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In vitro priming of T cell with horse antilymphocyte globulin (HALG) results in cytokine release, and this has been associated with its clinical efficacy in patients with severe aplastic anaemia (SAA). Rabbit antithymocyte globulin (RATG) has been studied less extensively. In this study we compare the in vitro priming effect of HALG and RATG on purified normal marrow T cells: end-points of the study were 1) levels of TNF-alpha (TNF-alpha), IFN-gamma (IFN-gamma) GM-CSF in T cell supernatants, and 2) effect of T cell supernatants on colony formation with or without exogenous GM-CSF TNF-alpha, IFN-gamma and GM-CSF levels were comparable for HALG, RATG and phytohaemagglutinin (PHA). T cell supernatants showed comparable enhancement of colony formation in the presence of recombinant human GM-CSF (rhGM-CSF) and supported colony forming unit granulomacrophage (CFU-GM) growth in the absence of growth factor. This study shows that horse and rabbit derived ALG/ATG and PHA have a comparable in vitro priming effect on T cells: both agents should probably be tested for their clinical efficacy in SAA patients.

Objective: Multipotent mesenchymal stromal cells (MSCs) are endowed with multilineage differentiative potential and immunomodulatory properties. It is still a matter of debate whether donor MSCs have sustained engraftment potential in host bone marrow (BM) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The aim of this study was to analyze the donor/recipient origin of MSCs in children receiving allogeneic either BM or cord blood (CB) transplantation. Methods: Thirty-seven pediatric patients undergoing allo-HSCT for either a malignant or a nonmalignant disorder were enrolled in the study; 19 received CB and 18 BM transplantation. Results were compared with those obtained in 14 adults given BM transplantation for either malignant or nonmalignant disorders. MSCs were grown from BM aspirates obtained 1-17 and 2-192 months after allo-HSCT in pediatric and adult patients, respectively. MSC samples at the third-fourth passage were phenotypically characterized. Donor/recipient origin of MSCs was assessed by amelogenin assay and microsatellite analysis. Results: MSCs could be grown from 30 of 37 children; at the third-fourth passage MSCs resulted positive (≥98%) for CD73, CD105, CD106, CD29, CD13, CD44 and negative (≤1%) for CD34, CD45, CD14. Mixed chimerism with donor cells was observed in 4 BM and 5 CB transplantation recipients, respectively; full recipient chimerism was detected in the remaining children. Full recipient MSC chimerism was observed also in all assessable (12/14) adult patients. Conclusions: BM of pediatric patients might be a more favorable milieu than that of adults for sustained engraftment of transplanted MSCs. MSCs able to engraft in the host can be transferred with cryopreserved CB units. © 2006 International Society for Experimental Hematology.

A bone marrow harvest is filtered either in the operating room, in the laboratory or during infusion to the patient. Filters are usually discarded. Little is known of haemopoietic progenitor cells (HPCs) trapped in the filters. The aim of the study was to evaluate HPC content in the filters and to assess the outcome of transplants with filter-discarded or filter-recovered cells. Haemopoietic progenitors were grown from filters of 19 marrow transplants. We then compared the outcome of 39 filter-recovered transplants from HLA-identical siblings (years 2001-2004) with a matched cohort of 43 filter-discarded marrow grafts (years 1997-2000). Filters contained on average 21% long-term culture-initiating cells (LTC-IC) and 15% fibroblasts colony-forming units (CFU-F) of the total progenitor cell content. Filter-discarded transplants had significantly more grade II-IV graft-versus-host disease (GvHD) (42 vs 15%, P=0.008) as compared to filter-recovered transplants, and more transplant-related mortality (TRM) (20 vs 3%, P=0.04). The actuarial survival at 5 years is 69 vs 87%, respectively (P=0.15). This study suggests that a significant proportion of LTC-IC is lost in the filters together with CFU-F. Recovery and add back of progenitors trapped in the filters may reduce GvHD and TRM.

Objective Intravenous (IV) injection is currently the normal method for transplanting hematopoietic cells. However, the problem of seeding efficiency and homing is relevant especially when a limited number of stem cells is available. Intra–bone marrow (IBM) injection of bone marrow cells (BMCs) may overcome this problem. Materials and methods Irradiated (750 cGy) C57BL/6J mice were transplanted with 1 × 10 5 BMCs harvested from transgenic mice expressing an enhanced version of the green fluorescent protein (EGFP + ) via IBM or with 1 × 10 6 EGFP + BMCs via IV. Irradiated (320 cGy) NOD/SCID mice were transplanted with 1 × 10 6 human cord blood (CB) cells via IBM or with 1 × 10 7 human CB cells via IV. Results In C57BL/6J mice after 90 days, the fraction of EGFP + cells harvested was 37% and 53% in IV-treated and IBM-treated (contralateral tibia and femur in the IBM) mice, respectively: the expansion folds were 114 and 1760, respectively. In NOD/SCID mice, the percentages of CD45 + cells and CD45 + /CD34 + cells were, at 30 days, 3.3% and 0.3% in IV-treated mice, and 4.4% and 1.1% in IBM-treated mice. At 60 days, the percentages of CD45 + cells and CD45 + /CD34 + cells were 2.1% and 0.3% in IV-treated mice and 1.4% and 0.4% in IBM-treated mice. At day 90 the percentages of CD45 + cells and CD45 + /CD34 + cells were 3% and 0.3% in IV-treated mice and 2.3% and 0.4% in IBM-treated mice. Conclusion Our data demonstrate that IBM transplantation is associated with a seeding efficiency 15 times greater than IV transplantation. IBM transplantation may improve the results of transplant and may be useful in several settings: 1) when a limited number of hematopoietic progenitors are available; and 2) in experiments aiming to place in the bone marrow stem cells of other lineages (CNS, muscle, etc.).

+HOX genes are expressed in the hematopoietic system and increasing data point to their involvement in the control of proliferation and/or differentiation. Genes belonging to the C cluster are preferentially expressed in developing and differentiated lymphoid lineages. However, recent studies demonstrated, by RT-PCR, that the HOXC4 gene is also actively transcribed in the most undifferentiated hematopoietic cells (CD34(+)38(low)) and in more mature myeloid and erythroid progenitors. We evaluated the expression of HOXC4 protein on human CD34(+) cells and the in vitro effect of its overexpression on proliferation and differentiation.We assessed the expression of HOXC4 on human CD34(+) cells using a polyclonal antibody raised against the C-terminal portion of the protein expressed using the baculovirus system. Overexpression of HOXC4 in human CD34(+) cells was obtained by retroviral gene transfer; its effect on clonogenic (CFU-GM, BFU-E, and CFU-GEMM) and early progenitors (LTC-IC) was evaluated.The HOXC4 protein is indeed expressed in human CD34(+) cells, and its overexpression in human CD34(+) cells increases the proliferation potential of clonogenic and early progenitors. CFU-GM showed a median threefold expansion (range: 1.1-19.4; p0.002) compared with control transduced with the vector alone. The increment of BFU-E was higher (median ninefold, range 2.5-35; p0. 0009) and erythroid colonies presented a larger size with normal morphology. An even more marked effect was observed on LTC-IC (median 13, onefold; range 4.1-102.1, p0.0001).We demonstrate that HOXC4 is expressed in CD34(+) cells and that its overexpression induces an in vitro expansion of committed as well as very early hematopoietic progenitors. The most striking effect was obtained on LTC-IC with an expansion of 13.1-fold. The enforced expression of HOXC4 induced a significant increase (p0.009) in the number of erythroid colonies compared with CFU-GM, although without perturbing, at least in vitro, the maturation program of the cells. On the other hand, the effect of the gene overexpression did not induce any skewing in the colony types derived from the myeloid lineage.

