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The effect of G-CSF, GM-CSF, IL-1 and IL-3 on the proliferation of acute leukemia cells (evaluated as (3)HTdR uptake) was investigated in short-term liquid cultures and compared with that observed on normal bone marrow (BM) populations enriched for immature cells. In acute myeloid leukemias (AML), a marked leukemic proliferation was induced in 10/18 cases by IL-3, in 9/18 by GM-CSF, in 7/18 by IL-1 and in 4/18 by G-CSF. In acute lymphoid leukemias (ALL), marked stimulation was observed in 7/11 cases with IL-3 and in 5/11 with GM-CSF, whereas IL-1 and G-CSF were ineffective. Both in AML and ALL, the combination of several factors did not result in an additive synergistic effect. Purified normal BM cells responded to all four growth factors and their combinations produced an additive effect on cell proliferation which probably relates to the heterogeneity of the cell populations studied. The effect of a G-CSF, GM-CSF, IL-1 and IL-3 on the proliferation of acute leukemia cells by different growth factors suggests that caution should be exercised in their clinical use in these diseases.

The use of granulocyte colony-stimulating factor (G-CSF) primed bone marrow (G-BM) has been recently considered as an alternative to mobilized hematopoietic stem cells from peripheral blood (G-PB), especially in the haploidentical transplant setting. The purpose of this study was to compare the effect of in vivo G-CSF priming on BM and PB hematopoietic, mesenchymal (MSC), and immune cells. Forty healthy donors undergoing BM harvest for haploidentical transplant were given subcutaneous recombinant human G-CSF for 7 days. BM and PB samples were harvested on days -7 and 0. The hematopoietic stem/progenitor cells increased significantly after G-CSF priming in both BM and PB with a selective rise of BM CD34(+)CD38(-) cell subset. A striking enhancement of the mesenchymal progenitors was detected in G-BM. CD3(+), CD4(+), CD8(+), and CD19(+) cell fractions; the naive CD4(+) and CD8(+) subpopulations; and natural killer and regulatory T cells increased in G-BM, whereas only slight changes were detected in PB. Myeloid dendritic cells (DC1) were significantly up-regulated in both G-BM and G-PB, whereas DC2 increased only in G-BM. In conclusion, our results show substantial differences in the biologic effects exerted by G-CSF at BM and PB levels on hematopoietic cells and immune cell fractions. Furthermore, the impressive rise of MSC progenitors in G-BM might also be relevant to provide MSCs for several clinical use.

Ex vivo amplification of human hematopoietic stem cells (HSC) without loss of their self-renewing potential represents an important target for transplantation, gene and cellular therapies. Valproic acid is a safe and widely used neurologic agent that acts as a potent inhibitor of histone deacetylase activities. Here, we show that valproic acid addition to liquid cultures of human CD34+ cells isolated from cord blood, mobilized peripheral blood, and bone marrow strongly enhances the ex vivo expansion potential of different cytokine cocktails as shown by morphologic, cytochemical, immunophenotypical, clonogenic, and gene expression analyses. Notably, valproic acid highly preserves the CD34 positivity after 1 week (range, 40-89%) or 3 weeks (range, 21-52%) amplification cultures with two (Flt3L + thrombopoietin) or four cytokines (Flt3L + thrombopoietin + stem cell factor + interleukin 3). Moreover, valproic acid treatment increases histone H4 acetylation levels at specific regulatory sites on HOXB4, a transcription factor gene with a key role in the regulation of HSC self-renewal and AC133, a recognized marker gene for stem cell populations. Overall, our results relate the changes induced by valproic acid on chromatin accessibility with the enhancement of the cytokine effect on the maintenance and expansion of a primitive hematopoietic stem cell population. These findings underscore the potentiality of novel epigenetic approaches to modify HSC fate in vitro.

