Antonio Tabilio

Full Haplotype-Mismatched Hematopoietic Stem-Cell Transplantation: A Phase II Study in Patients With Acute Leukemia at High Risk of Relapse

PURPOSE Establishment of hematopoietic stem-cell (HSC) transplantation from mismatched relatives is feasible for patients with acute leukemia. As our original method of graft processing was unsuitable for large-scale clinical studies, we use automated devices for CD34+ cell purification. PATIENTS AND METHODS Sixty-seven patients with acute myeloid leukemia (AML; 19 complete remission [CR] 1, 14 CR 2, nine CR > 2, 25 in relapse) and 37 with acute lymphoid leukemia (ALL; 14 CR 1, eight CR 2, two CR > 2, 13 in relapse) were conditioned with total-body irradiation, thiotepa, fludarabine, and antithymocyte globulin. Peripheral-blood progenitor cells were mobilized with recombinant human granulocyte colony-stimulating factor and depleted of T-cells using CD34+ cell immunoselection. No post-transplantation graft-versus-host disease (GvHD) prophylaxis was administered. RESULTS Primary engraftment was achieved in 94 of 101 assessable patients. Six of the seven patients who rejected the primary graft, engrafted after a second transplantation. Overall, 100 of 101 patients engrafted. Acute GvHD developed in eight of 100 patients, and chronic GvHD, in five of 70 assessable patients. Thirty-eight patients died of nonleukemic causes. Relapse occurred in nine of 66 patients receiving transplantation in remission and in 17 of 38 receiving transplantation in relapse. Median follow-up of the 40 patients who survived event-free was 22 months (range, 1 to 65 months). Event-free survival (+/- standard deviation) rate was 48% +/- 8% and 46% +/- 10%, respectively, for the 42 AML and 24 ALL patients receiving transplantation in remission. CONCLUSION Our transplantation procedure provides reliable, reproducible CD34+ cell purification, high engraftment rates, and prevention of GvHD. The mismatched-related transplant emerges as a viable, alternative source of stem cells for acute leukemia patients without matched donors and/or those who urgently need transplantation.

Mesenchymal cells recruit and regulate T regulatory cells

OBJECTIVE Despite much investigation into T regulatory cells (Tregs), little is known about the mechanism controlling their recruitment and function. Because multipotent mesenchymal stromal cells (MSCs) exert an immune regulatory function and suppress T-cell proliferation, this in vitro study investigated their role in Treg recruitment and function. MATERIALS AND METHODS Human MSCs and different T cell populations (CD3(+), CD3(+)/CD45RA(+), CD3(+)/CD45RO(+), CD4(+)/CD25(+), CD4(+)/CD25(+)/CD45RO(+), CD4(+)/CD25(+)/CD45RA(+)) from healthy donors were cocultured for up to 15 days. Harvested lymphocytes were analyzed by flow cytometry and FoxP3 and CD127 expressions were measured by real-time polymerase chain reaction. Their regulatory activity was assessed. RESULTS We demonstrate MSC recruit Tregs from a fraction of CD3(+) and from immunoselected CD3(+)/CD45RA(+) and CD3(+)/CD45RO(+) fractions. After culture with MSCs both immunoselected fractions registered increases in the CD4(+)/CD25(bright)/FoxP3 subset and CD127 expression was downregulated. When purified Treg populations (CD4/CD25(+), CD4/CD25(+)/CD45RA(+), and CD4/CD25(+)/CD45RO(+)) are used in MSC cocultures, they maintain FoxP3 expression and CD127 expression is downregulated. Treg suppressive capacity was maintained in Treg populations that were layered on MSC for up to 15 days while control Tregs lost all suppressive activity after 5 days culture. CONCLUSIONS In conclusion, our study demonstrates that MSCs recruit, regulate, and maintain T-regulatory phenotype and function over time.