Objective In many patients with chronic myeloid leukemia (CML), a residual population of primitive normal (Ph-negative) progenitors persists despite the marked expansion of the leukemic (Ph-positive) clone. These cells may be found in the blood of patients studied soon after diagnosis or during the period of endogenous hematopoietic recovery that follows myelo-reductive therapy. Based on those observations, we have developed a clinical protocol that allows collection of Ph-negative peripheral blood progenitor cells (PBPC) with transplantable hematopoietic regenerative potential. The aim of this study is to examine changes that occur in the percentage of Ph-negative- and Ph-positive-committed progenitor cells and to determine the relationship between changes and clinical outcome. Materials and Methods We followed 15 patients with CML, mobilized and autografted soon after diagnosis with 85%–100% Ph-negative PBPC for a median time of 28 months (range 18–50) after transplant. At 6 months, 1 year, 2 years, and last follow-up, cytogenetic analyses were performed on fresh bone marrow cells and on colony-forming cells (CFC). Results Autologous transplantation induces a reduction in the proportion of Ph-positive CFC, from 70%–100% to 0%–25% in the majority of patients (78%). After autografting, 8 of 15 patients achieved a long-lasting cytogenetic remission (median, 24 months; range, 21–43) with a Ph-positivity ranging between 0% and 20% at the level of mature mononuclear cells and colony-forming cells (CFC). In some patients, the majority of CFC remained Ph-negative, whereas the majority of the mature cells were Ph-positive. Other patients (5/15) developed cytogenetic relapse (100% Ph-positive), although they were in hematological remission. We found that detection of Ph-positive long-term- culture initiating cells (LTC-IC) in the marrow at diagnosis was the only factor significantly associated with recurrence of the disease (p 0.01); on the other hand, the number of Ph-negative LTC-IC infused showed a significant correlation with a better outcome (p 0.03). Conclusion We have shown that a prolonged period of complete or almost complete Ph-negative hemopoiesis can be achieved in patients with CML who undergo autografting with Ph-negative progenitors. Longer follow-up study will be needed to assess whether these changes are associated with improved survival.

Cytogenetic abnormalities and paroxysmal nocturnal haemoglobinuria (PNH) phenotype are frequent findings in aplastic anaemia patients treated with immunosuppressive therapy (IST). In this study we investigated whether the appearance of clonal haemopoiesis influences patient outcome and survival. 97 patients entered this study and were followed from the onset of the disease for a median follow-up (FU) of 53 months. 93% are alive, 56% achieved complete remission, 30% partial remission, both transfusion independent, and 14% did not respond. Three groups were identified: (A) patients without evidence of emerging clones (71/97); (B) patients who acquired chromosomal abnormalities (13/97); (C) patients who showed low expression of glycosyl phosphatidylinositol anchored proteins (GPI-AP) (PNH phenotype) at presentation or later (16/97). Three patients showed both PIG-AP deficiency and chromosomal abnormalities. The actuarial survival of patients without clonal haemopoiesis (n = 71) at 6 years was 95%, for patients with chromosomal abnormalities (n = 13), 88%, and for patients with PIG-AP deficiency (n = 16), 89%. There was no difference in the probability of becoming transfusion independent in the three groups (93%, 92% and 88% respectively). This study confirmed that a proportion of severe aplastic anaemia (SAA) patients exhibit clonal markers during the time after IST, often coexisting with cytogenetically or phenotypically normal haemopoiesis. There was no significant clinical impact of these abnormalities on transfusion independence and survival at the median follow-up of 4 years.

We carried out studies to quantify Ph-negative progenitors both in steady state and during regeneration after chemotherapy and G-CSF in 23 newly diagnosed chronic myeloid leukaemia (CML) patients (group A) and in 14 individuals more than a year from diagnosis (nine in chronic and five in accelerated phase, group B). In steady-state bone marrow, Ph-negative long-term culture initiating cells (LTC-IC) and Ph-negative colony-forming-cells (CFC) were detected in 18/23 and 14/23 patients of group A versus 3/14 and 3/14 patients of group B (P

We investigated the hematopoietic reservoir in 43 severe aplastic anemia (SAA) patients following immunosuppression (IS) (n = 15) or bone marrow transplantation (BMT) (n = 28), at a median interval of 5 years (range, 2-20) from treatment. All patients had normal blood counts, good marrow cellularity, and normal numbers of colony forming unit-granulocyte macrophages (CFU-GM). Burst forming unit-erythroid (BFU-E) and colony forming unit-granulocyte erythroid megakaryocyte macrophages (CFU-GEMM) numbers were reduced when compared with normal controls. However, the most pronounced defect was observed at the level of long-term culture-initiating cells (LTC-IC), which significantly differed from controls (P

Allogeneic bone marrow transplantation (BMT) for refractory Behcet's disease with severe CNS involvement

This trial was designed to test the use of CD34 + selected haemopoietic stem cells (HSC) in HLA-mismatched donor-recipient pairs, following intensive conditioning with thiotepa, antilymphocyte globulin (ALG), cyclophosphamide and single-dose total-body irradiation (sTBI). 10 patients aged 16-50 with advanced malignancies and a two- or three-antigen mismatched family donor entered this study. Donor marrow and G-CSF primed peripheral blood cells were processed separately on CD34 columns (Ceprate). The median number of infused CD34 + cells were 5.66 x 10 6 / kg, with 0.55 x 10 6 /kg CD3 + cells. Nine patients received cyclosporin for graft-versus-host disease (GVHD) prophylaxis. Median neutrophil counts on day 21 were 2 x 10 9 /l with a median platelet count of 60 x 10 9 /l, but CD4 counts remained extremely depressed throughout the study. Acute GvHD was scored as grade ()-I in two patients, as grade II in seven, and grade III in one. Eight patients died at a median interval of 72d from HSCT (range 20-144) due to cytomegalovirus (CMV) associated interstitial pneumonitis (IP) (n = 5), renal failure (n = 1), GvHD (n = 1) and Aspergillus meningitis (n = 1). Two patients are alive 365-495 d post transplant, one in remission and one in relapse. This study suggests that large numbers of positively selected mismatched HSC can rapidly engraft after intensive conditioning regimen: however, profound post-transplant immunodeficiency leads to a high risk of lethal infectious complications.

Thirty-one patients (median age, 44 years) with advanced hematologic malignancies were given thiotepa 15 mg/kg, and cyclophosphamide 120 (n = 14) or 150 (n = 17) mg/kg followed by unfractionated peripheral blood stem cell transplants (PBSCT) from genotypically identical siblings (n = 28) or one antigen mismatched family donor (n = 3). Donors were mobilized with granulocyte colony-stimulating factor 5 to 10 microgram/kg/d for 6 days and underwent two to three leukapheresis on days +5, +6, +7. The median cell yield per donor expressed/kg of recipients body weight was as follows: nucleated cells 13 x 10(8)/kg; CD34+ cells 6 x 10(6)/kg; colony-forming unit-granulocyte macrophage 38 x 10(4)/kg, and CD3+ cells 449 x 10(6)/kg. The diagnoses were chronic myeloid leukemia (n = 4), acute myeloid (n = 9) or lymphoid leukemia (n = 2), acute myelofibrosis (n = 2), multiple myeloma (n = 1), lymphoma (n = 6), chronic lymphocytic leukemia (n = 1) myelodysplasia (n = 6). Twenty-eight patients had advanced disease, 29 patients were first grafts, and 2 were second transplants 3 and 9 years after the first. Neutrophil counts of 0.5 x 10(9)/L and platelet counts of 30 x 10(9)/L platelets were both achieved on day +14 (median). Engraftment could be proven by sex markers or DNA polymorphism in 29 of 31 patients: one had early leukemia relapse and one patient was unevaluable because of early death. Acute graft-versus-host disease (GVHD) was scored as minimal or absent (grade 0 to 1) in 14 patients, moderate (grade II) in 13, and severe (grade III to IV) in four. Causes of death were leukemia (n = 4), acute GVHD (n = 4, with associated cytomegalovirus infections in three), sepsis (n = 1), liver failure (n = 1), multiorgan failure (n = 1), and hemorrhage (n = 1). The actuarial transplant mortality is 29%, the actuarial relapse rate 22%. Nineteen patients survive with a median follow up of 288 days (100–690). The actuarial 2-year survival is 57%. Three patients received PBSCT from family donors mismatched for one class II antigen: all engrafted, one developed grade I aGVHD; one died of leukemia on day +155; two are alive disease free 267 to 290 days postgraft. This study suggests that thiotepa cyclophosphamide followed by unfractionated PBSC allograft may be an alternative form of transplant for adults with advanced leukemia, also in the setting of one antigen mismatched donor. The engraftment is rapid with acceptable GVHD and relatively low transplant-related mortality.

