infected cells inhibited 74% of cell surface uPA activity. Similar results were obtained in a cell invasion assay. In vivo, intratumoral injection of the adenoviruses into LLC tumors on day 24 postinjection induced lower but significant tumor growth suppression by AdhmATF (tumor volume was 1185 ± 128 mm3), whereas suppression by AdmATF was greater (407 ± 147 mm3). In the MDA-MB-231 tumor model, on day 52 postinjection, tumor size was 187 ± 47 mm3 in the AdhmATF-treated group and 468 ± 65 mm3 in the AdmATF-treated group. The LLC and MDA-MB- 231 cell lines transfected by mATF or hmATF genes showed growth inhibition In vivo equivalent to the results obtained by adenovirus treatment. These results demonstrate the strong anticancer activity of ATF even when its uPAR-binding affinity has been suppressed, and indicate that ATF exerts an antitumor effect via dual mechanisms: essentially through targeting the uPA–uPAR system via the EGF-like domain and partially through targeting a uPAR-independent interaction via the kringle domain.

The use of monoclonal antibodies against interleukin-2 receptor (IL-2R)-α chains could be an effective treatment of acute graft-versus-host disease (GvHD). Experimental model and clinical studies have reported various results.

No abstract

In a previous randomized study, the authors reported that granulocyte colony-stimulating factor (G-CSF) increased the chemotherapy dose-intensity delivered during the consolidation therapy of high-risk childhood acute lymphoblastic leukemia (ALL). The aim of the current study was to perform an economic evaluation in the same cohort.

New concepts may prove necessary to profit from the avalanche of sequence data on the genome, transcriptome, proteome and interactome and to relate this information to cell physiology. Here, we focus on the concept of large activity-based structures, or hyperstructures, in which a variety of types of molecules are brought together to perform a function. We review the evidence for the existence of hyperstructures responsible for the initiation of DNA replication, the sequestration of newly replicated origins of replication, cell division and for metabolism. The processes responsible for hyperstructure formation include changes in enzyme affinities due to metabolite-induction, lipid-protein affinities, elevated local concentrations of proteins and their binding sites on DNA and RNA, and transertion. Experimental techniques exist that can be used to study hyperstructures and we review some of the ones less familiar to biologists. Finally, we speculate on how a variety of in silico approaches involving cellular automata and multi-agent systems could be combined to develop new concepts in the form of an Integrated cell (I-cell) which would undergo selection for growth and survival in a world of artificial microbiology.

Cerivastatin is used in the treatment of hypercholesterolemia to inhibit 3-hydroxy 3-methylglutaryl coenzyme A reductase and thus prevent the synthesis of cholesterol precursors, such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), responsible, respectively, for translocation of Ras and Rho to the cell membrane, a step required for their cell signaling, leading to cell proliferation and migration. Recently, it has been suggested that non lipid-related effects of statins could play a beneficial role in cancer therapy. In this study, we have investigated the mechanisms by which statins inhibit cancer and the types of cancers which could benefit from this therapy. In MDA-MB-231 cells, an aggressive breast cancer cell line with spontaneous activation of Ras and NFκB and overexpression of RhoA, cerivastatin induced inhibition of both cell proliferation and invasion through Matrigel. This anti-proliferative effect was related to G1/S arrest due to an increase in p21Waf1/Cip1. The anti-invasive effect was observed from 18 h and could be explained by RhoA delocalization from the cell membrane, resulting in disorganization of the actin fibers and disappearance of focal adhesion sites. The importance of RhoA inactivation in both these inhibitory effects was proved by their reversion by GGPP but not by FPP. Moreover, cerivastatin was also shown to induce inactivation of NFκB, in a RhoA inhibition-dependent manner, resulting in a decrease in urokinase and metalloproteinase-9 expression, two proteases involved in cell migration. The participation of Ras inactivation is considered a subsidiary mechanism for the effects of cerivastatin, as they were not rescued by FPP. Prolonged treatment of MDA-MB-231 cells with high doses of cerivastatin induced a loss of cell attachment. Interestingly, the effect of cerivastatin was considerably lower on poorly invasive MCF-7 cells. In conclusion, our results suggest that cerivastatin inhibits cell signaling pathways involved in the invasiveness and metastatic properties of highly invasive cancers.

Recent studies have suggested that inhibitors of 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A reductase (statins) can play a role in protection against vascular risk, which is independent of cholesterol reduction. It could act by inhibiting the synthesis of isoprenoids (farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP)), which are respectively essential for membrane attachment and biological activity of GTPases Ras and RhoA. This study demonstrates that a statin (cerivastatin) inhibits angiogenesis. This effect was due to a decrease in endothelial cell locomotion which was reversed by GGPP. It was mainly related to delocalization of RhoA from cell membrane to cytoplasm, responsible for the disorganization of actin stress fibers. Furthermore, a decrease in MMP‐2 secretion, involved in cell invasion, was also observed. This effect is rather due to Ras inhibition as it was reversed by FPP. This anti‐angiogenic activity could explain the beneficial effect of statins on atherosclerosis and on cancer prevention as shown by clinical studies.