Constitutively activated Notch signaling is involved in survival and apoptosis resistance of B-CLL cells

Notch signaling is involved in tumorigenesis, but its role in B-chronic lymphocytic leukemia (B-CLL) pathogenesis is not completely defined. This study examined the expression and activation of Notch receptors in B-CLL cells and the role of Notch signaling in sustaining the survival of these cells. Our results show that B-CLL cells but not normal B cells constitutively express Notch1 and Notch2 proteins as well as their ligands Jagged1 and Jagged2. Notch signaling is constitutively activated in B-CLL cells, and its activation is further increased in B-CLL cells, which resist spontaneous apoptosis after 24-hour ex vivo culture. Notch stimulation by a soluble Jagged1 ligand increases B-CLL cell survival and is accompanied by increased nuclear factor-kappa B (NF-kappaB) activity and cellular inhibitor of apoptosis protein 2 (c-IAP2) and X-linked inhibitor of apoptosis protein (XIAP) expression. In contrast, Notch-signaling inhibition by the gamma-secretase inhibitor I (GSI; z-Leu-Leu-Nle-CHO) and the specific Notch2 down-regulation by small-interfering RNA accelerate spontaneous B-CLL cell apoptosis. Apoptotic activity of GSI is accompanied by reduction of NF-kappaB activity and c-IAP2 and XIAP expression. Overall, our findings show that Notch signaling plays a critical role in B-CLL cell survival and apoptosis resistance and suggest that it could be a novel potential therapeutic target.

Expression of platelet membrane glycoproteins and alpha-granule proteins by a human erythroleukemia cell line (HEL).

We demonstrate that HEL, a human erythroleukemic cell line, has numerous megakaryocytic markers which were markedly enhanced following the addition of the inducers dimethyl sulfoxide or 12‐O‐tetradecanoylphorbol‐13‐acetate to the culture medium. Ultrastructural and cytochemical studies showed: (i) the presence of organelles morphologically resembling the platelet alpha‐granules; and (ii) a peroxidase activity with the same characteristics as that specifically found in platelets. The platelet alpha‐granule proteins (von Willebrand factor, platelet factor‐4 and beta‐thromboglobulin) were immunologically detected in the HEL cell cytoplasm and their amounts increased after induction. Of particular interest was the presence of platelet membrane proteins. A monoclonal antibody specific for glycoprotein Ib bound to HEL cells. Platelet membrane glycoproteins IIb and IIIa were identified on intact cells using specific antibodies in a binding assay or in cell lysates using either crossed immunoelectrophoresis or an immunoblotting procedure following SDS‐polyacrylamide gel electrophoresis. Most HEL cells also expressed the platelet alloantigen PIA1. All of the platelet membrane proteins were present in higher amounts after induction. Glycophorin A, specific for the erythroid lineage, was also detected on HEL cells. Thus, while confirming the presence of erythroid markers, our studies provide evidence that the HEL cell line also expresses platelet antigens. As such, HEL cells represent a unique system with which to study the biosynthesis of platelet‐specific proteins and glycoproteins.

Immunophenotype of adult and childhood acute promyelocytic leukaemia: correlation with morphology, type of PML gene breakpoint and clinical outcome. A cooperative Italian study on 196 cases

Acute promyelocytic leukaemia (APL), characterized by a specific PML‐RARα fusion gene resulting from translocation t(15;17) and by a high response rate to differentiation therapy with all‐trans retinoic acid, presents clinical (varying WBC counts, age and treatment outcome), morphological (hypergranular M3 and hypogranular M3V) and molecular (three isoforms of PML breakpoint) heterogeneity.

A randomised clinical trial comparing idarubicin and cytarabine to daunorubicin and cytarabine in the treatment of acute non-lymphoid leukaemia