The aim of this study was to test whether prolonged administration of granulocyte colony-stimulating factor (G-CSF) would allow the collection by leukapheresis of PBHP in patients with SAA. For this purpose, nine SAA patients, 7 to 46 years old, six of whom were enrolled at diagnosis of their disease and three after previous immunosuppression had failed, were treated with antilymphocyte globulin (ALG) (day 1 to 5), cyclosporin A (5 mg/kg/d orally) (day 6 to 90) and G-CSF 5 micrograms/kg/d (day 6 to 90). A total of 40 leukaphereses were performed, (range 2 to 7 per patient), between days +10 and +168 from G- CSF treatment. White blood cell count at the time of harvest ranged from 1.2 to 18.1 x 10(9)/L. Results can be summarized as follows: the median number of cells collected per patient was 5.0 x 10(8)/kg (range 2.6 to 18.7), the median number of CD34+ cells was 1.8 x 10(6)/kg (range 0.27 to 3.8) and the median number of colony-forming units granulocyte-macrophage (CFU-GM) was 3.9 x 10(4)/kg (range 0 to 39). Twenty leukaphereses performed between days +33 and +77 of G-CSF treatment grew granulocyte macrophages and erythroid colonies in vitro. No colony growth was obtained from 20 leukaphereses performed before day +33 or after day +80. In six patients the total number of CFU-GM recovered were in the range described for autologous peripheral blood stem cell grafts. (2.6 to 39 x 10(4)/kg). In conclusion, this study suggests that circulating hematopoietic progenitors can be recovered after ALG priming and after at least 1 month of G-CSF treatment in a proportion of patients with SAA. Whether these cells will be suitable for autologous transplantation remains to be determined.

Two putative types of circulating endothelial progenitor cells have been recently identified in vitro: (1) endothelial colony-forming cell (ECFC) and (2) colony-forming unit–endothelial cell (CFU-EC). Only the former is now recognized to belong to endothelial lineage. We have used the ECFC and CFU-EC assays to readdress the issue of the clonal relation between endothelial progenitor cells and hematopoietic stem cells in patients with Philadelphia-positive and Philadelphia-negative chronic myeloproliferative disorders. Both ECFCs and CFU-ECs were cultured from peripheral blood mononuclear cells, and either BCR-ABL rearrangement or JAK2-V617F mutation were assessed in both types of endothelial colonies. We found that ECFCs lack the disease-specific markers, which are otherwise present in CFU-ECs, thus reinforcing the concept that the latter belongs to the hematopoietic lineage, and showing that in chronic myeloproliferative disorders the cell that gives rise to circulating ECFC has a distinct origin from the cell of the hematopoietic malignant clone.

'.the leukemic stem line is a small minority within the total cell mass;. when the leukemic stem line is not exceeding the normal stem cell numbers, its proliferation may still be under partial control.' LG Lajtha, Blood Cells 1981; 7: 45-62 We performed cytogenetic analysis on fresh bone marrow cells and on progenitor cell colonies in a patient who relapsed after allogeneic BMT for CML and was subsequently treated with donor lymphocyte infusions (DLI). Two Philadelphia-positive clones were identified at relapse. One clone displayed an additional chromosomal abnormality most likely induced by radio-chemotherapy and therefore arising in a single cell. This cell displays the characteristics of a stem cell, since it was able to support 20% of Ph-positive hemopoiesis for 5 months. If the progeny of a single Ph-positive stem cell account for 20% of hemopoiesis, a very low number of leukemic stem cells may sustain relapse after allogeneic BMT. This is in keeping with two observations: (1) at relapse, long-term culture initiating cells (LTC-IC) were all donor-derived and Ph-negative; (2) on average, the pace of the disease is very slow after relapse following allogeneic-BMT. Therefore, we hypothesize that a small number of leukemic stem cells may be involved in the initial events of relapse following BMT for CML.

Minimal residual disease (MRD) was monitored in 80 patients with acute lymphoid (ALL, n=44) or myeloid (AML, n=36) leukemia, undergoing allogeneic haemopoietic stem cell transplantations. MRD markers were IgH-VDJ and TCR gene re-arrangement for ALL, and Wilm's Tumor (WT1) expression for AML. The overall cumulative incidence (CI) of MRD was positive in 45% and the CI of hematologic relapse was 24% (36% in MRD+ vs. 16% in MRD patients, p=0.03). The median interval from transplant to first MRD positivity was 120 days and to hematologic relapse 203 days. Patients were divided in 3 MRD groups: MRD (n=44), MRD+ given donor lymphocyte infusions (DLI) (n=17) and MRD+ not given DLI (n=19): leukemia relapse rates in these 3 groups were 16%, 6% and 63%, respectively (p

Objective Endothelial progenitor cells (EPCs) are involved in neovessel formation. So far, therapeutic angiogenesis is hampered by the low frequency and limited proliferative potential of these cells isolated from peripheral blood. Recently, it has been shown that cord blood–derived EPCs (CB EPCs) can be ex vivo expanded on a clinical scale. In this study, we evaluated the expansion potential of CB EPCs together with their phenotypic, functional, and chromosomal stability over time. Materials and Methods Flow cytometry, in vitro tube formation, and proliferation assays were performed to characterize CB EPC–derived cells. Chromosomal stability was evaluated by karyotype analysis. In vitro and in vivo tumorigenicity was evaluated by soft agar assay and injection into nonobese diabetic/severe combined immunodeficient mice, respectively. Results We showed that CB EPC–derived cells displayed phenotypic and functional features of EPCs, although a process of maturation was observed over time. Although we confirmed that CB EPCs have a greater expansion potential compared to peripheral blood EPCS, we observed a high incidence of cytogenetic alterations (71%) in the expanded endothelial cell population, even at early times of culture. In two cases, spontaneous transformation in vitro was documented, but none of the samples tested showed tumorigenic potential in vivo. Conversely, no karyotype alterations have been observed on peripheral blood EPCs–derived cells. Conclusions We confirm that CB represents a good source for clinical ex vivo expansion of EPCs. However, because of high frequency of karyotype alterations, these cells cannot be considered free of risk in clinical application.

Summary Background Cord-blood transplants are associated with delayed or failed engraftment in about 20% of adult patients. The aim of this phase I/II study was to establish the safety and efficacy of a new administration route (intrabone) for cord-blood cells, measured by the donor-derived neutrophil and platelet engraftment. Methods Adult patients with acute leukaemia, for whom an unrelated stem-cell transplantation was indicated and no suitable unrelated human leucocyte antigen (HLA)-matched donor had been identified, were included in the study and underwent a cord-blood transplant in San Martino Hospital, Genoa, Italy. Eight patients were in first complete remission, ten in second complete remission, and 14 had advanced-stage, refractory disease. HLA matching was 5/6, 4/6, and 3/6 for 9, 22, and one patient, respectively. Cord-blood cells were concentrated in four 5-mL syringes, and were infused in the superior-posterior iliac crest under rapid general anaesthesia. Median transplanted cell dose was 2·6×10 7 /kg (range 1·4–4·2). The primary endpoint was the probability of neutrophil and platelet recovery after intrabone cord-blood transplantantion. Secondary endpoints included the incidence of acute graft-versus-host disease, relapse, and overall survival. This trial is registered on the ClinicalTrials.gov website, number NCT 00696046. Findings Between March 31, 2006, and Jan 25, 2008, 32 consecutive patients with acute myeloid leukaemia (n=20) or acute lymphoblastic leukaemia (n=12) underwent a cord-blood transplant (median age 36 years [range 18–66]). No complications occurred during or after the intrabone infusion of cells. Four patients with advanced-stage disease died within 12 days of the procedure. Median time to recovery of neutrophils in 28 patients (≥0·5×10 9 /L) was 23 days (range 14–44) and median time to recovery of platelets in 27 patients (≥20×10 9 /L) was 36 days (range 16–64). All patients were fully chimeric from 30 days after transplantation to the last follow-up visit, suggesting an early complete donor engraftment. No patient developed grade III–IV acute graft-versus-host disease. Causes of death were transplant related (n=5), infection (n=7), and relapse (n=4). 16 patients were alive and in haematological remission at a median follow-up of 13 months (range 3–23). Interpretation Our preliminary data suggest that direct intrabone cord-blood transplantation overcomes the problem of graft failure even when low numbers of HLA-mismatched cord-blood cells are transplanted, thus leading to the possibility of use of this technique in a large number of adult patients. Funding This work was supported by grants from the Associazione Italiana Ricerca contro il Cancro (FF), Compagnia di San Paolo Torino (FF), Progetto CARIGE Cellule Staminali (FF), the EUROCORD III (QLRT 2001-01918) (FF), Ministero della Salute (Ricerca Finalizzata Ministeriale 2005) (FF), and the Associazione Italiana Leucemie, Sezione Ligure.

