ALBERT

Description: The use of umbilical cord blood as a source of marrow repopulating cells for the treatment of pediatric malignancies has been established. Given the general availability, the ease of procurement, and progenitor content, cord blood is an attractive alternative to bone marrow or growth factor mobilized peripheral blood cells as a source of transplantable hematopoietic tissue. However, there is a major potential limitation to the widespread use of cord blood as a source of hematopoietic stem cells for marrow replacement and gene therapy. There may be enough hematopoietic stem cells to reconstitute children, but the ability to engraft an adult might require ex vivo manipulations. We describe an in vitro system in which the growth of cord blood CD34+ cells is sustained and greatly expanded for more than 6 months by the simple combination of two hematopoietic growth factors. Progenitors and cells belonging to all hematopoietic lineages are continuously and increasingly generated (the number of colony-forming unit–granulocyte-macrophage [CFU-GM] present at the end of 6 months of culture are well over 2,000,000-fold the CFU-GM present at the beginning of the culture). Very primitive hematopoietic progenitors, including long-term culture-initiating cells (LTC-ICs) and blast cell colony-forming units, are also greatly expanded (after 20 weeks of liquid culture, LTC-IC number is over 200,000-fold the initial number). The extremely prolonged maintenance and the massive expansion of these progenitors, which share many similarities with murine long-term repopulating cells, suggest that extensive renewal and little differentiation take place. This system might prove useful in diverse clinical settings involving treatment of grown-up children and adults with transplantation of normal or genetically manipulated hematopoietic stem cells.

Description: During development, mice with mutations of stem cell factor (SCF) or its receptor c-kit exhibit defects in melanogenesis, as well as hematopoiesis and gonadogenesis. Consequently, accumulating evidence suggests that the c-kit/SCF system plays a crucial role in all of these processes and in tumors which derive from them. Especially in neuroblastoma (infant tumors of neuroectoderm crest derivation such as melanocytes) it would appear that an autocrine loop exists between c-kit and SCF, and that the functional block of the c-kit receptors with monoclonal antibodies (MoAbs) results in a significant decrease in cellular proliferation. We studied the expression and role of c-kit and SCF in cell lines of soft tissue sarcoma of neuroectodermic origin, such as Ewing's sarcoma (ES) and peripheral neuro-ectodermal tumors (PNET). Using flow cytometry with MoAb CD117 PE, c-kit expression was highlighted in all six of the cell lines examined. This receptor was specifically and functionally activated by SCF, as shown by the binding experiments and the intracellular phosphotyrosine and immunoprecipitation studies that were performed. Using reverse transcriptase polymerase chain reaction analysis, five of the six cellular lines expressed the mRNA of SCF. In the medium measured by using an enzyme- linked immunosorbent assay, low concentrations of SCF were found: only the TC32 cellular line produced significantly higher levels (32 pg) than control. In serum-free culture the addition of SCF reduced the percentage of apoptotic cells from 25% to 90% in five out of the six cellular lines. This observation was confirmed by (1) the functional block of c-kit with MoAb: after 7 days of culture more than 30% of the cells were apoptotic (range 31.5% to 100%) in five out of six cell lines and there was also a decrease in the percentage of cells in phase S, and (2) c-kitantisense oligonucleotides: in the cellular lines treated with oligonucleotides (in relation to the untreated lines) there was a notable reduction (P 

Description: During development, mice with mutations of stem cell factor (SCF) or its receptor c-kit exhibit defects in melanogenesis, as well as hematopoiesis and gonadogenesis. Consequently, accumulating evidence suggests that the c-kit/SCF system plays a crucial role in all of these processes and in tumors which derive from them. Especially in neuroblastoma (infant tumors of neuroectoderm crest derivation such as melanocytes) it would appear that an autocrine loop exists between c-kit and SCF, and that the functional block of the c-kit receptors with monoclonal antibodies (MoAbs) results in a significant decrease in cellular proliferation. We studied the expression and role of c-kit and SCF in cell lines of soft tissue sarcoma of neuroectodermic origin, such as Ewing's sarcoma (ES) and peripheral neuro-ectodermal tumors (PNET). Using flow cytometry with MoAb CD117 PE, c-kit expression was highlighted in all six of the cell lines examined. This receptor was specifically and functionally activated by SCF, as shown by the binding experiments and the intracellular phosphotyrosine and immunoprecipitation studies that were performed. Using reverse transcriptase polymerase chain reaction analysis, five of the six cellular lines expressed the mRNA of SCF. In the medium measured by using an enzyme- linked immunosorbent assay, low concentrations of SCF were found: only the TC32 cellular line produced significantly higher levels (32 pg) than control. In serum-free culture the addition of SCF reduced the percentage of apoptotic cells from 25% to 90% in five out of the six cellular lines. This observation was confirmed by (1) the functional block of c-kit with MoAb: after 7 days of culture more than 30% of the cells were apoptotic (range 31.5% to 100%) in five out of six cell lines and there was also a decrease in the percentage of cells in phase S, and (2) c-kitantisense oligonucleotides: in the cellular lines treated with oligonucleotides (in relation to the untreated lines) there was a notable reduction (P 

