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Introduction:The prevention ofdisease transformation which typically occurs during the first years of treatment, is one of the critical goals of the treatment of CP-CML with TKI. In addition, treatment free remission (TFR) represents now another major challenge. In vitrodata show that ponatinib is one of the most powerful inhibitors of wild-type as well as mutated BCR::ABL1 TK and this has been translated in heavily pre-treated CML patients (pts) intolerant or resistant to other compounds. In this trial we assessed the safety and efficacy of a 6-months debulking strategy using ponatinib as front-line therapy in newly diagnosed CP-CML, followed by a maintenance with imatinib, in order to bring a high proportion of pts in TFR.

Cytotoxic T lymphocytes kill target cells via the polarized secretion of cytotoxic granules at the immune synapse. The lytic granules are initially recruited around the polarized microtubule-organizing center. In a dynein-dependent transport process, the granules move along microtubules toward the microtubule-organizing center in the minus-end direction. Here, we found that a kinesin-1–dependent process is required for terminal transport and secretion of polarized lytic granule to the immune synapse. We show that synaptotagmin-like protein 3 (Slp3) is an effector of Rab27a in cytotoxic T lymphocytes and interacts with kinesin-1 through the tetratricopeptide repeat of the kinesin-1 light chain. Inhibition of the Rab27a/Slp3/kinesin-1 transport complex impairs lytic granule secretion. Our data provide further molecular insights into the key functional and regulatory mechanisms underlying the terminal transport of cytotoxic granules and the latter's secretion at the immune synapse.

Cytotoxic T lymphocytes kill target cells via the polarized secretion of cytotoxic granules at the immune synapse. The lytic granules are initially recruited around the polarized microtubule-organizing center. In a dynein-dependent transport process, the granules move along microtubules toward the microtubule-organizing center in the minus-end direction. Here, we found that a kinesin-1–dependent process is required for terminal transport and secretion of polarized lytic granule to the immune synapse. We show that synaptotagmin-like protein 3 (Slp3) is an effector of Rab27a in cytotoxic T lymphocytes and interacts with kinesin-1 through the tetratricopeptide repeat of the kinesin-1 light chain. Inhibition of the Rab27a/Slp3/kinesin-1 transport complex impairs lytic granule secretion. Our data provide further molecular insights into the key functional and regulatory mechanisms underlying the terminal transport of cytotoxic granules and the latter's secretion at the immune synapse.

Cytotoxic T lymphocytes (CTLs) and natural killer cells help control infections and tumors via a killing activity that is mediated by the release of cytotoxic granules. Granule secretion at the synapse formed between the CTL and the target cell leads to apoptosis of the latter. This process involves polarization of the CTL's secretory machinery and cytotoxic granules. The small GTPase Rab27a and the hMunc13-4 protein have been shown to be required for both granule maturation and granule docking and priming at the immunologic synapse. Using a tandem affinity purification technique, we identified a previously unknown hematopoietic form of Slp2a (Slp2a-hem) and determined that it is a specific effector of the active form of Rab27a. This interaction occurs in vivo in primary CTLs. We have shown that (1) Rab27a recruits Slp2a-hem on vesicular structures in peripheral CTLs and (2) following CTL-target cell conjugate formation, the Slp2a-hem/Rab27a complex colocalizes with perforin-containing granules at the immunologic synapse, where it binds to the plasma membrane through its C2 domains. The overexpression of a dominant-negative form of Slp2a-hem markedly impaired exocytosis of cytotoxic granules—indicating that Slp2a is required for cytotoxic granule docking at the immunologic synapse.

Cytotoxic T lymphocytes (CTLs) and natural killer cells help control infections and tumors via a killing activity that is mediated by the release of cytotoxic granules. Granule secretion at the synapse formed between the CTL and the target cell leads to apoptosis of the latter. This process involves polarization of the CTL's secretory machinery and cytotoxic granules. The small GTPase Rab27a and the hMunc13-4 protein have been shown to be required for both granule maturation and granule docking and priming at the immunologic synapse. Using a tandem affinity purification technique, we identified a previously unknown hematopoietic form of Slp2a (Slp2a-hem) and determined that it is a specific effector of the active form of Rab27a. This interaction occurs in vivo in primary CTLs. We have shown that (1) Rab27a recruits Slp2a-hem on vesicular structures in peripheral CTLs and (2) following CTL-target cell conjugate formation, the Slp2a-hem/Rab27a complex colocalizes with perforin-containing granules at the immunologic synapse, where it binds to the plasma membrane through its C2 domains. The overexpression of a dominant-negative form of Slp2a-hem markedly impaired exocytosis of cytotoxic granules—indicating that Slp2a is required for cytotoxic granule docking at the immunologic synapse.