BACKGROUND. Leukemias are dependent on Akt/NF-κB activation and angiogenesis. METHODS. The antiangiogenic Akt/NF-κB inhibitor xanthohumol (XN) has in vitro activity against acute and chronic myelogenous leukemia cell lines (AML, CML) and fresh samples from patients were investigated. RESULTS. Inhibition of cell proliferation is associated with induction of apoptosis and reduced VEGF secretion. Decreased cell invasion, metalloprotease production, and adhesion to endothelial cells observed in the presence of XN could prevent in vivo life-threatening complications of leukostasis and tissue infiltration. CONCLUSIONS. As endothelial cells and hematopoietic cells are mutually correlated in their development and growth, targeting both tumor cells and endothelial cells with agents possessing cytotoxic and antiangiogenic activities may lead to synergistic antitumor effects interrupting a reciprocal stimulatory loop between leukemia and endothelial cells. Cancer 2007. © 2007 American Cancer Society.

We assessed the long-term outcome of patients with relapsed acute myeloid (n=86) or acute lymphoid leukemia (n=66), undergoing an allogeneic hemopoietic stem cell transplantation in our unit. The median blast count in the marrow was 30%. Conditioning regimen included total body irradiation (TBI) (10-12 Gy) in 115 patients. The donor was a matched donor (n=132) or a family mismatched donor (n=20). Twenty-two patients (15%) survive disease free, with a median follow-up of 14 years: 18 are off medications. The cumulative incidence of transplant related mortality is 40% and the cumulative incidence of relapse related death (RRD) is 45%. In multivariate analysis of survival, favorable predictors were chronic graft-versus-host disease (GvHD) (P=0.0003), donor other than family mismatched (P=0.02), donor age less than 34 years (P=0.02) and blast count less than 30% (P=0.07). Patients with all four favorable predictors had a 54% survival. In multivariate analysis of relapse, protective variables were the use of TBI (P=0.005) and cGvHD (P=0.01). This study confirms that a fraction of relapsed leukemias is cured with an allogeneic transplant: selection of patients with a blast count

To the editor: Hematologic relapse after an allogeneic hemopoietic stem cell transplantation (HSCT) in patients with acute leukemia is associated with a poor outcome, despite further cell therapy in the form of donor lymphocyte infusions (DLIs).[1][1] This has been taken as evidence that acute

A proportion of patients develop poor graft function (PGF) following an allogeneic hemopoietic stem cell transplant (HSCT). It is uncertain whether a boost of donor marrow or blood cells is beneficial in terms of trilineage recovery and non-relapse-related mortality (NRM).The aim of this study was to compare outcomes in patients with PGF and full donor chimerism following an allogeneic HSCT who did or did not receive a boost of donor stem cells. The study included patients with primary PGF--i.e. those failing to achieve sustained graft function- and secondary PGF--i.e. those developing PGF after complete hematologic recovery. We studied 54 patients with PGF: 20 patients received no further donor cell infusion (group A), 14 received a boost of unmanipulated marrow or blood cells from the original donor, without further conditioning (group B), and 20 received donor cells after CD34 selection without conditioning (group C). The three groups were comparable for disease phase, patients' age, donor type, primary or secondary PGF, full donor chimerism and duration of PGF.Trilineage recovery was seen in 40%, 36% and 75% of the patients in, respectively, groups A, B and C (p=0.02). In multivariate Cox analysis trilineage recovery was more frequent in patients with secondary PGF (RR of complete recovery 2.82, p=0.01) and in patients receiving CD34+-selected cells (RR of complete recovery 3.0; p=0.007). There was no effect of donor type on hematologic recovery. The rate of NRM was 55%, 64%, 20% in groups A, B and C, respectively (p=0.06) and was highly correlated with trilineage recovery (RR 0.36, p0.0001). PGF was the primary cause of death in 30%, 21% and 10% of the patients in the three groups, graft-versus-host disease (GVHD) in 5%, 36%, and 10%.In patients with poor graft function (a) a boost of CD34+-selected peripheral blood donor cells is associated with a high chance of trilineage recovery and a low risk of acute GVHD; (b) a boost of unmanipulated donor cells does not seem to offer a survival advantage over no infusion of cells; and (c) NRM is lower when using peripheral blood cells for the boost. These data may be useful when discussing second stem cell donations for patients with poor graft function.

Vascular endothelial growth factor165 (VEGF165) and semaphorin3A (SEMA3A) elicit pro- and antiangiogenic signals respectively in endothelial cells (ECs) by binding to their receptors VEGFR-2, neuropilin-1 (NRP1), and plexin-A1. Here we show that the VEGF165-driven angiogenic potential of multiple myeloma (MM) ECs is significantly higher than that of monoclonal gammopathy of undetermined significance (MGUS) ECs (MGECs) and human umbilical vein (HUV) ECs. This is probably due to a constitutive imbalance of endogenous VEGF165/SEMA3A ratio, which leans on VEGF165 in MMECs but on SEMA3A in MGECs and HUVECs. Exogenous VEGF165 induces SEMA3A expression in MGECs and HUVECs, but not in MMECs. Moreover, by counteracting VEGF165 activity as efficiently as an anti-VEGFR-2 antibody, exogenous SEMA3A restrains the over-angiogenic potential of MMECs. Our data indicate that loss of endothelial SEMA3A in favor of VEGF165 could be responsible for the angiogenic switch from MGUS to MM.

Objective To compare the suppressive effect of mesenchymal stem cells (MSC), derived from normal individuals or severe aplastic anemia patients (SAA), on T-cell activation. Patients and Methods We studied bone marrow MSC from 19 healthy donors and 23 SAA patients in different phases of the disease: at diagnosis (n=3), following immunosuppressive therapy (IS) (n=16), or after a bone marrow transplant (BMT) (n=4). MSC were tested for T-cell suppression in the following assays: mixed lymphocyte reaction (MLR), phytohemaglutinin (PHA)-primed cultures, activation surface markers, γ-IFN production, hematopoietic colony formation (CFC), production of cyclic ADP–ribose (cADPR). Results The abnormalities of SAA MSC included: 1) significantly lower suppression of T-cell proliferation induced by alloantigens ( p =0.009) or PHA ( p =0.006); 2) impaired capacity to suppress CD38 expression on PHA-primed T cells ( p =0.001); 3) impaired ability to suppress γ-IFN production in PHA cultures, resulting in an 11-fold higher γ-IFN concentration; 4) no preventive effect on T cell–mediated inhibition of CFC; and 5) significantly reduced ( p =0.009) production of cADPR, a universal calcium mobilizer. MSC-mediated suppression of PHA-induced T-cell proliferation was restored to control levels in 3 of 4 patients post-BMT. Conclusion The ability of MSC to downregulate T-cell priming, proliferation, and cytokine release is deficient in patients with SAA, persists indefinitely after immunosuppressive therapy, but seems to be restored after BMT. Whether these abnormalities are relevant to the pathogenesis of aplastic anemia remains to be determined.

Delayed hematopoietic recovery is the main factor precluding a wider use of cord blood (CB) transplants. We hypothesized that this delayed engraftment might not be related to an insufficient number of stem cells in the graft, but to an intrinsic difficulty of these cells to undergo differentiation. To test our hypothesis, 2 groups of children were compared; 12 received a CB transplant and 12 an adult bone marrow (BM) transplant. We studied neutrophil and platelet recovery and, at a median time of approximately 1 year after transplantation, the frequency of colony-forming cells (CFCs) and long-term culture initiating cells (LTC-ICs) in the BM of the 2 groups. Recipients of BM transplants received 1-log more cells and had significantly faster neutrophil and platelet recovery. Conversely, the frequency of committed and early progenitors was significantly higher in the BM of children given CB cells compared with BM transplant recipients (median count of CFC/2 × 104 BM mononuclear cells, 20 versus 11, P = .007; median count of LTC-IC/106 BM mononuclear cells, 8.2 versus 0.2 P = .001). CB, but not adult BM stem cells, can better restore the host hematopoietic progenitor cell reservoir; the delayed engraftment after CB transplantation may reflect the difficulty of CB progenitors to reprogram themselves toward differentiation.