Description: Cord blood transplant (CBT) is associated with delayed or failed engraftment in about 20% of patients. We present here the results of intra-bone (IB) transplant of cord blood cells from two Institutions. Fifty-eight consecutive adult patients were included in this study. Patients were: 29 AMLs (10 in first, 3 in second remission and 15 in advanced stage,1 secondary), 15 ALLs (1 in first, 5 in second remission and 9 in advanced stage) 4 CMLs (2 Acc. Phase, 2 Blastic Transformation), 3 refractory Hodgking Disease, 4 refractory Non-Hodgking Lymphomas (HD), 1 Atypical myeloproliferative disease (after HD), 1 SAA and 1 Diskeratosis Congenita (DC). Patients’ median age was 36 years (18–66). Human Leukocyte Antigen (HLA) matching was 6/6, 5/6, 4/6 and 3/6 for one, 10, 46 and one patient, respectively. Conditionig Regimen consisted in: fractionated TBI (10–12 Gy)/cyclophosphamide(CTX) (n=42), Fludarabin (Flu)-CTX-TBI 2 Gy (n=6), Treosulfan (Treo)-Thiotepa (TT)-Flu (n=10). Median transplanted cell dose was 2.6 x107/kg (1.4–5.4) and CD34+ cell dose was 0.83 x105/kg (0.42–4.53). Cord blood cells were gently infused in the supero-posterior iliac crest (SPIC) under rapid general anaesthesia. In 21 patients the procedure was performed in both (right and left) SPIC while in 37 patients in either left or right SPIC. No complications were observed during or after the IB infusion of cells. Eight patients with advanced disease died within 12 days from transplant. Two patients did not engraft; one (SAA) had a second IB CBT but engrafted only after mesenchymal stem cell infusion; the other patient (CML-AP) is being re-transplanted. All other patients engrafted. Median time to neutrophils (PMN) and platelet (Plt) recovery was day +23 (14–44) and day +38 (16–64) respectively. Kaplan-Meier probabilities were then 100% for both PMN and Plt. at day +44 and day +64, respectively. Cumulative incidence were 87% (95% CI: 73–97) at day +44 for PMN and 82% (95% CI: 67–95) at day +64 for Plt. At day +30 haematopoietic progenitors Colony Forming Cells frequency were within the limit of normal range both in injected and un-injected site; this shows a rapid recirculation and seeding in the entire hematopoietic system. Chimerism was 100 % donor in all patients from day +60. In 47 patients at risk for acute Graft-versus-Host Disease (GVHD) the score was: grade 0 (n=37) grade I (n=3) and grade II (n=6), grade III (n=1). Seven patients had moderate and two patients extensive chronic GVHD. Causes of death were multiorgan failure(n=8), infections (n=12), PTLD (n=1) and relapse/progression (n=5). Thirty-two patients are alive: 4 in relapse/progression; one with graft failure; and 27 in haematological remission at a median follow-up of 20 months (2–29). Three pediatric patients (age 1– 7 years) were also included in this study: 1 JMML in graft failure after BMT; 1 ALL with t (9;22) in first CR and 1 Hemophagocytic Lymphohistiocytosis (HLH) with active disease following three unrelated BMT. HLA matching was 5/6 (1pt) and 4/6 (2 pts); Conditioning Regimen were: TT+Flu+L-PAM, TBI 12 Gy +CTX+TT, Treo+TT+Flu; median cell dose injected IB was 4.6 x107 TNC (3.0–7.0) and 0.76 x105 CD34+ (0.3–2) respectively. Patients achieved PMN and Plt. recovery at 12-27-47 and 31-34-63 days from IB-injection. All patients are alive, disease-free and full donor chimerism at 249, 172 and 85 days following IB CBT. Our data suggest that direct i.b. cord blood transplant is associated with a very high rate of engraftment even when low numbers of HLA mismatched cord blood cells are transplanted, thus, rendering this transplant possible in a greater number of patients with hematologic diseases. Moreover, high risk pediatric patients may also benefit of IB transplant as salvage procedure.