Osteoclasts from a patient affected by osteopetrosis were examined in vivo and in vitro. Iliac crest biopsy revealed an osteosclerotic pattern, with prominent numbers of osteoclasts noted for hypernuclearity and incomplete adherence to the bone surface. A population comprising tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated and mononuclear cells, and alkaline phosphatase-positive stromal fibroblasts was obtained in vitro from bone marrow. Mononuclear TRAP-positive precursors spontaneously fused in culture to form giant osteoclast-like cells. These cells expressed the osteoclast marker MMP-9 and calcitonin receptor, and lacked the macrophage marker, Fc receptor. Expression and distribution of c-src, c-fms, and CD68, and response to steroid hormones relevant to osteoclast differentiation and function were apparently normal, whereas cell retraction in response to calcitonin was impaired. TRAP-positive multinucleated cells did not form osteoclast-specific adhesion structures (clear zone, podosomes, or actin rings). Bone resorption rate was severely reduced in vitro. Focal adhesions and stress fibers were observed en lieu of podosomes and actin rings. Adhesion structures contained low levels of immunoreactive vitronectin receptor, most of this integrin being retained in cytoplasmic vesicles. These data provide the first characterization of abnormal differentiation and function of human osteopetrotic osteoclast-like cells.

We prospectively studied the chimaerism status in the bone marrow (BM) and peripheral blood (PB) of 23 patients receiving umbilical cord (UCB, 14 cases) or BM (nine cases) transplants from unrelated donors by PCR amplification of four individual-specific VNTR genetic loci. Haematological engraftment, with persistent full donor pattern. was observed in 10/14 (72%) patients receiving UCB and in 9/9 (100%) patients transplanted with marrow from an unrelated donor (MUD). In contrast, the remaining four patients converted to an autologous pattern. Three out of these four patients had an early autologous haematological reconstitution reaching a neutrophil level >0.5 x 10(9)/l at days 27, 33 and 37 after transplant, respectively. In all three of these patients, chimaerism analysis demonstrated an early appearance of donor cells (i.e. within 35 d after UCB transplant) showing a transient full donor (one case) or mixed chimaerism condition (two cases). Despite the early autologous haemopoietic reconstitution, one of the three patients died of GVHD at day 60, which was explained by the demonstration of low levels of donor lymphoid cells. In the MUD group all nine patients converted to a persistent full donor pattern with haematological reconstitution, accompanied in two of them by transient mixed chimaerism lasting to days 60 and 270 after transplant. Our data show that monitoring of chimaerism may predict graft failure with or without early autologous haemopoietic reconstitution in patients receiving unrelated UCB transplants. Furthermore, chimaerism analysis may identify, in patients with autologous reconstitution, those at risk of severe GVHD in whom immunosuppressive therapy should not be discontinued.

Eighty patients with high-risk hematologic malignancies underwent unmanipulated, G-CSF–primed BM transplantation from an haploidentical family donor. Patients were transplanted in first or second complete remission (CR, standard-risk: n =45) or insecond CR or active disease (high-risk: n =35). The same regimen for GVHD prophylaxis was used in all cases. The cumulative incidence (CI) of neutrophil engraftment was 93% 0.1%. The 100-day CIs for II-IV and III-IV grade of acute GVHD were 24% 0.2% and 5% 0.6%, respectively. The 2-year CI of extensive chronic GVHD was 6% 0.1%. The 1-year CI of treatment-related mortality was 36% 0.3%. After a median follow-up of 18 months, 36 of 80 (45%) patients are alive in CR. The 3-year probability of overall and disease-free survival for standard-risk and high-risk patients was 54% 8% and 33% 9% and 44% 8% and 30% 9%, respectively. In multivariate analysis, disease-free survival was significantly better for patients who had standard-risk disease and received transplantations after 2007. We conclude that unmanipulated, G-CSF–primed BM transplantation from haploidentical family donor provides very encouraging results in terms of engraftment rate, incidence of GVHD and survival and represents a feasible, valid alternative for patients with high-risk malignant hematologic diseases, lacking an HLA identical sibling and in need to be urgently transplanted.Haploidentical, unmanipulated, G-CSF-primed bone marrow transplantation. Haploidentical hematopoietic stem cell transplantation for hematologic malignancies.