The absolute content of CD34+ cells in the peripheral blood of 84 patients with myelofibrosis with myeloid metaplasia (MMM) and 23 patients with other Philadelphia-negative (Ph−) chronic myeloproliferative disorders (CMDs) was investigated. In MMM, the median absolute number of circulating CD34+cells was consistently high (91.6 × 106/L; range, 0-2460 × 106/L). Receiver operating characteristic curve analysis showed that 15 × 106/L as a decision criterion for CD34+cells produced an almost complete discrimination between MMM patients out of therapy and other Ph− CMDs (positive predictive value, 98.4%; negative predictive value, 85.0%). MMM patients with higher numbers of CD34+ cells had a significantly longer disease duration (P = .019) and higher spleen volume index (P = .014), liver volume (P = .000), percentage of circulating immature myeloid cells (P = .020), and percentage of myeloid blasts (P = .000). When CD34+ cells were correlated with the use of Dupriez risk stratification, CD34+ cells increased significantly from low-risk (median, 68.1 × 106/L) to intermediate-risk (median, 112.8 × 106/L) and high-risk patients (median 666.1 × 106/L) (F = 4.95;P = .009). When CD34+ cells were correlated with a severity score on the basis of both myeloproliferative and myelodepletive characteristics of the disease, only the myeloproliferation index was significantly associated with CD34+ cell level (F = 5.7;P = .000). Overall survival and interval to blast transformation from the time of CD34+ cell analysis were significantly shorter in patients with more than 300 × 106/L CD34+ cells (P = .005 and .0005, respectively). In conclusion, the absolute number of CD34+ circulating cells allows MMM to be distinguished from other Ph− CMDs; it is strongly associated with the extent of myeloproliferation and predicts evolution toward blast transformation.

The aim of this study was to compare the in vitro growth of cord blood-derived progenitors with that of bone marrow and peripheral blood.We analyzed 192 umbilical cord blood (UCB), 35 normal bone marrow (NBM), and 35 granulocyte colony-stimulating factor (G-CSF)-primed normal peripheral blood (NPB) samples. Standard clonogenic assays (colony-forming unit granulocyte-macrophage [CFU-GM], burst-forming unit erythroid [BFU-E], CFU-granulocyte erythroid megakaryocyte macrophage [GEMM]) and standard long-term culture-initiating cell (LTC-IC) assay were performed. LTC-IC frequency also was tested under modified culture conditions. The variables tested were incubation temperature (37 degrees C and 33 degrees C) and supportive stromal cell lines (NIH3T3 and M210-B4).The CFU-GM and CFU-GEMM frequencies of UCB samples were similar to NPB and higher compared to NBM samples (p10(-4) and p0.007 respectively). On the other hand, the BFU-E frequency was lower in cord blood samples (5.2 +/- 5.6/10(4) MNC) compared to bone marrow (7 +/- 3.8/10(4) MNC; p0.005) and peripheral blood (15.2 +/- 11.1/10(4) MNC; p10(-4)). All colony types (CFU-GM, BFU-E, CFU-GEMM) generated from cord blood progenitors were larger with respect to the other tissues. The LTC-IC frequency was markedly decreased (8.8 +/- 3.8/10(6) MNC) in cord blood with respect to bone marrow (40.7 +/- 7.4/10(6) MNC; p10(-4)) and peripheral blood (28.8 +/- 3.8/10(6) MNC; p0.04). However, when culture conditions (temperature, stromal layers) were modified, UCB-LTC-IC frequency significantly increased, while the growth of early progenitors derived from adult tissues (BM and PB) did not show any variation. Whatever culture conditions were used, the proliferative potential of UCB LTC-IC was significantly higher with respect to bone marrow and G-CSF-primed PB (10.6 +/- 7.7 colonies vs. 5.9 +/- 5 vs 3.2 +/- 2.2 colonies; p0.02 and p0.001 respectively).Optimal conditions for estimation of the LTC-IC frequency in cord blood samples seem to be different from those usually applied to PB and BM progenitors. Although UCB hemopoietic progenitors have a higher proliferative potential than those from bone marrow and G-CSF-primed peripheral blood, their quantitation depends on the culture conditions, which makes it difficult to establish their exact number. This problem and the fact that a significant proportion of UCB samples grew poorly in culture make it necessary to develop suitable and standardized functional assays to test UCB progenitor content before the transplantation procedure.

In the majority of newly diagnosed patients with chronic myeloid leukaemia (CML), the bone marrow contains consistent numbers of normal Ph-negative surrogate stem cells (LTC-IC) which seem to decline rapidly with time. This is confirmed by mobilization studies showing that early after diagnosis is the optimal time to collect Ph-negative progenitor to be utilized for restoring Ph-negative haematopoiesis. In the marrow of the majority of CML patients at diagnosis Ph-positive LTC-IC are found at a lower frequency than Ph-negative LTC-IC and, unexpectedly, they do not show a tendency to increase with time as long as patients remain in chronic phase. Therefore, the decline of normal haematopoiesis does not seem related to a parallel increase in Ph-positive stem cells.

Normal immature hematopoietic progenitors are relatively well preserved in most patients newly diagnosed with chronic myeloid leukemia, but tend to decline rapidly with time. Such exhaustion could reflect a suppressive effect of the Philadelphia positive clone expansion and/or be induced by Interferon-alpha treatment.A total of 51 CML patients were classified into three groups. Newly diagnosed untreated patients were group A (n=30). Of the 21 treated individuals with Interferon-alpha, for at least 12 months, 15 showed no cytogenetic response (group B) while six showed persisting major/complete response (group C). Patients belonging to groups A and B were mobilized with chemotherapy plus G-CSF while patients of group C received a short course of G-CSF only.Patients responding to IFN-alpha (group C) showed comparable numbers of bone marrow Ph- long-term culture initiating cells to those of newly diagnosed individuals (group A): 8.5 (1-65)/10(6) MNC vs 10.5 (1-30), while non-responders had markedly lower numbers:1 (1-5). The amount of Ph- LTC-IC collected was significantly lower in patients of group B 1.8 (0-325)x10(2)/kg than in patients of either group A 31.3 (0-952)x10(2)/kg (P0.002) or group C 109 (8-259)x10(2)/kg (P0.01). Interestingly, five patients of group B who had 100% Ph+ metaphases, but Ph- progenitors in their bone marrow, mobilized normal amounts of Ph(-) progenitors.These findings suggest that the decline of normal hematopoietic progenitors, currently observed in the majority of CML patients, is not induced by IFN-alpha treatment, but it is likely due to the expanding leukemic clone. They also indicate that normal hematopoietic reservoir is consistently preserved in patients given IFN-alpha early after diagnosis and achieving a stable cytogenetic response.

The Philadelphia (Ph) translocation t(9;22) results in the creation of the BCR-ABL gene, which is now regarded as central to the mechanism that underlies the chronic phase of chronic myelogenous leukemia (CML). From a clinical point of view, BCR-ABL mRNA detection has become the basis for the study of minimal residual disease in CML, particularly when a complete cytogenetic remission is achieved after interferon-alpha (IFN-alpha) therapy or allogeneic stem cell transplantation. We have recently demonstrated that it is possible to mobilize normal peripheral blood progenitor cells (PBPC) in higher rates if this procedure is performed during the early chronic phase. In an attempt to monitor the leukemic cell content of PBPC collections, we used quantitative-competitive RT-PCR (QC-RT-PCR). Thirty consecutive Philadelphia (Ph) chromosome positive patients were enrolled in this study. After chemotherapy and G-CSF, 14 patients achieved 100% Ph-negative metaphases, nine patients hador =34% and seven patients34% leukemic metaphases. A total of 116 collection samples were studied. For each sample, BCR-ABL transcript numbers and BCR-ABL/ABL ratio were evaluated. A highly significant correlation between Ph-positive metaphases and BCR-ABL transcript numbers (r = 0.84, P0.0001) or BCR-ABL/ABL ratio (r = 0.86, P0.0001) was found. For patients that underwent the procedure in early chronic phase, Ph-negative collections showed different levels of BCR-ABL expression. BCR-ABL transcript numbers varied from a median of 100/microg RNA in the first and second leukaphereses, to 500/microg RNA in the third and fourth leukaphereses, and 1500/microg RNA in the fifth leukapheresis (P = 0.002). BCR-ABL/ABL ratio values showed similar kinetics. We have also demonstrated that there is a correlation between low values in BCR-ABL/ABL ratio (or =0.01) in the reinfused PBPC and the achievement of cytogenetic remission after autografting (chi2 test, P = 0.01). In conclusion, this study demonstrates that QC-RT-PCR for BCR-ABL is a reliable and helpful method for monitoring residual leukemic load in mobilized PBPC, particularly in Ph-negative collections. Moreover, QC-RT-PCR allows selection of the best available collections for reinfusion into patients after myeloablative therapy.