Recent studies have suggested that inhibitors of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (statins) can play a role in protection against vascular risk, which is independent of cholesterol reduction. It could act by inhibiting the synthesis of isoprenoids (farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP)), which are respectively essential for membrane attachment and biological activity of GTPases Ras and RhoA. This study demonstrates that a statin (cerivastatin) inhibits angiogenesis. This effect was due to a decrease in endothelial cell locomotion which was reversed by GGPP. It was mainly related to delocalization of RhoA from cell membrane to cytoplasm, responsible for the disorganization of actin stress fibers. Furthermore, a decrease in MMP-2 secretion, involved in cell invasion, was also observed. This effect is rather due to Ras inhibition as it was reversed by FPP. This anti-angiogenic activity could explain the beneficial effect of statins on atherosclerosis and on cancer prevention as shown by clinical studies.

This is the first report of the long-term results of CD34⁺ cell transplantation in children with neuroblastoma. We investigated the hematologic and immune recovery, posttransplant morbidity, and clinical outcome of these children. Twenty-three children with advanced neuroblastoma had PBPCs (20 patients) or BM (3 patients) collected, followed by CD34⁺ cell selection on Ceprate column. The purge of residual neuroblastoma cells was evaluated using an RT-PCR for tyrosine hydroxylase (TH) mRNA assay. Reinfusion of CD34⁺ cells followed busulfan + melphalan myeloablative chemotherapy. A median of 2.9 × 10⁶ CD34⁺ cells/kg was reinjected. Median days to achieve ANC > 0.5 × 10⁹/liter and platelets > 50 × 10⁹/liter were 13 (range 9–33) and 59 (range 22–259), respectively. Circulating T cells were primarily CD4⁻/CD8⁺ with fewer than 0.2 10⁹CD4⁺ cells/liter throughout the first 6 months. CD19⁺ cells and CD56⁺ cells were not detectable up to day +35 posttransplant. At 1 year posttransplant, 16 evaluable patients had stable hematopoiesis with 2.3 × 10⁹ ANC/liter (range 0.8–4.1), 1.4 × 10⁹ lymphocytes/liter (range 0.5– 2.0) and 251 × 10⁹ PLT/liter (range 35–490). After the completion of hematopoietic reconstitution, six events of severe septicemia/septic shock were noted. Six children had severe VZV infections, and 2 had EBV-associated lymphoproliferation. Thirteen patients are alive with a median follow-up of 40 months (range 2–54). Ten patients have died; 8 relapsed or developed progressive disease, 1 died from nondocumented pneumopathy at day 56, and 1 developed AML-M4 at 3 years posttransplant. In children, CD34⁺ cell transplantation can be accomplished with a reduction of neuroblastoma cell inoculum in the selected graft as assessed by RT-PCR analysis. CD34⁺ cell grafts provide successful neutrophil reconstitution. However, delayed platelet recovery, persistent decrease in CD4⁺ lymphocyte levels and a high incidence of serious and life-threatening late infections were observed in these children. There remains a critical need to evaluate any real clinical benefit of CD34⁺ cell autografts in neuroblastoma patients. Med. Pediatr. Oncol. 35:1–7, 2000. © 2000 Wiley-Liss, Inc.

In bone marrow, haematopoietic stem cells (HSC) rely on close contact with stromal cells for proliferation and differentiation. Stromal cell-derived factor (SDF-1) is a chemokine produced by bone marrow stromal cells and has been reported to be a chemoattractant for CD34+cells. SDF-1 was evaluated for effects on proliferation of both mature and immature human progenitor cells in vitro. Neither proliferation nor maturation of peripheral blood cells was stimulated by SDF-1 alone. Moreover, we have previously demonstrated that 5-fluorouracile (5-FU) resistant HSC require a combination of interleukin 12 (IL-12), IL-6 and SCF for the production of morphologically recognizable clonogenic elements at day 14 in semisolid medium. Our data reported a strong enhancement of the IL-6, IL-12, SCF-induced synergism (172%) by SDF-1 (296.5%). Furthermore, our data suggest that this chemokine alone had no effect on triggering quiescent cells and may preserve these cells from 5-FU cell damage or upregulate early-acting cytokine receptors. Thus, SDF-1 might play a key role in early human haematopoiesis through its potent synergistic effects in combination with early-acting cytokines. These results suggest that a programmed response to sequential cytokine stimulation may be part of a control mechanism required for maintenance of proliferation of primitive HSC.