Abstract 255 patients with acute non-lymphoid leukaemia (ANLL), observed between October 1984 and June 1987, entered a chemotherapy regimen consisting of induction therapy with cytarabine in combination with idarubicin (IDA/ARA) or daunorubicin (DNR/ARA), followed by consolidation with four courses of IDA+ARA plus 6-thioguanine (6-TG) or DNR+ARA+6-TG and a 6 month maintenance therapy with 6-TG and ARA. The median age was 62 years (range 55–78 years) and 33 were aged more than 70 years. The treatment groups were comparable for median age, FAB type, performance status and initial blood counts. 249 patients were randomised, 124 to the IDA/ARA arm and 125 to the DNR/ARA arm. Complete remission was achieved in 50 patients (40%) on the IDA/ARA treatment program and 49 patients (39%) on DNR/ARA. No definite differences were found between patients receiving IDA/ARA and those treated with DNR/ARA as far as complete response (CR), overall survival, failure free and relapse free survival are concerned. 74% of the complete responders in the IDA/ARA arm and 51% in the DNR/ARA arm achieved CR after a single course of treatment. Resistant leukaemia was observed in 13.7% of the patients in the IDA/ARA arm and in 31.2% in the DNR/ARA one, whereas hypoplastic death occurred in 29% and 14.4%, respectively. In conclusion, our data failed to show any advantage of idarubicin over daunorubicin even though there is some evidence that IDA, despite the higher toxicity, is more rapid in eradicating leukaemia as proved by the higher CR rate obtained after one course of induction.

Downmodulation of ERK activity inhibits the proliferation and induces the apoptosis of primary acute myelogenous leukemia blasts

MAP kinase/ERK kinase (MEK)-extracellular signal-regulated kinase (ERK) kinases are frequently activated in acute myelogenous leukemia (AML), and can have prosurvival function. The purpose of this study was to induce downmodulation of MEK-ERK activation in AML primary blasts in order to detect the effect on cell cycle progression and on the apoptosis of leukemic cells. We investigated 14 cases of AML with high ERK 1/2 activity and four cases with undetectable or very low activity. After 24 h incubation of the AML blasts with high ERK activity using PD98059 (New England BioLabs, Beverly, MA, USA), a selective inhibitor of MEK1 phosphorylation, at concentrations of 20 and 40 μM, we observed a strong decrease in the levels of ERK1/2 activity. A significant decrease of blast cell proliferation compared with untreated controls was found. In contrast, the proliferation of blast cells that expressed low or undetectable levels of ERK activity was not inhibited. Time-course analysis demonstrated that the downmodulation of MEK1/2, ERK1 and ERK2 dual-phosphorylation was evident even after 3 h of treatment with 20 and 40 μM. The cleavage of poly(ADP-ribose) polymerase (PARP), an early sign of apoptosis, appeared after 18 h of PD98059 treatment at concentrations of 20 and 40 μM in eight of the 14 cases. After 24 h of treatment, cleaved PARP appeared in all 14 cases. Time-course analysis of cell cycle progression and apoptosis showed that PD98059 induced a G1-phase accumulation with low or undetectable levels of apoptosis after 24 h incubation; after 48 and 72 h incubation, a significant increase of apoptosis was observed. Thus, the primary effect of ERK downmodulation was a cell cycle arrest followed by the apoptosis of a significant percentage of the leukemic blasts.The preclinical model of leukemia treatment reported in this paper makes further comment with regard to MEK1 inhibition as a useful antileukemic target, and encourages the conducting of in vivo studies and clinical investigations.

The hypoxia-inducible factor is stabilized in circulating hematopoietic stem cells under normoxic conditions

The hypoxia‐inducible factor (HIF) transcriptional system enables cell adaptation to limited O2 availability, transducing this signal into patho‐physiological responses such as angiogenesis, erythropoiesis, vasomotor control, and altered energy metabolism, as well as cell survival decisions. However, other factors beyond hypoxia are known to activate this pleiotropic transcription factor. The aim of this study was to characterize HIF in human hematopoietic stem cells (HSCs) and evidence is provided that granulocyte colony stimulating factor‐mobilized CD34+‐ and CD133+‐HSCs express a stabilized cytoplasmic form of HIF‐1α under normoxic conditions. It is shown that HIF‐1α stabilization correlates with down‐regulation of the tumour suppressor von Hippel‐Lindau protein (pVHL) and is positively controlled by NADPH‐oxidase‐dependent production of reactive oxygen species, indicating a specific O2‐independent post‐transcriptional control of HIF in mobilized HSCs. This novel finding is discussed in the context of the proposed role of HIF as a mediator of progenitor cell recruitment to injured ischemic tissues and/or in the control of the maintenance of the undifferentiated state.