Description: Abstract 806 Objective: The analysis of donor safety and early side effects related to hematopoietic stem cells (HSC) collection from bone marrow (BM) or peripheral blood (PB) in pediatric HLA-identical sibling donors. Methods: From 2005 to 2009, data regarding pediatric (

Description: Introduction Using the EBMT registry, we retrospectively analyzed outcomes for 373 pediatric patients who underwent second allogeneic transplant for relapsed acute leukemia at 120 centers in 32 countries, between the years 2004 and 2013, in an attempt to assess relapse, survival, GVHD and other outcomes, as well as identify factors correlating with prognosis in this cohort of patients. To our knowledge, this is the largest analysis of pediatric patients undergoing second allogeneic HSCT for relapsed acute leukemia to date. This allowed for an independent analysis of each disease, including 214 patients with ALL and 159 with AML. Patients and Methods Centers received a questionnaire completing data already available in the ProMISe database on patients between 0-18 years of age treated between 2004 and 2013. Results A total of 387 patients received a second SCT after relapse. 373 have been included in the analysis, 214 for ALL and 159 for AML. Detailed data were available for 201 patients from 48 centers; for the remainder, analysis was based on the registry. For the entire cohort overall survival (OS) at 2 and 5 years were 38% and 29%, and leukemia free survival (LFS) 30% and 25% respectively. ALL: With a median follow up from 2nd SCT of 36.4 months, OS at 1 and 5 years were 47% and 28% respectively. LFS was 39% and 28% respectively. NRM at 2 years was 22%. In multivariate analyses favorable prognostic factors for both OS and LFS were: CR prior to 2nd SCT (p=0.0001), interval 〉 12 months between transplants (p=0.0007), use of myeloablative conditioning (p=0.039) and the presence of cGvHD after the first SCT (p=0.0001). Good prognostic factor for low NRM was interval of more than 12 months between transplants (p=0.0002). AML: With a median follow up from 2nd SCT of 50 months, OS at 1 and 5 years were 44% and 15% respectively. LFS was 28% and 15% respectively. NRM at 2 years was 18%. In multivariate analyses, favorable prognostic factors for OS as well as LFS were: CR prior to 2nd SCT (p=0.031;0.044 respectively), interval 〉 6 months between transplants (p=0.0003;0.0001 respectively), and having cGvHD after the first SCT (p=0.0001). Most patients experience disease relapse or NRM within the first year after their second transplant. This observation seems to be more consistent in patients transplanted for ALL, with more changes over time in patients with AML. For ALL in particular, the 2-year incidences of relapse, NRM and LFS were not different from those at 5-years. Even in the relapse setting, survival rates for patients with ALL remain superior to patients with AML, consistent with the prognostic differences at diagnosis. Our findings, consistent for the AML and ALL subgroups, suggest that cGHVD prior to second HSCT is associated with better outcome. The identification of cGHVD prior to second transplant has not been heretofore described as a favorable prognostic factor. This strong correlation merits further study, specifically as to the underlying biology for this association. Conclusion Children with relapsed acute leukemias have a substantial chance to become long term survivors following a second SCT. CR prior to second SCT, longer interval between transplants and the presence of cGvHD after the first transplant, are favorable prognostic factors for ALL and AML. Our findings may help physicians in discussing the risk-benefit of a second transplant. These results are particularly relevant in an era where an explosion of new therapies, specifically targeted therapies and those that modulate the immune response, behoove us to carefully identify subpopulations of patients for whom specific therapies are appropriate. Novel approaches are needed to minimize relapse risk as well as short and long term morbidity in these pediatric patients while considering a second SCT for relapsed acute leukemia. Disclosures Corbacioglu: Jazz Pharmaceuticals: Consultancy, Honoraria. Bader: Novartis, Medac, Amgen, Riemser, Neovii: Consultancy, Honoraria, Research Funding.