Hematopoietic stem and progenitor cells (HSPC) can improve the long-term outcome of transplanted individuals and reduce the relapse rate. Valproic acid (VPA), an inhibitor of histone deacetylase, when combined with different cytokine cocktails, induces the expansion of CD34 + cell populations derived from cord blood (CB) and other sources. We evaluated the effect of VPA, in combination with thrombopoietin (TPO), on the viability and expansion of CB-HSPCs and on short- and long-term engraftability in the NOD/SCID mouse model. In vitro , VPA + TPO inhibited HSPC differentiation and preserved the CD34 + cell fraction; the self-renewal of the CD34 + TPO + VPA-treated cells was suggested by the increased replating efficiency. In vivo , short- and long-term engraftment was determined after 6 and 20 weeks. After 6 weeks, the median chimerism percentage was 13.0% in mice transplanted with TPO-treated cells and only 1.4% in those transplanted with TPO + VPA-treated cells. By contrast, after 20 weeks, the engraftment induced by the TPO + VPA-treated cells was three times more effective than that induced by TPO alone, and over ten times more effective compared to the short-term engraftment induced by the TPO + VPA-treated cells. The in vivo results are consistent with the higher secondary plating efficiency of the TPO + VPA-treated cells in vitro .

Background. Persistent mixed chimerism represents a state wherein recipient and donor cells stably co-exist after haematopoietic stem cell transplantation. However, since in mostly of the studies reported in literature the engraftment state was observed in the nucleated cells, in this paper we determined the donor origin in the mature erythrocytes of patients with persistent mixed chimerism after transplantation for haemoglobinopathies. Results were compared with the engraftment state observed in singularly picked-up burst-forming unit-erythroid colonies and in the nucleated cells collected from the peripheral blood and from the marrow. Design and Methods. The donor origin of the erythrocytes was determined analyzing differences on the surface antigens of the erythrocytes suspension after incubation with anti-ABO and/or anti-C, -c, -D, -E and -e monoclonal antibodies by a flow cytometer. Short tandem repeats analysis was used to determine the donor origin of nucleated cells and burst-forming unit-erythroid colonies singularly picked up after 14 days incubation. Results. A proportion of donor-derived nucleated cells of 71%, 46%, 15% and 25% was observed at day 1364, 1385, 1314 and 932 respectively, in four transplanted patients affected by haemoglobinopathies. Similar results were also obtained in the erythroid precursors, analyzing the donor/recipient origin of the burst-forming unit-erythroid colonies, while on the contrary, at the same days of observation, a proportion of 100%, 100%, 73% and 90% donor-derived erythrocytes was observed in the four patients with persistent mixed chimerism. Conclusions. Our results showed that mostly of the erythrocytes present in four long-term transplanted patients affected by haemoglobinopathies and characterized by the presence of few donor engrafted nucleated cells were of donor origin. The indication that small proportions of donor engrafted cells might be sufficient to clinical control the disease in patients affected by haemoglobinopathies is relevant, although the biological mechanisms underlying these observations need to be further investigated.

The aim of this single center study was to assess the impact of pre-transplant factors on long-term follow-up in young patients affected by high-risk acute lymphoblastic leukemia (ALL) who underwent an unrelated cord blood transplant (CBT). The conditioning regimens, graft-versus-host disease (GVHD) prophylaxis and supportive policies were uniform for all patients.We analyzed the results of CBT performed in 30 patients, aged18 years, affected by high risk ALL. As conditioning regimen, all patients received 12 Gy fractionated total body irradiation, etoposide, cyclophosphamide and horse anti-lymphocyte globulin. GVHD prophylaxis consisted of 6-methylprednisolone and cyclosporine A. RESULTS The cumulative incidence of engraftment was 93% (95% CI:0.85-0.93). The cumulative incidence of grade III-IV acute and chronic GVHD was 7% (95% CI:0.01-0.19) and 33% (95% CI: 0.17-0.64), respectively. The 9-year cumulative incidence of transplant-related mortality and relapse was 34% (95% CI:0.13-0.45) and 31% (95% CI:0.16-0.61), respectively. The 9-year overall survival, leukemia-free survival and event-free survival were 42% (95% CI:0.52-0.93), 47% (95% CI:0.25-0.61) and 46% (95% CI:0.33-0.61), respectively. A number of CFU-GM1 x 10(4)/Kg of recipient body weight was the only factor that negatively affected all outcome parameters both in univariate and multivariate analyses.The infused cell dose expressed as in vitro progenitor cell growth represents the most important pre-transplant factor affecting the long-term outcome after an unrelated CBT in young patients with high risk ALL. The number of CFU-GM should thus be considered in the selection process of cord blood units for transplant.