Macrophages play a major role in angiogenesis. We recently reported that oncostatin M (OSM), a cytokine of the interleukin (IL)‐6 family secreted by macrophages, has a potent angiogenic activity on human microvascular endothelial cells (HMEC‐1), but has no effect on macrovascular cells (human umbilical vein endothelial cells (HUVECs)). In this work, we show that in HMEC‐1, OSM (0.5–2.5 ng/ml), leukemia inhibitory factor (LIF) (25 ng/ml), bFGF (25 ng/ml) and IL‐1β (5 ng/ml) induced production of cyclooxygenase (COX)‐2. In contrast, in HUVECs, neither OSM nor LIF induced COX‐2 mRNA, suggesting that COX‐2 might be implicated in the angiogenic activity of OSM. This was confirmed by the inhibiting effect on OSM‐induced HMEC‐1 proliferation of specific COX‐2 inhibitors. In vivo studies confirmed this findings. We conclude that induction of COX‐2 by OSM is necessary for its angiogenic activity, but is not sufficient since IL‐1β, which also induces COX‐2 in HMEC‐1, has only a poor proliferative effect.

Abstract: To clarify the factors that may affect the peripheral blood stem cell (PBSC) collection in children weighing ≤ 15 kg, a consecutive registry of 109 leukapheresis procedures was analyzed. Collections were performed on a COBE Spectra separator. In 65.1% of the procedures, the peripheral vein, together with a central catheter inserted routinely at diagnosis, or 2 peripheral veins were used to access/return. For 84.4% of the procedures, the extracorporeal line was primed with red blood cells. The median granulocyte‐macrophage colony forming unit (CFU‐GM) number derived from 1 patient's blood volume processed was 13.8 times 104/kg. Six times, a collection series failed, al ways in children treated for ≥ 26 weeks and 4 of those times in children weighing ≤ 11 kg. The patient's age, diagnosis, duration of preleukapheresis treatment, and mobilization regimens did not significantly affect the collection yield. Twenty‐four transplantations were performed. The median times to neutrophils > 0.5× 109/L and platelets > 20 × 109/L were 13 and 20 days, respectively. We conclude that even in very small children, leukapheresis can be performed safely, allowing adequate PBSC collection for transplantation and/or in vitro manipulations.

Human interleukin 3 receptor (IL-3R) is constitutively expressed on committed haematopoietic stem cells, where it mediates proliferation and differentiation. This receptor is also expressed by monocytes and may induce functional activation. Interleukin (IL)-4, IL-10, IL-13, and transforming growth factor β (TGF-β) have different effects on human monocytes. As IL-3R may be regulated by different cytokines, whether the above-mentioned cytokines were able to modulate the α chain of IL-3R (IL-3Rα) on monocytes was examined. Effects on IL-3Rα antigen (Ag) expression were analysed by direct immunofluorescence and flow cytometry. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect variations in IL-3Rα mRNA expression. IL-10 and TGF-β were found to downregulate IL-3Rα Ag. In contrast, IL-4 and IL-13 both caused a dose- and time-dependent increase. A maximum effect was observed at 1 ng/ml of IL-4 for 18 h. Furthermore, in RT-PCR, IL-4 was found to slightly up-regulate IL-3Rα mRNA expression. These observations suggest that IL-4 and IL-13 play a role in the regulation of IL-3Rα expression and the effects of cytokines on human monocytes may be mediated, in part, through the regulation of IL-3R.

Macrophages play a major role in angiogenesis. We recently reported that oncostatin M (OSM), a cytokine of the interleukin (IL)-6 family secreted by macrophages, has a potent angiogenic activity on human microvascular endothelial cells (HMEC-1), but has no effect on macrovascular cells (human umbilical vein endothelial cells (HUVECs)). In this work, we show that in HMEC-1, OSM (0.5–2.5 ng/ml), leukemia inhibitory factor (LIF) (25 ng/ml), bFGF (25 ng/ml) and IL-1β (5 ng/ml) induced production of cyclooxygenase (COX)-2. In contrast, in HUVECs, neither OSM nor LIF induced COX-2 mRNA, suggesting that COX-2 might be implicated in the angiogenic activity of OSM. This was confirmed by the inhibiting effect on OSM-induced HMEC-1 proliferation of specific COX-2 inhibitors. In vivo studies confirmed this findings. We conclude that induction of COX-2 by OSM is necessary for its angiogenic activity, but is not sufficient since IL-1β, which also induces COX-2 in HMEC-1, has only a poor proliferative effect.