A PHASE II STUDY OF INTERLEUKIN-2 IN 49 PATIENTS WITH RELAPSED OR REFRACTORY ACUTE LEUKEMIA

In this report we present the results of a multicenter phase II study of high-dose recombinant Interleukin-2 (rIL-2) in patients with refractory or relapsed acute leukemia. Forty-nine patients with acute myeloid leukemia (AML: 30 patients) or acute lymphoblastic leukemia (ALL: 19 patients) were included. Median age was 30 years (range: 4-71). Four patients were treated for primary refractory disease and 45 for relapsed disease (16 patients > 2nd relapse). Twenty-four patients (49%) had previously received bone marrow transplantation (allogeneic: 5, autologous: 19). Patients were scheduled to receive three 5-day cycles of rIL-2 given every other week. rIL-2 was administered as bolus I.V. infusion of 8 x 10(6) UI/m2 every 8 hours during cycle I and every 12 hours during cycles 2 and 3. Patients received a mean of 76% of rIL-2 planned dose. Main toxicity was hematologic (grade IV thrombopenia: 84%). Hemodynamic and metabolic toxicities lead to treatment discontinuation in 10 patients (20%). Strong immune activation was achieved including a significant increase in activated T-cells and Lymphokine-Activating-Killer cell (LAK) activity. Twenty-seven out of 30 AML patients could be evaluated for response: 2(7%) achieved complete remission (CR) which lasted 3 and 4 months. No response was observed in the 18 assessable ALL patients, most of whom (77 %) presented absolute drug resistance. These results show that this high dose rIL-2 regimen induces significant biological effects and provides some anti-leukemic activity in patients with advanced leukemia. Considering the severe toxicity observed and the limited remission rate achieved here, rIL-2 does not appear to be a valuable therapeutic option for such patients. However, the undoubted anti-leukemic activity of this cytokine invites further investigation especially in the minimal residual disease situation.

Immunomagnetic isolation of CD4+CD25+FoxP3+ natural T regulatory lymphocytes for clinical applications.

Although CD4+/CD25+ T regulatory cells (Tregs) are a potentially powerful tool in bone marrow transplantation, a prerequisite for clinical use is a cell‐separation strategy complying with good manufacturing practice guidelines. We isolated Tregs from standard leukapheresis products using double‐negative selection (anti‐CD8 and anti‐CD19 monoclonal antibodies) followed by positive selection (anti‐CD25 monoclonal antibody). The final cell fraction (CD4+/CD25+) showed a mean purity of 93·6% ± 1·1. Recovery efficiency was 81·52% ± 7·4. The CD4+/CD25+bright cells were 28·4% ± 6·8. The CD4+/CD25+ fraction contained a mean of 51·9% ± 15·1 FoxP3 cells and a mean of 18·9% ± 11·5 CD127 cells. Increased FoxP3 and depleted CD127 mRNAs in CD4+CD25+FoxP3+ cells were in line with flow cytometric results. In Vβ spectratyping the complexity scores of CD4+/CD25+ cells and CD4+/CD25‐ cells were not significantly different, indicating that Tregs had a broad T cell receptor repertoire. The inhibition assay showed that CD4+/CD25+ cells inhibited CD4+/CD25‐ cells in a dose‐dependent manner (mean inhibition percentages: 72·4 ± 8·9 [ratio of T responder (Tresp) to Tregs, 1:2]; 60·8% ± 20·5 (ratio of Tresp to Tregs, 1:1); 25·6 ± 19·6 (ratio of Tresp to Tregs, 1:0·1)). Our study shows that negative/positive Treg selection, performed using the CliniMACS device and reagents, enriches significantly CD4+CD25+FoxP3+ cells endowed with immunosuppressive capacities. The CD4+CD25+FoxP3+ population is a source of natural Treg cells that are depleted of CD8+ and CD4+/CD25‐ reacting clones which are potentially responsible for triggering graft‐versus‐host disease (GvHD). Cells isolated by means of this approach might be used in allogeneic haematopoietic cell transplantation to facilitate engraftment and reduce the incidence and severity of GvHD without abrogating the potential graft‐versus‐tumour effect.