Chronic graft-versus-host disease (GVHD) remains the most common late complication of allogeneic stem cell transplantation, producing significant long-term morbidity and contributing to a substantial risk of late mortality. Chronic GVHD may be more common, more protracted and less responsive to current treatments after peripheral-blood stem cell (PBSC) transplantation than after bone marrow transplantation. The purpose of this retrospective cohort study was to determine whether the hazard of extensive chronic GVHD after allogeneic PBSC transplantation could be decreased by prolonging cyclosporine A (CsA) prophylaxis over 12 months.Fifty-seven consecutive patients with hematologic malignancies who had received a PBSC transplant from an HLA-identical sibling were evaluable for chronic GVHD. All patients began CsA tapering at day 50 but 2 different durations of immunosuppression were used: the first 36 patients were allocated to receive a 6-month course with tapering by 5% at weekly intervals (group A), while the following 21 received a 12-month course with tapering by 5% every 2 weeks (group B).The cumulative incidence of extensive chronic GVHD at 2 years was 69% (95% CI, 53-85%) for group A and 25% (95% CI, 3-47%) for group B with a significantly lower hazard in group B than in group A (HR=0.2; 95% CI, 0.07-0.57; p=0.0009). In multivariate analysis, the 12-month CsA tapering schedule was associated with a significantly decreased risk of extensive chronic GVHD (HR=0.2; 95% CI, 0.06-0.66; p=0.008). The hazard of transplant-related mortality, relapse and failure to survive in remission was not significantly different among the 2 groups.One-year CsA prophylaxis seems to be more effective than the standard six-month CsA regimen at preventing extensive chronic GVHD after PBSC transplant from an HLA-identical sibling. Conclusive assessment of the benefits of such prolonged immunosuppression, in terms of better quality of life and minor morbidity, requires both long-term follow-up to evaluate the rates of relapse and secondary tumors and a randomized setting.

Peripheral blood stem cells (PBSC) are widely used in the setting of dose-intensive chemotherapies in patients with multiple myeloma (MM). Although the granulocyte colony-stimulating factor (G-CSF), following chemotherapy or not, is considered the standard growth factor for mobilizing PBSC, the optimal chemotherapeutic regimen still remains to be defined. Cyclophosphamide (CTX) is an effective drug in the treatment of MM which is capable of mobilizing PBSC if followed by growth factors, even though administration of high-dose CTX (7 g/m2) results in severe toxicity requiring hospitalization and increasing costs. We have retrospectively analyzed the results obtained in 38 newly diagnosed MM patients treated with 1.2 g/m2 CTX on days 1 and 3 combined with 40 mg dexamethasone daily for 4 days. The results were compared with those obtained in 25 newly diagnosed MM patients treated with 7 g/m2 CTX. A higher number of CD34+ cells/kg was collected during the first leukapheresis and a statistically significant lower consumption of G-CSF was observed following two doses of 1.2 g/m2 CTX compared to the 7 g/m2 CTX dose. The possibility of treating patients with day-hospital regimens, with a satisfactory yield of hematopoietic cells harvested, may have relevant economic implications for treatment strategies in MM patients.

Summary Several studies have demonstrated that G-CSF, GM-CSF and, in particular, IL-3 can effectively recruit acute myeloid leukaemia (AML) blasts into the cell cycle, resulting in a significant increase in cytosine-arabinoside (Ara-C) mediated cytotoxicityin vitro. Since IL-3 has shown biological and clinical activity, we investigated the cell kinetic effects of rIL-3 and high-dose Ara-C/idarubicin in three patients with refractory AML selected for the presence of chromosome 7 monosomy; this enabled differentiation between the effects of IL-3 on leukaemic and on normal cells. Thein vivo administration of rhIL-3 (250μ/m2 d s.c. for 6–10 d) recruited AML blasts into the cell cycle in two of the three patients, and this effect resulted in an increase inin vitro growth of clonogenic cells (CFU-L) and of their S-phase fraction. The percentage of leukaemic cells with monosomy 7 increased only in the two cases who showed a proliferative response. Normal cells were not recruited, even when rhIL-3 was administered for up to 10 d.In vitro studies showed an increased Ara-C cytotoxicity on clonogenic AML cells, in particular with LL-3 plus GM-CSF, thus confirming the priming effects of IL-3 in the two responding cases. The results of this study suggest that rhIL-3 can selectively recruit leukaemic cells into the cell cycle. Although leukaemic blasts can be sensitized to Ara-C, other mechanisms of primary blast resistance may limit the clinical benefit of kinetic-based approaches.