Seven of 21 patients with sickle cell anemia developed neurologic complications 5 to 243 days (median, 33 days) after allogeneic marrow transplantation. Among these 7 patients, indications for transplantation included either a past history of stroke (4 patients) or recurrent severe vaso-occlusive events (3 patients). All received marrow from an HLA-identical sibling after preparation with busulfan and cyclophosphamide, and in 4 patients with antithymocyte globulin. Five of 6 patients developing seizures received anticonvulsant and supportive treatment with resolution of neurologic abnormalities. Three patients experienced intracranial bleeding, which was fatal in two. Of the 14 patients free of neurologic complications, 4 patients had experienced stroke before transplantation. However, among all patients with prior stroke, the incidence of intracranial hemorrhage was 38% (3/8), whereas none of the 13 patients without prior stroke developed posttransplant intracranial bleeding (P= .026). We conclude that patients with sickle cell anemia are at increased risk for neurologic complications after marrow ablative therapy and that patients with prior stroke are at increased risk for intracranial hemorrhage. Transplantation of patients before the onset of overt stroke may reduce this risk.

We update our experience on large-volume leukapheresis (LVL) in very small patients with malignancies. LVLs were performed with the aim of reducing the psychological impact of leukaphereses by reducing the number of procedures while collecting large numbers of cells. Seventeen LVLs were performed using a Cobe Spectra separator in 14 patients weighing 15 kg. A median of 3.8 patient's blood volumes corresponding to 296 mL/kg (range, 202–565) of blood was processed per session of 190 minutes (120–279) duration. A femoral catheter was installed specially for collection for 88% LVL (vs. 35% for standard leukaphereses). A median volume of 16.9 mL/kg was collected with 5.4 × 10⁸ MNC/kg (range, 0.6–16.3) and 8.2 × 10⁶ CD34+ cells/kg (range, 1.3–31.7). No signs of complications due to citrate toxicity were encountered. No hypotensive or hypothermic episodes were observed. Platelet counts were significantly diminished after each procedure (median: −59%). When the extracorporal line was not primed with red blood cells (RBC), the difference between pre-LVL and post-LVL hemoglobin levels was significant with a median 32 g/L decrease. The LVL approach for peripheral blood progenitor cells (PBPC) collection in very small children may expose them to the risk of anemia and thrombocytopenia and an excess of special central line installation. The application of this technique in these patients should be reserved for special cases when a very large number of cells must be collected and should be performed by an experienced team. Med. Pediatr. Oncol. 32:7–10, 1999. © 1999 Wiley-Liss, Inc.

No abstract

Background:Hematopoietic stem cell transplant (HSCT) from an HLA identical (HLAid) sibling donor is a well-established curative therapy for sickle cell disease (SCD). However, the ideal age to perform HSCT in SCD patients remains controversial. We report the outcomes after HLAid sibling HSCT for SCD according to patient's age at the time of HSCT as well as their pre-transplant characteristics.

De Montalembert: Addmedica: Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau. Guerci-Bresler:ARIAD: Speakers Bureau; BMS: Speakers Bureau; Novartis: Speakers Bureau; PFIZER: Speakers Bureau.

Walters: ViaCord and AllCells, Inc: Other: Medical director. Bertrand:ERYTECH Pharma: Consultancy. Peters:Medac: Research Funding; Fresenius: Research Funding; Amgen: Research Funding; Jazz: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Sanovi: Research Funding; Pierre-Fabre: Research Funding.

Introduction:

L’OMS définit une réaction leucémoïde par la présence d’une blastose périphérique chez le nouveau-né trisomique. Or, une blastose sanguine est présente chez la plupart des nouveau-nés trisomiques, d’autant plus en cas de prématurité, et seule une blastose sanguine>20 % serait vraiment prédictive d’une réaction leucémoïde. Cependant, il est maintenant acquis que le paramètre diagnostique le plus spécifique est la présence d’une mutation acquise du gène GATA1 dans les blastes sanguins.

No relevant conflicts of interest to declare.

Ruminy: Centre Henri Becquerel: Patents & Royalties. Marchand:Centre Henri Becquerel: Patents & Royalties.

In acute leukemia, recurrent chromosomal translocations which result in the fusion of two genes are frequent. Some of these markers have a well established prognostic and therapeutic impact and are routinely screened at diagnosis by cytogenetics and RT-PCR. Yet, due to the limitations of these methods, only a few among the dozens of known rearrangements are systematically tested. Many abnormalities which could provide important clinical information thus remain ignored, mainly due to the impossibility of performing a cost effective multi-targeted screening. To overcome this obstacle, we have developed a simple and parsimonious assay which allows a reliable detection of dozens of fusion genes in only a few hours.