CD4+CD25+ regulatory T (Treg) cells play a critical role in the induction and maintenance of peripheral immune tolerance. In experimental transplantation models in which tolerance was induced, donor-specific Treg cells could be identified that were capable of transferring the tolerant state to naive animals. Furthermore, these cells appeared to have indirect allospecificity for donor antigens. Here we show that in vivo alloresponses can be regulated by donor alloantigen-specific Treg cells selected and expanded in vitro. Using autologous dendritic cells pulsed with an allopeptide from H2-Kb, we generated and expanded T-cell lines from purified Treg cells of CBA mice (H2k). Compared with fresh Treg cells, the cell lines maintained their characteristic phenotype, suppressive function, and homing capacities in vivo. When cotransferred with naive CD4+CD25− effector T cells after thymectomy and T-cell depletion in CBA mice that received CBK (H2k+Kb) skin grafts, the expanded Treg cells preferentially accumulated in the graft-draining lymph nodes and within the graft while preventing CBK but not third-party B10.A (H2k+Dd) skin graft rejection. In wild-type CBA, these donor-specific Treg cells significantly delayed CBK skin graft rejection without any other immunosuppression. Taken together, these data suggest that in vitro–generated tailored Treg cells could be considered a therapeutic tool to promote donor-specific transplant tolerance.

CD4+CD25+regulatory T (Treg) cells play a critical role in the induction and maintenance of peripheral immune tolerance. In experimental transplantation models in which tolerance was induced, donor-specific Treg cells could be identified that were capable of transferring the tolerant state to naive animals. Furthermore, these cells appeared to have indirect allospecificity for donor antigens. Here we show that in vivo alloresponses can be regulated by donor alloantigen-specific Treg cells selected and expanded in vitro. Using autologous dendritic cells pulsed with an allopeptide from H2-Kb, we generated and expanded T-cell lines from purified Treg cells of CBA mice (H2k). Compared with fresh Treg cells, the cell lines maintained their characteristic phenotype, suppressive function, and homing capacities in vivo. When cotransferred with naive CD4+CD25−effector T cells after thymectomy and T-cell depletion in CBA mice that received CBK (H2k+Kb) skin grafts, the expanded Treg cells preferentially accumulated in the graft-draining lymph nodes and within the graft while preventing CBK but not third-party B10.A (H2k+Dd) skin graft rejection. In wild-type CBA, these donor-specific Treg cells significantly delayed CBK skin graft rejection without any other immunosuppression. Taken together, these data suggest that in vitro–generated tailored Treg cells could be considered a therapeutic tool to promote donor-specific transplant tolerance.

To modulate alloreactivity after hematopoietic stem cell transplantation, suicide gene–expressing donor T cells can be administered with an allogeneic T-cell–depleted bone marrow graft. Immune competence of such cells is a critical issue. The impact of the ex vivo gene transfer protocol (12-day culture period including CD3/interleukin-2 [IL-2] activation, retroviral-mediated gene transfer, and G418-based selection) on the anti–Epstein-Barr virus (EBV) potential of gene-modified cells has been examined. Cytotoxic (pCTL) and helper (pTh) cell precursor limiting dilution assays, interferon-γ enzyme-linked immunospot, or fluorescence-activated cell sorter analysis after tetrameric HLA-A2/EBV peptide complexes revealed that the frequency of anti-EBV T cells was lower in gene-modified cells (GMCs) than in similarly cultured but untransduced T cells and was even lower than in fresh peripheral blood mononuclear cells, demonstrating both an effect of the culture and of the transduction or selection. The culture-dependent loss of EBV-reactive cells resulted from the preferential induction of activation-induced cell death in tetramer+ cells. Replacing the initial CD3/IL-2 activation by CD3/CD28/IL-2 partially restored the anti-EBV response of GMCs by reducing the initial activation-induced cell death and enhancing the proliferation of EBV-tetramer+cells. Moreover, the G418 selection, and not the transduction, was directly toxic to transduced tetramer+ cells. Replacing the G418 selection by an immunomagnetic selection significantly prevented the selection-dependent loss of EBV-specific cells. Overall, ex vivo gene modification of primary T cells can result in a significant reduction in EBV-reactive T cells through both culture-dependent and selection-dependent mechanisms. Improving immune functions of GMCs through modifications of the cell culture conditions and transduction/selection processes is critical for further clinical studies.