Abstract 2350 In this study we investigated the feasibility and clinical value of non T-cell depleted bone marrow transplantation (BMT) from HLA haploidentical related donor in patients with high risk hematological malignancies. Materials and Methods. Between August 2005 and May 2010, 71 patients were transplanted for acute myeloid leukemia (AML) (n=42), acute lymphoblastic leukemia (n=13), chronic myeloid leukemia (n=5), Hodgkin lymphoma (n=5), plasmacell leukemia (n=3), myelofibrosis (n=2) and myelodisplastic syndrome (n=1). Their median age was 35 years (5-71). At time of BMT, all patients were at very high risk: 39 were in early (complete remission 1 or 2) and 31 in more advanced stage of disease. Seventeen of them were given a previous transplant either autologous (n=14) or allogenic (n=3). All donors were HLA identical at 1 haplotype and mismatched for 2 (n=24) or 3 (n=47) loci on the unshared haplotype. As pretransplant regimen, 10 patients received a reduced intensity conditioning consisting of Fludarabine (Flu) alone (n=1), Flu + Thiotepa (Thio) + Melphalan (n=2) or Flu + Thio + i.v.Busulphan (Bu) (n=7), and 61 patients received a myeloablative therapy consisting of Aracytin + Cyclophosphamide combined with TBI (n=7) or Treosulphan (n=11) or Bu (n=11), whereas the last 32 consecutive patients underwent transplant after conditioning with the association of Thio + Flu + i.v. Bu. All patients received an identical graft-versus-host disease (GvHD) prophylaxis consisting of Fresenius Antithymocyte Globulin (5 mg/Kg/d from day -4 to -1) combined with Cyclosporine (1,5-3 mg/Kg/day i.v. from day -7 to +28 and orally 5 mg/Kg until day +365), Methotrexate (15 mg/sqm on day +1 and 10 mg/sqm on day +3, +6 and +11), Mycophenolate Mofetil (1 g/d from day +7 to +100) and the anti-CD 25 monoclonal antibody Basiliximab (20 mg i.v. on day 0 and +4). Bone marrow cells were harvested from all donors after priming with Filgrastim at 3–4 microg/Kg/d from day -7 to -1. Bone marrow cells were infused fresh and unmanipulated on day 0. Results. The median dose of total nucleated, CD34+ and CD3+ cells infused was 7.8 (1-28) ×108/kg, 2.1 (0.8-11) ×106/Kg and 28 (10-98) ×106/Kg, respectively. One patient had a primary graft failure and 5 patients died too early to be evaluated for engraftment. Results in terms of cumulative incidence (CI) of PMN engraftment, acute and chronic GvHD, relapse, transplant related mortality (TRM) and overall survival (OS) estimated with the Kaplan Meyer method are given in the Table. After a median follow-up of 16 (3-56) months, the 3 years probability of OS and disease-free survival (DFS) was 43% and 37% respectively for all patients (see Figure). The 3 years OS for the 42 patients with AML was 48% (61% for 30 patients transplanted in early stage and 18% for 12 patients transplanted in advanced stage, P=0.01). The 1 year OS for the 32 patients transplanted with the conditioning therapy employed in the last 2 years (Thio + Flu + i.v. Bu) was 72% in 18 patients transplanted in early stage and 52% in 14 patients transplanted in advanced stage (P=0.47). Early stage Advanced stage All patients P value N. 39 32 71 CI of PMN engraftment at day 30 87±0.3 81±0.5 88+0.7 NS CI of acute GvHD grade II - IV 34±0.7 16±0.6 26±0.3 NS CI of acute GvHD grade III - IV 9±0.4 3±0.1 6±0.1 NS CI of chronic GvHD 8±0.2 14±0.8 11±0.1 NS CI of relapse at 2 years 30±0.7 37±1 33±0.4 NS CI of TRM at 1 year 21±0.4 41±0.5 30±0.3 NS CI of TRM at 2 years 24±0.5 48±1.3 36±0.6 NS OS at 3 years 54±9.2 26±9.9 43±7 0.03 Download : Download high-res image (91KB) Download : Download full-size image Conclusions. BMT from haploidentical donor using G-CSF primed, unmanipulated bone marrow cells and an intensive regimen for GVHD prophylaxis is correlated with high engraftment rate, low incidence of acute and chronic GVHD, acceptable TRM and favourable patient outcome. The results seem particularly encouraging for AML patients grafted at an early disease stage. In alternative to transplant from matched unrelated donor or cord blood, this approach can be offered to high risk patients with hematological malignancies particularly for those who are on urgency for a transplant. Results are given as % ± standard error Disclosures: No relevant conflicts of interest to declare.