Sickle Cell Anemia (SCA) remains a disease with high risk of morbidity and early death. HSCT is currently the only curative treatment for SCA, but its use has been limited by the risks of transplant-related mortality (TRM) and GVHD. We have previously demonstrated that antithymocyte globulin (ATG) efficiently decreases the rejection rate in HSCT for SCA; however, its optimal dose has not been defined, prompting us to retrospectively analyse outcomes in patients from the SFGM-TC cohort receiving different ATG doses.The cohort includes 236 SCA-patients (109F, 127M), (229 SS, 5 Sb0, 2 SDPunjab) transplanted in France (1988-2012) with geno-identical donors after homogeneous myeloablative conditioning regimen (CR; BU-CY), and rabbit ATG (n=215) at 5-15 mg/kg (n=35), 20 mg/kg (n=160) or no ATG (n=20). Cell sources were bone marrow (BM, n=197), cord blood (CB, n=30), CB+BM (n=8), and peripheral blood cells (PBC, n=1). Chimerism was assessed by quantitative real-time PCR from PBC at Day-30, 60, 90, Month-6, 12 and every year post-transplant, and defined as total donor chimerism (TDC): >95% donor (D); low (5-50%D) or high (50-95%D) mixed chimerism (MC),; or rejection (< 5%D). Disease-free survival (DFS) was defined as the absence of HSCT-related death and rejection. GVHD prophylaxis consisted of CSA-short MTX for BMT and CSA alone for CBT. Results Mean age at transplant was 9.7yr (range: 2.2-28.9), with 33 patients older than 15. With a mean follow-up of 5.6yr (range 0.5-22.5), TRM was not different with or without ATG (3.4%; 0.8-6%) whereas DFS was higher (p=0.004) with ATG (95.4%; 92-4-98.4) than without (72.7%; 51.7-93.7), due to differences in rejection rates (p<0.001). DFS was similar whether the cell source was CB or BM. No lymphoma was observed, and only one post-transplant death at 5m was virus-related (adenoviral meningo-encephalitis). Incidences of AGVH ≥2, 3-4 and chronic-GVHD (cGVHD) were 19%, 6% and 8.7%, respectively. Univariate Cox analysis showed that significant risk factors affecting DFS were low dose of ATG, AGVH grade, and cGVHD, whereas cell source, ABO compatibility, donor’s and recipient’s (DR) age, and CMV status were not. Risk factors associated with cGVHD (Logistic regression) were ATG dose (20% incidence with no ATG, 15.6% with 5-15 mg/kg and only 6.4% with 20), DR age (even after exclusion of CBT), whereas DR genders, DR CMV status, and cell source doses (NC, CD34, CD3+) were not. Multivariate analysis only retained ATG dose (OR=0.9/mg/kg increase, 95% CI:0.8-1, p=0.007) and donor’s age>15 (OR=7.9, 95%CI: 2.5-25.6,p<0.001) as significant independent risk factors for cGVHD. Chimerisms (n=165) (Figure) were significantly associated with ATG dose and cGVHD occurrence. Among the 6 patients with low MC at 1yr, 3 had not received ATG and rejected at 2, 2.2 and 8yr post-HSCT whereas the 3 others who had received ATG remained stable. All patients with high MC at 1yr (n=54) and TDC (n=80) are doing well with the same electrophoretic profile as their donor, excepted one patient who experienced appearance of MC with hemolysis (D/R group U+/U-) 5yr post-CBT, requiring donor lymphocyte infusion (DLI), which reversed chimerism to TDC but induced cGVHD. This study confirms that myeloablative CR with ATG offers 95% DFS to SCA-patients and that stable MC in absence of SCA-symptoms does not require intervention such as DLI. It reports for the first time in HSCT for SCA that donor’s age significantly increases GVHD risk, suggesting to choose the youngest sibling when possible, and that high ATG dose (20 mg/kg), which significantly reduces cGVHD risk without enhancing viral risk, should be recommended.Bernaudin: Novartis: Research Funding. Bertrand:ERYTECH Pharma: Principal Investigator Other. Gluckman:Cord use: Honoraria; gamida: Honoraria.

Sickle Cell Anemia (SCA) remains a disease with high risk of morbidity and early death. HSCT is currently the only curative treatment for SCA, but its use has been limited by the risks of transplant-related mortality (TRM) and GVHD. We have previously demonstrated that antithymocyte globulin (ATG) efficiently decreases the rejection rate in HSCT for SCA; however, its optimal dose has not been defined, prompting us to retrospectively analyse outcomes in patients from the SFGM-TC cohort receiving different ATG doses.