We report our experience of high-dose cyclophosphamide (HDCY) followed by high-dose therapy (HDT) and peripheral blood progenitor cell (PBPC) autografting in patients with diffuse, intermediate and high-grade non-Hodgkin's lymphomas who have failed conventional treatment. From 1991 to 1996, 54 consecutive patients pre-treated with a median of two chemotherapy lines entered the study. Eighteen patients (33%) were still responders to conventional chemotherapy (sensitive relapse), and 20 patients (37%) were in partial response (PR) after chemotherapy (CT). Sixteen patients (30%) were resistant to conventional CT either at presentation (non responder) or in relapse (resistant relapse). Thirty-nine patients had bone marrow involved by disease and fifteen had an hypoplastic marrow following conventional treatment. Patients received HDCY (7gr/m2) and G-CSF or GM-CSF in order to collect PBPC. Median collected CD34+ cells was 12.3 x 10(6)/Kg (range 0.7-197). After HDT (BEAM or Melphalan + TBI) 50 patients underwent PBPC autografting. According to intention to treat, 44 (81%) of 54 patients achieved complete remission (CR) (50% after HDCY and 31% after HDT). Procedure related death occurred in 6 patients (11%), one after HDCY and 5 after autografting. Twenty-nine (66%) of 44 patients are still in CR, 7 to 63 months (median 27 months) after the procedure. Three-year probability of survival, disease-free survival and progression-free survival are 63%, 64% and 52% respectively. In conclusion, HDCY is an effective procedure not only in mobilizing PBPC, but also in reducing tumour burden. HDT with PBPC support may further improve the outcome in this category of high-risk non-Hodgkin's lymphomas.

Thirty-five aggressive non-Hodgkin's lymphomas (NHL) with marrow involvement received high-dose cyclophosphamide (7 g/m2) and G-CSF in order to collect peripheral blood progenitor cells (PBPC). Fourteen patients were in partial remission, 16 patients were in relapse (“sensitive”, 12; “resistant”, 4) and 5 patients were in refractory to conventional treatment. A good yield of PBPC was obtained in 30 patients, while a low number of CD34+ cells and of CFU-GM was seen in two cases. Two patients entered progression and one patient died. Thirty patients underwent PBPC autografting. Twenty-nine out of 35 (83%) patients entered complete remission (CR). Two patients died in CR of infection following marrow aplasia 3 and 6 months after autografting. At 3 years the probability of survival and disease-free survival (DFS) are of 62% and 51% respectively.

Allogeneic bone marrow transplant recipients maintain normal peripheral blood counts long term, suggesting durable support from engrafted stem cells. In order to investigate late hemopoietic reconstitution at the level of committed and early progenitors (LTC-IC), we studied 64 long-term survivors at a median interval of 6 years (range: 2-20) after allogeneic bone marrow transplant. CFU-GM and BFU-E numbers did not differ from normal controls; CFU-GEMM were found to be significantly decreased (1.2 +/- 0.2/10(5) vs 3.1 +/- 0.4, P = 0.001). The most remarkable defect was however, the low frequency of LTC-IC (3.2 +/- 0.6/10(6) vs 54.2 +/- 9.3, P = 0.0001) that did not improve with time and did not correlate with phase of the disease, conditioning regimen, CMV infections or GVHD. Number of infused cells and CFU-GM content of marrow grafts did not seem to influence the number of LTC-IC. This study documents a significantly reduced number of early progenitors in BMT patients despite normal numbers of committed progenitors and normal peripheral blood counts. This finding may suggest a permanent reduction of the stem cell reservoir after allogeneic bone marrow transplantation.

Elevated white blood cell counts are frequently found in patients with chronic myeloid leukaemia (CML). Although some studies have disclosed that bone marrow of CML patients may contain some normal Philadelphia-negative early progenitor cells, it has been assumed that the dramatic increase of white blood cells was entirely related to the leukaemic cell expansion. In this study we attempted to quantify the number of normal and leukaemic progenitor cells in the bone marrow and peripheral blood of newly diagnosed CML patients. Bone marrow and peripheral blood cells of eight newly diagnosed CML patients were analysed for clonogenic colony-forming cells (CFC) and very early progenitor cells, i.e. long-term culture initiating cells (LTC-IC). The leukaemic (Ph-positive) or normal (Ph-negative) origin of progenitor cells was revealed by cytogenetic analysis performed on single colonies arising from in-vitro assays. In 6/8 patients the marrow CFC frequency ranged from 400 to 9300/10(6) mononuclear cells (MNC), 0-50% being Philadelphia chromosome negative; the LTC-IC frequency ranged from 0 to 11/10(6) MNC, and were 80-100% Ph-negative. The corresponding absolute values into peripheral blood were: CFC = 1-35.5 x 10(3)/ml, 0-50% Ph-negative, and LTC-IC = 0-2.5 x 10(3)/ml, 0-100% Ph-negative. In one patient, no LTC-IC were detected in either the marrow or the peripheral blood. In conclusion, in the peripheral blood of some CML patients, the number of normal LTC-IC is more than 3 times the number of leukaemic progenitor cells, and is much higher (50 times) than the corresponding value found in normal subjects in steady state (2.5/ml v 124/ml). These data support the concept that leukaemic 'stem cells', with respect to normal ones, may be considerably fewer than previously thought. In addition it is shown that at the beginning of CML high numbers of normal LTC-IC are spontaneously mobilized into the blood. Finally, the presence of Ph-negative early progenitors into the blood may represent a potential source of normal stem cells available for autografting providing they can be separated from leukaemic cells.

Abstract 5178 Several reports established an association between iron chelation therapy with deferasirox and hematopoietic improvement in patients with myelodysplastic syndromes. Data in β-thalassemia major (TM) patients is absent. We evaluated levels (frequency and aboslute number) of several hematopoietic peripheral progenitors (HPP: Colony Forming Unit-Granulocyte/Macrophage [CFU-GM], Erythroid Burst-Forming Unit [BFU-E], Colony Forming Unit-Granulocyte, Erythrocyte, Macrophage, Megakaryocyte [CFU-GEMM], and Long Term Culture-Initiating Cells [LTC-IC]) in 26 TM patients (median age 28. 5 years, 42. 3% males) and 12 age-matched controls. All TM patients had to be using the same iron chelator for at least 6 months with >80% compliance. The levels of all HPP were significantly higher in TM patients than controls, and varied between splenectomized and nonsplenectomized patients (lower CFU-GM and BFU-E, and higher LTC-IC in splenectomized patients). Patients using deferasirox (n=9) showed significantly higher levels of BFU-E compared with both deferoxamine (n=10) and deferiprone (n=7) treated patients (p Disclosures: Forni: Novartis Pharmaceuticals: Research Funding; Ferrokin: Research Funding. Musallam:Novartis Pharmaceuticals: Honoraria.

The aim of this study was to evaluate the effect of stem cell factor (SCF) on the in vitro growth of bone marrow hematopoietic progenitors from patients with acquired severe aplastic anemia (AA) or Fanconi's anemia (FA). For this purpose, we studied 11 patients with acquired AA (5 at diagnosis, 6 after ALG treatment), 12 patients with FA, and nine normal controls. Bone marrow cells were plated in vitro for colony- forming unit granulocyte-macrophage (CFU-GM) (in the presence of granulocyte-macrophage colony-stimulating factor [GM-CSF]), and for burst-forming unit-erythroid (BFU-E) and CFU-granulocyte, erythroid, monocyte, megakaryocyte (CFU-GEMM) colonies (in the presence of erythropoietin and interleukin-3 [IL-3]), with or without 20 ng/mL of SCF. In normal controls, SCF enhanced the growth of CFU-GM colonies from 103 to 263 (median), of BFU-E from 168 to 352, and of GEMM colonies from 6 to 38/10(5) cells plated. In patients with acquired AA, SCF induced a significant enhancement of BFU-E growth (8 to 29; P = .01) and allowed the formation of GEMM colonies that were not scored in baseline culture conditions (0 to 8; P = .01). CFU-GM growth was enhanced (4 to 20), but not significantly (P = .3). This was true both for patients at diagnosis and after antilymphocyte globulin treatment. By contrast, 10 of 12 FA patients grew no CFU-GM, BFU-E, or CFU-GEMM colonies, with or without SCF. In two FA patients (one transfusion- dependent and one transfusion-independent), an enhancement of CFU-GM and/or BFU-E was observed. The lack of response of hematopoietic progenitor cells from FA patients to GM-CSF+SCF or IL-3+SCF was not dependent on a defective expression of cytokine receptor messenger RNAs. Northern blot analysis showed in marrow cells from acquired AA and FA patients the presence of normal transcripts for alpha- and beta- chains of GM-CSF/IL-3 receptor and for c-kit protein. In conclusion, SCF promotes the in vitro growth of hematopoietic progenitors in patients with acquired AA, but not in patients with FA, pointing out the intrinsic nature of the defect in the latter disorder.