Two AML patients, whose leukemic clonogenic cells totally reacted to the anti-lactofucopentaose III S4-7 monoclonal antibody (MoAb), underwent autologous bone marrow transplantation, in first complete remission, after in-vitro purging with S4-7 MoAb and complement. After ablative chemotherapy (BAVC regimen) and reinfusion of S4-7 purged cells, regeneration of marrow cells occurred with prompt recovery of granulopoiesis and erythropoiesis. A more delayed platelet recovery was observed. The two patients are in complete remission at 20 and 11 months from ABMT. The results indicate that immunologic purging with S4-7 MoAb is safe and suitable for selected AML patients undergoing ABMT.

Between January 1984 to June 1985, 18 Ph1 positive chronic myeloid leukemia (CML) patients in chronic phase (CP) underwent allogeneic bone marrow transplantation (BMT) from HLA identical and MLC negative siblings. The median age was 32.5 yr and median disease duration of CML at time of BMT was 19.3 months. The pretransplant conditioning regimen consisted of cyclophosphamide (CTX) (120 mg/kg) and 10.20 Gy total body irradiation (TBI) at 6 doses of 1.7 Gy each, administered in 3 daily fractions over 2 days at a dose rate of 15-20 cGy/min. To prevent graft-vs-host disease (GvHD) we used methotrexate (MTX) in one patient and cyclosporin-A (CYA) in the other 17 patients. In addition to CYA, given until day +365, 10 patients received donor marrow depleted of T cells with CAMPATH-1. The residual marrow lymphocytes were always less than 1%. The rate of engraftment was significantly correlated with the number of nucleated cells infused. Neither GvHD nor graft failure were observed among CAMPATH-1 patients. In this group one cytogenetic and one hematologic relapse occurred. The overall actuarial survival at 24 months is 78%. Of the 10 patients treated with donor marrow depleted of T cells, 9 are alive after a median follow-up of 9 months (range 5-18), with an actuarial survival of 90%. Of the other 8 patients transplanted with untreated marrow, 5 are alive after a median follow-up of 19.3 months (range 3.7-24) and the actuarial survival is 63.8%. This pilot study seems to demonstrate that T-cell depletion of donor bone marrow with CAMPATH-1 is effective to prevent GvHD, while the risk of graft failure can be avoided using a "standard" conditioning regimen including a fractionated TBI with a fast dose rate and a prolonged administration of CYA at the maximum tolerable dosage. While the high frequency of relapses suggests the employ of more aggressive anti-leukemic conditioning regimens in CAMPATH-1 treated marrow recipients.

Lonidamine (LND), previously reported as a useful antitumor substance in combination with physical or chemical agents, has been studied for its capacity in increasing pharmacological elimination in vitro of residual tumor cells from human bone marrow. Different drugs were tested in association with LND against mixtures of human bone marrow and a tumor cell line, clonogenic human leukemic blast progenitors, and normal human bone marrow precursors. The results demonstrated that LND increased the efficacy of anthracycline derivatives (Adriamycin, Mitoxantrone) both on the tumor cell line and on the leukemic blast progenitors, while VP-16 or ASTA-Z 7654 was not affected by the same substance. The toxicity on normal stem cells reflected that of each drug and was not modified by the addition of LND. While a consistent dose-dependent CFU-GM reduction was observed immediately after treatment with the different drugs, a complete recovery was reached after 7 and 14 days of long-term marrow cultures. Because of the low toxicity and the efficacy demonstrated in association with certain agents in increasing tumor cell elimination in vitro, LND could play an important role in in vitro purging prior to autologous bone marrow transplantation.