The long term prognosis of ETV6/RUNX1 -positive acute lymphoblastic leukemia (ALL) remains to be evaluated with regard to the frequency of late relapses and the possible existence of a preleukemic stem cell. We performed a retrospective study based on a long-term follow up of the FRALLE 93 ALL relapses to address the issue of the outcome of ETV6/RUNX1 -positive ones.1395 patients aged 0 to 20 years with untreated ALL (except L3) were included between 01-Jan-1993 and 31-Dec-1999. From 1995, children were systematically screened for four fusion transcripts (ETV6-RUNX1, BCR-ABL, E2A-PBX1, MLL-AF4). The FRALLE 93 study population was stratified into three groups (low-risk [LR], intermediate-risk [IR], and high-risk [HR]) based on the following prognostic factors: age, white-cell count at diagnosis, haemoglobin level, immunophenotype, karyotype, and response to steroids. Patients received an initial treatment comprised of a prednisone prophase and a triple-drug intrathecal injection. Induction treatment then included prednisone, vincristine, L-asparaginase, daunorubicin (except for the LR group), and one or two more triple-drug intrathecal injections (TIT). The main treatment features of the SR and IR protocol were induction, consolidation, delayed intensification, and maintenance (total treatment duration of 26 and 38 months for girls and boys respectively). Treatment of the HR patients consisted of induction, consolidation, two delayed intensifications, and maintenance with a total treatment time of 2 years. Depending on subgroups, CNS-directed therapy included intrathecal injections +/− high-dose methotrexate +/− cranial irradiation. Following factors influencing survival after first relapse were analyzed: age, leukocytosis, gender, duration of first remission (CR1), risk groups defined in the REZ-BFM 95/96 study, sites of relapse and post CR2 consolidation treatment (AlloSCT or not). Results. ETV6/RUNX1 status was defined for 724 patients B lineage ALL. Overall, 162 of the 713 children who reached CR1 (45 % of boys) relapsed, including 43 with t(12;21). Cumulative incidence of relapses did not differ between ETV6/RUNX1 -positive and negative ALL (p=0.94), with a 5-year estimate at 19.4% and 19.9%, respectively nor according to gender and type of relapse. Nevertheless, 11 out of 26 relapses in the ETV6/RUNX1 -positive ALL males (43%) were testicular (4 testis isolated) versus 16 out of 70 (23%) in ETV6/RUNX1 -negative ALL cases (p= 0.04). Thirty three (77%) had been stratified as LR (n=6) or IR (n=27) group and 32 displayed good early response at initial diagnosis. Thirty five (81.4%) patients were classified as S1/S2 and 8 (18.6%) as S3/S4. All but three received second line salvage therapy (37/40 were included in the COOPRALL 97) and 16 underwent an AlloSCT (6 S3/S4). Based on univariate analyses, the overall survival of ETV6-RUNX1 -positive ALL after relapse was significantly affected by the duration of the first remission with a OS that was significantly improved when relapse occurred after 36 months (5-year OS: 80.6+/−7.9% versus 34.7+/− 12.3, p=0.002). Female gender was also associated with a poor survival (p= 0.015), whereas the site of relapse (p= 0.13), age at initial diagnosis (p= 0.81), leukocytosis (p=0.42), and consolidation strategy (p=0.18) had no affect on survival. In multivariate Cox-regression analysis, only the duration of first remission remained associated with the outcome (Figure 1).We found a high rate of testicular relapse without any increase of other extramedullary sites and an excellent outcome for ETV6/RUNX1 -positive leukemia relapses occurring over 36 months post-diagnosis. These findings highlight the interest of a primary treatment able to cross the testicular barrier. They also support the hypothesis that ETV6/RUNX1 “late relapses” are due to a novel leukemic clone, sensitive to a novel cycle of ALL chemotherapy.No relevant conflicts of interest to declare.