The aim of the present study was to test the effect of cyclosporin A (CyA) in vitro on CFU-GM growth from patients with severe aplastic anemia (SAA). For this purpose, bone marrow (BM) cells from 9 SAA patients and 5 healthy individuals were incubated with or without CyA and then cultured for CFU-GM growth in the presence of exogenous recombinant human GM-CSF (30 ng/ml). SAA patients were tested before or after treatment with CyA, or after treatment with antilymphocyte globulin (ALG). In 3 patients responding to CyA, the addition of CyA in vitro enhanced colony growth from 13 +/- 10 to 40 +/- 20/10(5) BM cells (p = 0.01) - the median increment of colony formation was 2.4-fold. In 5 ALG responders, CyA produced no increment of CFU-GM growth (from 14 +/- 26 to 15 +/- 16/10(5) BM cells, p = 0.1). CyA did not enhance significantly CFU-GM growth in normal controls (from 57 +/- 45 to 58 +/- 81/10(5) BM cells, p = 0.9). In conclusion, it would appear that some patients with SAA can respond to CyA in vivo and in vitro, and ALG responders are not necessarily among these. This is in keeping with different mechanisms of action of CyA and ALG and possibly with the existence of distinct pathogenetic pathways in SAA.

Background. Cord blood transplants (CBT) are associated with delayed or failed engraftment in a significant proportion of patients (pts). Two of our previous observations suggested (i) that, in the animal model, direct intra-bone (i.b.) injection improves seeding efficiency and (ii) that the delayed engraftment was not related to an insufficient number of hematopoietic stem cells but rather to some difficulties to differentiate and maturate. Methods. Unrelated CB cells were selected for 29 consecutive pts (18 CB units were 4/6 HLA antigen matched, 10 were 5/6 and one 3/6 antigen matched). Median cell dose infused was 2.3 x10^7/kg (range 1.4 – 4.2). CB cells were concentrated in 4 syringes of 5 ml each and injected in the supero-posterior iliac crest (SPIC) under rapid general anesthesia (10 min. with propofol). Pts’ median age was 38 years (18–63); 25 had acute leukaemia (21 with refractory or relapsed disease and 4 high risk first remission leukemia); 2 chronic myeloid leukemia in advanced phase; 2 refractory Hodgkin’s disease. Most pts (n=24) were prepared with conventional conditioning regimen (TBI-cyclophosphamide). Results. The infusion of cells i.b. in SPIC (11 pts bilaterally; 18 pts monolaterally) was uneventful. Five pts are not evaluable because they died within 14 days from transplant. All pts surviving more than 14 days engrafted (100%). Median time for PMN (>0.5x10^9/l) and platelets (>20x10^9/l) engraftment was day +23 (14–40) and +38 (range 22–60) respectivelly. Four pts died of infection; one patient died of PTLD on day +140. Four patients relasped and 3 died of relapse. Fifteen out of 16 alive patients are in hematologic or molecular remission at a median follow up of 7.5 months (range 2–17). From day +30 full donor chimerism was documented in CD3, bone marrow cells and progenitor cells from both the injected and in non-injected SPIC; from day +30, CFC progenitors had already reached the lower values of the range of normal individuals in bilateral sites. These findings document the colonization of the hematopoietc system and the recovery of stem cell reservoir possibly due to an improvement of seeding efficiency. Only 3 pts (8%) experienced acute GvHD (2 grade II and 1 grade I); 4 pts. have moderate chronic GVHD. It is known that lymphocyte trafficking is one of the crucial factor in immunity. Two combined factors might contribute to the low incidence of acute GvHD: few of the transplanted T cells do not reach/circulate primarily in the lymphatic organs, where they would be immediately confronted with host antigen presenting cells as probably occurs after i.v. injection; injected T cells come immediately in contact with mesenchymal stem cells (MSC) and osteoblasts, known to be potent immunosuppressants. Conclusion. Direct intra-bone transplant of CB cells overcomes the problem of graft failure and is associated with reduced incidence of acute GvHD even when low numbers of HLA mismatched CB cells are transplanted. Nearly all patients searching for a CB unit were able to undergo CBT. This approach may change our policy of hemopoietic cell transplants.

We are reporting the outcome of 39 patients with idiopathic myelofibrosis (IMF) who received an allogeneic hemopoietic stem cell transplant (HSCT) at a single Institution. Patients. The conditioning was a thiotepa - cyclophosphamide based reduced intensity regimen, and graft versus host disease (GvHD) prophylaxis was cyclosporin and methotrexate. 30 patients received marrow, 9 peripheral blood. The donor was an HLA identical sibling (ISD) (n=22) or an alternative related or unrelatd donor (UD) (n=17). The median age was 51 (32–67), the median interval diagnosis HSCT was 1015 days (225–7178) and the Lille risk score at the time of transplant was intermediate in 23 and high in 16 patients. Splenectomy was performed in 25 patients as a preparation for transplant. Results. The overall actuarial survival is 50% with a median follow up of 823 days (221– 4356). In univariate analysis favourable factors for survival were a Karnowsky score of 100% (93% vs 37%), an interval diagnosis transplant of less than 1 year (85% vs 55%), an HLA identical sibling donor (68% vs 47%) and splenectomy pre-transplant (60% vs 50%). When combined together, 17 and 22 patients has 0–1 or 2–3 favourable factors pre-transplant: their 4 year actuarial survival is respectively 0% vs 83% (p=.0001). Patients with 0–1 favourable factors, had higher transplant related mortality (41% vs 0%; p=0.009) and higher relapse related death (35% vs 4%, p=0.01). Conclusions. We conclude that allogeneic transplantation is a curative option for patients with idiopathic myelofibrosis. We have identified factors which predict a favourable outcome and may be used to select patients for this procedure. Download : Download high-res image (208KB) Download : Download full-size image Figure

Background. Hematologic relapse after an allogeneic HSCT for acute leukaemia is associated with poor outcome: in our Unit, 5 year survival for acute lymphoblastic leukaemia (ALL) relapsing after an allograft is 10% and for acute myeloid leukaemia (AML) this figure is 15%. Most relapses (79%) occur within the first year after transplant. Aim of the study. Prospectively assess minimal residual disease (MRD) after allogeneic HSCT to test (a) incidence of MRD positivity, (b) predictive value on hematologic relapse and (c) whether MRD positivity can be used to initiate treatment with donor lymphocyte infusions (DLI). Patients and methods. Seventy three patients with ALL (n=29) or AML (n=44) received an allogeneic HSCT, most of them after a conventional (CY-TBI) conditioning regimen. Re-arrangement for JH regions and fusion genes (bcr/abl) (RQ-PCR) were used to monitor MRD in ALL, whereas Wilms Tumour 1 (WT1) expression (RQ-PCR) was used in AML. Monitoring was done on bone marrow samples taken pre-transplant, and on day+30, +60, +90, +120, +150, +180. At the time of a positive MRD patients would be eligible for DLI in escalating doses, starting at 10^6/kg CD3+ cells for matched related donors, increasing by ½ log every 30 days up to a maximum dose of 5x10^7/kg CD3+ cells. For unrelated and 1 antigen family mismatched transplants the dose of DLI was reduced by 1 log. Number of MRD+ patients post-transplant. Overall 30 patients of 73 patients (41%) were MRD positive after transplant, 66% for ALL and 25% for AML. Predictive value of MRD positivity on relapse. Hematologic relapse occurred in 15 of 30 MRD+ patients and in 2 of 43 MRD negative patients (p=0.0001). For ALL patients these figures were 10/19 vs 2/10 (p=0.09) and for AML it was 5/11 vs 0/33 (p=0.0004). Preventing relapse with DLI in MRD+ patients. Of the 30 MRD positive patients 16 were given no DLI because the donor was not available or unsuitable for second donation at the time of MRD positivity: 14 patients relapsed (87%) and 3 (19%) survive. Fourteen patients were given DLI, one relapsed (7%) and 12 survive (86%). The difference of hematologic relapse in MRD positive patients receiving DLI (8%) or not receiving DLI (57%) is highly significant (p=0.0001). This particularly true for ALL patients receiving DLI (n=10) (1 relapse and 8 survivors) as compared to ALL patients not receiving DLI (n=9) (9 relapses and 1 survivor). Conclusions. This study shows that (a) MRD positivity is frequent after allogeneic HSCT, (b) MRD positivity predicts hematologic relapse and (c) treatment of MRD with DLI protects against hematologic relapse, and improves survival. This study also confirms our previous data on the efficacy of DLI in acute lymphoblastic leukemia, when the tumour burden is low.