To modulate alloreactivity after hematopoietic stem cell transplantation, suicide gene–expressing donor T cells can be administered with an allogeneic T-cell–depleted bone marrow graft. Immune competence of such cells is a critical issue. The impact of the ex vivo gene transfer protocol (12-day culture period including CD3/interleukin-2 [IL-2] activation, retroviral-mediated gene transfer, and G418-based selection) on the anti–Epstein-Barr virus (EBV) potential of gene-modified cells has been examined. Cytotoxic (pCTL) and helper (pTh) cell precursor limiting dilution assays, interferon-γ enzyme-linked immunospot, or fluorescence-activated cell sorter analysis after tetrameric HLA-A2/EBV peptide complexes revealed that the frequency of anti-EBV T cells was lower in gene-modified cells (GMCs) than in similarly cultured but untransduced T cells and was even lower than in fresh peripheral blood mononuclear cells, demonstrating both an effect of the culture and of the transduction or selection. The culture-dependent loss of EBV-reactive cells resulted from the preferential induction of activation-induced cell death in tetramer+cells. Replacing the initial CD3/IL-2 activation by CD3/CD28/IL-2 partially restored the anti-EBV response of GMCs by reducing the initial activation-induced cell death and enhancing the proliferation of EBV-tetramer+cells. Moreover, the G418 selection, and not the transduction, was directly toxic to transduced tetramer+cells. Replacing the G418 selection by an immunomagnetic selection significantly prevented the selection-dependent loss of EBV-specific cells. Overall, ex vivo gene modification of primary T cells can result in a significant reduction in EBV-reactive T cells through both culture-dependent and selection-dependent mechanisms. Improving immune functions of GMCs through modifications of the cell culture conditions and transduction/selection processes is critical for further clinical studies.

The herpes simplex virus thymidine kinase gene type 1 (HSV-Tk) ganciclovir (GCV) system is a novel therapeutic strategy for the modulation of graft-versus-host disease (GVHD), a major complication of allogeneic stem cell transplantation (allo-SCT). Retroviral-mediated gene transfer of the HSV-Tkgene into donor T lymphocytes before allo-SCT may allow their in vivo selective depletion after treatment with GCV. The expression of theHSV-Tkgene was analyzed in vitro in CEM cells, a human lymphoblastoid cell line, transduced with 2 different vectors, each containing the HSV-Tkgene and a selectable marker gene. GCV-resistant clones were identified within the clones expressing the marker gene. Characterization of the molecular events leading to this resistance revealed a 227-bp deletion in the HSV-Tkgene due to the presence of cryptic splice donor and acceptor sites within the HSV-Tkgene sequence. Furthermore, it was confirmed that this deletion was present in human primary T cells transduced with either vector and in 12 patients who received transduced donor T cells, together with a T-cell–depleted allo-SCT. In vivo circulating transduced T cells containing the truncated HSV-Tkgene were identified in all patients immediately after infusion and up to 800 days after transplantation. In patients who received GCV as treatment for GVHD, a progressive increase in the proportion of transduced donor T cells carrying the deleted HSV-Tkgene was observed. These results suggest that the limitations within the HSV-Tk/GCV system can be improved by developing optimized retroviral vectors to ensure maximal killing ofHSV-Tk–transduced cells.