Abstract 1508

There is increasing information about reduced intensity conditioning regimen AlloHSCT (Allogeneic Hematopoietic Stem Cell Transplantation) in children. The safety of this approach is now well established but data regarding efficacy are limited and the role in pediatric cancer has yet to be defined.We report results of a French pediatric AlloHSCT protocol with ATG-fludarabine (180 mg/m2) - Busilvex (3.2 at 4.8 mg/kg/d for 2 days) conditioning regimen. Related, unrelated bone marrow (BM) and Peripheral blood stem cell (PBSC) donors and Cord blood units (CB) were allowed. In case of CB a TBI 2 grays, Cyclophosphamide 50 mg/kg, Fludarabine 100 mg/m2 conditioning regimen was recommended. GVH prophylaxis consists in cyclosporine alone. A rapid discontinuation of systemic immunosuppression and re-injecting donor lymphocytes to initiate graft-versus-tumor effect are based on tumor assessment and blood chimerism. Inclusion criteria are children with malignancies that can be potentially cure by allograft but a conventional conditioning regimen being impossible due to toxicity and children with solid tumor or hematological malignancy remaining unresponsive to the reference strategies according to French best practices in pediatrics.From April 2007 to April 2010, 40 RIC AlloHSCT were performed in 10 different French pediatric graft centers: 13 Hodgkin Lymphoma, 7 acute myeloblastic leukaemia, 2 acute lymphoblastic leukaemia, 6 neuroblastoma, 8 rhabdomyosarcoma, 3 desmoplastic tumor and 1 Ewing sarcoma. Median age at transplantation was 15 years and median time from diagnosis to transplant was 18 months. Before transplant, 15 patients are in complete response and 25 patients (14/18 solid tumors) have active disease (11 progressive, 14 partial response). 21 had already received a myeloablative therapy (18 autograft and 3 allograft). Graft source was PBSC in 17 cases (7 related and 10 unrelated), BM in 18 (10 related and 8 unrelated), and 5 CB. The RIC Bu-flu conditioning regimen permits rapid engraftment without major toxicity contrary to the Cy-TBI in CB. 1 patient had primary graft failure: 1 CB and 5 patients experienced secondary graft failure: 3 CB, 1 PBSC and 1 BM. Median time to reach an ANC of 0.5 109/l was 16 days. Median time to reach a platelet count of 20 × 109/l was 2 days. Platelet count did not decrease below 20 109/l in 10 allografts. At day 30 post-transplant, chimerism is mainly donor for 30 and partial for 6 children. At day 100 post transplant, 4 out of 6 with initial mixed chimerism were converted into full donor chimerism. 8 patients received DLI and 17 patients experienced acute graft versus host disease (GvH) (2 grade IV and 15 grade ≤ II). A low day 365 TRM of 5% is reported in these heavily pre-treated patients. With a median follow-up of 15 months, the estimated 2 yr overall survival (OS) was respectively 57 % (71% for hematological malignancies and 42% solid tumors) (fig 1) and event free survival (EFS) 36% (50% for hematological malignancies and 19% solid tumors). Univariate analysis of EFS and OS showed no effect of related versus unrelated stem cell sources and BM versus PBSC. Our analysis identified a group of patients, who had no measurable disease at transplant, with a 2 yr OS and EFS of 86%. In term of efficacy, we observe a graft versus lymphoma effect in patients with advanced active Hodgkin lymphoma. Concerning solid tumors, all children included had a very bad prognosis and detectable disease before transplant. Our results may suggest that an immune-mediated effect cannot be excluded in some refractory solid pediatric tumors particularly in neuroblastoma. The main cause of failure of this approach is disease progression. Immunologic approaches after transplantation may help cure more of these very-high-risk patients.Even if further follow up is needed, this prospective study suggest that RIC regimen provides promising outcome in children previously not eligible for myeloablative AlloHSCT.This study “RICE” was registered at www.clinicaltrials.gov as NCT 007 50 126No relevant conflicts of interest to declare.

Despite progress made in sickle cell anemia (SCA) management, such as the prevention of pneumococcal infections, introduction of hydroxyurea therapy and early cerebral vasculopathy detection with transcranial Doppler, SCA remains a disease with high risk of morbidity and early death. Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for SCA; nevertheless, its use has been limited by the risk of transplant-related mortality (TRM). Our first experience, reported in Blood 2007, included 87 consecutive severe SCA- patients transplanted in France between 1988 and Dec-2004. We showed that the introduction of rabbit anti-thymocyte globulin (ATG) in the conditioning regimen in 2000 allowed a significant reduction of the rejection rate from 22.6% to 3% and that the outcome improved significantly with time as the DFS rate among the 44 patients transplanted after January 2000 was 95.3%. These data have justified continuing to transplant symptomatic young sickle cell patients having a geno-identical donor with the same conditioning regimen (CR) consisting of intravenous Busulfan (BU), Cyclophosphamide (CY) and rabbit ATG.In France, from 1992 to 2010, 144 SCA-patients (84M, 60F) have now been transplanted with a geno-identical donor using BU-CY-ATG as CR at the median age of 9.0 years (range:3.2-27.5). Transplants were performed in 16 different centers but 60 were performed in Hopital St-Louis and 21 in Hopital Debré in Paris. All recipients were SS or Sb0 and 76% of them were CMV+. All had been transfused and 47% had received more than 20 units. The source of cells was the bone marrow (BM) (n=121), cord blood (CB) alone (n=21), CSP (n=1) or BM+CB (n=1). GvHD prophylaxis consisted of the association of cyclosporine (CSA)-short MTX for BMT and CSA alone for CBT.Engraftment was successful in 141/144; the time to absolute neutrophil count > 500/mm3 was significantly shorter after BMT compared to CBT (mean ± SD; 21.3 ± 6.7 vs 32.1 ± 9.8, respectively; p<0.001) and platelets reached 50,000/mm3 sooner after BMT (day 28.3 ± 16.6) than after CBT (day 48.5 ± 20.3; p<0.001). No engraftment occurred in 3 cases (1 BMT, 2 CBT) with gradual autologous reconstitution, and one rejection was observed 3 years post-transplant. GvHA grade ≥ II occurred in 23% of patients, GvHA ≥ III in 4.4%, chronic GvH in 9.6% (extensive in 3 cases). No GvHD ≥ II or chGvHD were observed after CBT. Death occurred in 6 cases (4 were GvHD-related, 1 hemorrhagic stroke in a patient with severe cerebral vasculopathy with Moya and 1 sepsis in aplasia). No veno-occlusive disease was observed, but hemorrhagic cystitis (n=4), EBV proliferative disease requiring anti-CD20 therapy (n=1), nephrotic syndrome (n=1), and cerebral vasculopathy not SCA-related (n=1) were. Despite preventive measures such as anticonvulsant prophylaxis, strict control of hypertension, swift magnesium replacement, and an increase in the red blood cell and platelet transfusion thresholds to 9 g/dL and 50,000/mm3, respectively, seizures and posterior leukoencephalopathy, albeit reversible, remained a particularly frequent adverse effect of CSA and steroid therapy. Replacing CSA in 2002 by mycophenolate mofetil in case of GvHD requiring steroid therapy resulted in a significant reduction of the rate of these complications. With a median follow-up of 3.1 years (range 0.2–15.5), the overall survival at 3 yr was 95% (95%CI:91-99%). Considering as events the non-engraftments, rejections and deaths, the event-free survival (EFS) was 92.9% (95%CI:88.3-97.5). However, comparing the results before (n=23) and after 2000 (n=121) showed significant improvement of EFS at 3 yr: 73.9% (95%CI: 55.5–92.3) for transplants performed before 2000 vs 96.8% (95%CI:93.2-100) after 2000.These results with 121 patients transplanted since 2000 confirm that it is possible to offer more than 95% chances of cure to SCA-children, indicating that HLA-geno-identical HSCT after myeloablative conditioning with ATG should be considered as standard of care for SCA children, not only for those at high risk of stroke but also for children experiencing crises or other complications requiring intensive therapy such as transfusion program or hydroxyurea. Sibling cord-blood cryopreservation should be systematically offered and pre-implantation genetic diagnosis coupled with HLA selection discussed with the parents.No relevant conflicts of interest to declare.