Experimental evidence and preliminary clinical studies have demonstrated that human mesenchymal stem cells (MSCs) display important immune modulatory function of potential relevant interest in the setting of allogeneic hematopoietic stem cell (HSC) transplantation. Effectiveness of MSCs in controlling severe GVHD seems to be related to the immune-regulatory role they play in suppressing alloantigen-specific T-cell activation. Aim of the present study was to extend the analysis of the mechanisms responsible for the immune regulatory effect of interaction between MSCs and alloantigen-specific immune response elicited in vitro in primary and in secondary mixed lymphocyte culture (MLC). At difference with most previously reported studies, we decided to employ non-irradiated MSCs, reasoning that irradiation might impair, beside the proliferative capacity, also the differentiation capability of MSCs and, consequently, alter their interaction pattern with lymphocyte subsets. MSC were added to primary MLC at different doses (MLC-responder-PBMC:MSC ratios = 1:1 and 10:1). Dendritic cell (DC) differentiation, lymphocyte proliferation, alloantigen-specific cytotoxic activity and differentiation of CD4+ T-cell subsets expressing CD25 and/or CTLA4 antigens were assessed in primary and secondary MLC, comparing the effect observed using third-party MSCs with that obtained employing autologous to the MLC-responder (autologous) MSCs. Results demonstrated that human MSCs: (1) strongly inhibit alloantigen-induced DC1 differentiation; (2) down-regulate, in a dose-dependent manner, alloantigen-induced lymphocyte expansion, especially that of CD8+ T cells and of NK lymphocytes; (3) favor the differentiation of CD4+ T cells co-expressing CD25 and/or CTLA4, a phenotype associated with regulatory/suppressive function of immune response; (4) cause a dose-dependent reduction of alloantigen-specific cytotoxic capacity mediated by either cytotoxic T lymphocytes or NK cells; (5) exert more effective suppressive activity on MLC-induced T-cell activation when they are allogeneic rather than autologous with respect to responder cells. In particular, higher percentages of CD4+ and of CD4+CD25+ T cells co-expressing CTLA4+ were detected when third-party, rather than autologous, MSCs were added to MLC. These data suggest that T-cell recognition of alloantigens expressed by MSCs may further facilitate the preferential differentiation of activated CD4+ T cells expressing CTLA4, a glycoprotein, known to deliver an inhibitory signal to T cells and to mediate apoptosis of previously activated T lymphocytes. Several studies previously demonstrated that MSCs exert inhibitory effect on lymphocyte activation through the release of soluble factors. Our data suggest that the preferential differentiation of CD4+CD25+ regulatory T-cell subsets may be favored by other mechanisms of MSC-mediated inhibition of alloantigen-induced effector cell activation and expansion, and, in turn, these CD4+CD25+ cells contribute to propagate and extend suppressor activity. Altogether, our results provide immunological support to the use of MSCs for prevention of immune complications related to both HSC and solid organ transplantation and to the theory that MSCs are “universal” suppressors of immune reactivity.

Normal bone marrow derived mesenchymal stem cells (MSC) suppress T cell function, and may provide an immunoprotected environment for hemopoietic stem cells. In this study we compare the suppressive activity on T cell function of MSC from 23 patients with severe aplastic anemia (SAA) patients, at diagnosis (n=3), following immunosuppressive therapy (IS) (n=16) or after an allogeneic bone marrow transplant (BMT) (n=4), and normal individuals. As shown in Figure 1 increasing numbers of control MSC (25, 50 and 100x10^3 cells) produced a dose dependent suppression of PHA induced T cell proliferation; when MSC were derived from SAA patients, they exhibited significantly less suppressive activity (37% vs 6%, 81% vs 24%, 96% vs 35%). The reduced ability of SAA MSC to suppress mitogen induced T cell proliferation, was seen irrespective of disease phase. Paired experiments on mixed lymphocyte reaction confirmed the inability of SAA MSC to suppress T cell function. Other abnormalities of SAA MSC included: (a) impaired capacity to down regulate CD38 expression on PHA primed T cells, (b) impaired ability to suppress gamma-IFN production in PHA coltures, which resulted in a 11 fold different gamma-IFN concentration in the cultures; (c) no effect on T cell mediated inhibition of hemopietic colony formation. Finally SAA MSC produced significantly (1 log) less adenosin diphosphate -ribosyl cyclase (cADPR), a promoter of in vitro hematopoiesis. MSC mediated suppression of PHA induced T cell proliferation, was restored to control levels in 3 of 4 patients post-BMT. In conclusion, the ability of MSC to down regulate T cell priming, proliferation and cytokine release is deficient in patients with SAA, persists indefinitively after immunosuppressive therapy, but may be restored after BMT. Whether this defect plays a role in the pathogenesis of marrow failure, remains to be determined. Download : Download high-res image (231KB) Download : Download full-size image Figure

Objective. An objective of autologous transplant in chronic myelogenous leukemia is to reinfuse the patient stem cells with the lowest amount of leukemic progenitors. We analyzed if the cytogenetic response after autografting was correlated with the amount of BCR ABL–positive cells present in the graft. Materials and Methods. By BCR-ABL mRNA quantification, we studied the serial pheresis product from 40 Philadelphia (Ph)-positive patients who received ICE/mini-ICE mobilization therapy and underwent autologous stem cell transplant in our unit. Therefore, we correlated the residual disease in the graft reinfused to the patients with the cytogenetic response after autografting, taking into consideration only those responses that lasted 12 months or more. Results. Thirty-two patients had a graft with 0–35% Ph-metaphases and 19 had a graft with BCR-ABL/ABL ratio ≤0.01. After a median of 27 months (range, 12–50) from transplant, 18 patients achieved complete or major cytogenetic response lasting at least 12 months and 14 of them (78%) had a graft with BCR-ABL/ABL ratio ≤0.01 (range, 0.0003–0.01). Twenty-two patients had short-lived response or showed to be >35% Ph-positive in the marrow after transplant, but only 5 of them (23%) had a graft with BCR-ABL/ABL ratio ≤0.01 (range, 0.001–0.01). Therefore, we found a strong association between BCR-ABL/ABL ratio ≤0.01 in the reinfused progenitor collections and the achievement of complete or major cytogenetic remission after autografting ( χ 2 test, p = 0.0001). Patients reinfused with low contaminated grafts also showed a longer duration of the response (logrank test, P = 0.0009). Eleven patients were reinfused with the lowest-contaminated stem cell collections, according to the BCR-ABL/ABL ratio. None of these patients had prolonged neutropenia or thrombocytopenia following stem cell reinfusion and 9 of them had long-lasting complete or major cytogenetic response after transplant. Conclusion. This study demonstrates that the amount of BCR-ABL positive cells present in the graft is an important predictive factor for the achievement and the duration of cytogenetic response after autografting.

Peripheral blood T-cell subpopulations were studied in eight patients undergoing bone marrow transplantation (BMT) for severe aplastic anemia (SAA) or acute leukemia (AL). Seven received an allogeneic graft and one a syngeneic graft: seven showed clinical and pathological signs of graft versus host disease (GvHD) and a marked imbalance in T-cell subpopulations. In spite of normal numbers of circulating unfractionated T cells, T lymphocytes with receptors for IgG (TG cells) were increased 2- to 5-fold, whereas T cells with receptors for IgM (TM cells) showed a 2- to 20-fold decrease. The TM-TG imbalance paralleled the development of GvHD. The patient who received the syngeneic graft showed no TM-TG imbalance and no signs of GvHD. In addition pokeweed mitogen (PWM)-induced B-cell differentiation in vitro was severely impaired in these patients in the presence of either autologous or normal allogeneic T cells. Patients' T cells did not provide adequate help for autologous or normal B cells either in the presence or absence of TG cells. The patients' purified TG cells were capable of suppressing normal B-cell responses to PWM but to a lesser degree than normal TG cells. The imbalance of T-cell subpopulations with defective helper activity and the impaired B-cell function may play an important role in the development of immunodeficiency after bone marrow transplantation and particularly during GvHD.