Gluckman: Cord-use: Membership on an entity's Board of Directors or advisory committees.

Abstract 534

Abstract 3518

Abstract 975

From 06/93 to 12/99, 1395 children and adolescents were included in the FRALLE 93 protocol: group A (Very Low Risk): 182, B (Standard risk): 672 and C (High risk including T-ALL): 541. The median FU is 9.5 yr. Inclusion criterias in group B were: age between 1 and 15 yr, WBC< 100,000, BCP phenotype and no poor-prognosis cytogenetic feature (MLL rearrangement or Ph1). Group B pts were randomised in a 2-steps schedule: at inclusion: daunorubicin (DNR: 40 mg/m2 × 2) vs idarubicin (IDA: 8 mg/m2 × 2), and at consolidation: high-dose IV methotrexate (HD-MTX): 4 × 8g/m2 at D1, D15, D29 and D42, vs low-dose po MTX (LD-MTX): 4 × 25 mg/m2; the number of triple IT was the same for both arms (N = 18). Pts with CNS leukemia were not randomized for MTX. During induction, pts were also classified on D21 bone marrow aspiration according to the level of residual blasts: M1 (< 5%): n=555, M2 (6–25%): n=71 and M3 (> 25%): n=41; M2/M3 pts received 1 more antracyclin infusion at D22 and M3 pts were subsequently treated according to the HR group and not randomized for MTX. Both M1 and M2 pts who reached CR were randomized for MTX. The overall group B EFS, DFS and OS were respectively 80±2, 81±2, and 91±1%. Globally there is no statistically significant impact of 1st or 2nd randomization on the outcome of patients. When we look at the outcome according to the D21 status, the EFS are very different: M1: 84±2%, M2: 63±6% and M3: 74±7%. The very poor EFS of M2 pts, who can not actually be classified as SR pts, and the fact that M1 pts represent 90% of pts randomized for MTX led us to analyse separately M1 pts. For M1 pts there is no interaction between 1st and 2nd randomization for survival, EFS and relapse rate (Gail and Simon test, p = 0.51); therefore pts were pooled for 2nd randomization analysis. Among M1 pts, 271 pts were randomized for HD-MTX and 264 for LD-MTX; there is no difference regarding age or leucocytosis between the 2 arms. Results are as follows: 5-yr EFS Relapses CI iBM relapses CI Other relapses CI 5-yr OS (EFS: event-free survival, CI: cumulative incidence, iBM: isolated bone-marrow, OS: overall survival) HD-MTX 87.82±1.99% 0.12±0.04 7.01±0.02 5.17±0.02 95.20±1.30 LD-MTX 79.92±2.47% 0.20±0.06 10.6±0.03 9.10±0.03 88.64±1.95 Test log-rank Gray Gray Gray log-rank p value 0.028 0.037 0.16 0.18 0.07 At the contrary, among M2 pts there is no difference in EFS for pts treated with HD-MTX (61±7%) and LD-MTX (65±9%) Conclusion: HD-MTX seems to benefit to pts with standard-risk BCP-ALL and good early response to chemotherapy, and the benefit is associated with a reduction in the number of relapses. No benefit is demonstrated in M2 pts with SER who have a very poor outcome and do require a more intensive treatment; the better EFS of M3 patients treated with double delayed intensification is in favour of this hypothesis.