The herpes simplex virus thymidine kinase gene type 1 (HSV-Tk) ganciclovir (GCV) system is a novel therapeutic strategy for the modulation of graft-versus-host disease (GVHD), a major complication of allogeneic stem cell transplantation (allo-SCT). Retroviral-mediated gene transfer of the HSV-Tk gene into donor T lymphocytes before allo-SCT may allow their in vivo selective depletion after treatment with GCV. The expression of theHSV-Tk gene was analyzed in vitro in CEM cells, a human lymphoblastoid cell line, transduced with 2 different vectors, each containing the HSV-Tk gene and a selectable marker gene. GCV-resistant clones were identified within the clones expressing the marker gene. Characterization of the molecular events leading to this resistance revealed a 227-bp deletion in the HSV-Tk gene due to the presence of cryptic splice donor and acceptor sites within the HSV-Tk gene sequence. Furthermore, it was confirmed that this deletion was present in human primary T cells transduced with either vector and in 12 patients who received transduced donor T cells, together with a T-cell–depleted allo-SCT. In vivo circulating transduced T cells containing the truncated HSV-Tk gene were identified in all patients immediately after infusion and up to 800 days after transplantation. In patients who received GCV as treatment for GVHD, a progressive increase in the proportion of transduced donor T cells carrying the deleted HSV-Tkgene was observed. These results suggest that the limitations within the HSV-Tk/GCV system can be improved by developing optimized retroviral vectors to ensure maximal killing ofHSV-Tk–transduced cells.

The standard enzyme-linked immunosorbent assay (ELISA) for anticardiolipin antibodies (ACA) detects a heterogenous group of antibodies against cardiolipin on its own, ß2-glycoprotein I (ß2GPI), and, potentially, other phospholipid-binding plasma proteins from bovine or human origin. In an attempt to identify new proteic targets of ACA, we selected 6 patients who possessed cofactor-dependent ACA but no antibody to human or bovine ß2GPI detectable in the ß2GPI-ELISA. Three of these samples proved to recognize ß2GPI in combination with cardiolipin, but not ß2GPI directly immobilized on ?-irradiated polystyrene or agarose beads. In the other cases, the component required for ACA binding was purified from adult bovine serum or plasma by means of ammonium sulfate precipitation and chromatography on Phenyl-Sepharose, diethyl aminoethyl (DEAE)-cellulose, heparin-Ultrogel, and Sephacryl S-300 columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis coupled to N-terminal amino acid microsequencing identified the cofactors of patients no. 4, 5, and 6 ACA as lipopolysaccharide binding protein (LBP), complement C4b-binding protein (C4BP), and the thrombin-antithrombin (AT) complex, respectively. Adsorption of each of these cofactor preparations with cardiolipin liposomes led to suppression of ACA reactivity, concomitant with the loss of bands from SDS gels corresponding to sequenced material. Bacterial lipopolysaccharide (which forms high-affinity complexes with LBP) specifically neutralized the cofactor activity of the LBP preparation in a concentration-dependent manner. Bovine serum and plasma, as well as the C4BP preparation, optimally supported the binding of a rabbit anti-C4BP antiserum to immobilized cardiolipin. The binding of a rabbit anti-AT antiserum to solid-phase cardiolipin was sustained by the thrombin-AT preparation and bovine serum, but neither by bovine plasma nor by native AT, thus reproducing the behavior of patient no. 6 ACA. Taking advantage of the restricted recognition by the latter ACA of a cofactor from bovine origin appearing upon clotting, we studied the generation of such activity in human plasma supplemented with bovine AT or bovine prothrombin before clotting. In these conditions, patient no. 6 antibody binding to cardiolipin required the addition of bovine AT, whereas addition of bovine prothrombin alone was ineffective. We therefore concluded that those ACA targeted bovine AT once it has been modified/cleaved by thrombin. These findings underline the wide heterogeneity of ACA and the links that may exist between various coagulation pathways, inflammation and the complement system.

The standard enzyme-linked immunosorbent assay (ELISA) for anticardiolipin antibodies (ACA) detects a heterogenous group of antibodies against cardiolipin on its own, β2-glycoprotein I (β2GPI), and, potentially, other phospholipid-binding plasma proteins from bovine or human origin. In an attempt to identify new proteic targets of ACA, we selected 6 patients who possessed cofactor-dependent ACA but no antibody to human or bovine β2GPI detectable in the β2GPI-ELISA. Three of these samples proved to recognize β2GPI in combination with cardiolipin, but not β2GPI directly immobilized on γ-irradiated polystyrene or agarose beads. In the other cases, the component required for ACA binding was purified from adult bovine serum or plasma by means of ammonium sulfate precipitation and chromatography on Phenyl-Sepharose, diethyl aminoethyl (DEAE)-cellulose, heparin-Ultrogel, and Sephacryl S-300 columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis coupled to N-terminal amino acid microsequencing identified the cofactors of patients no. 4, 5, and 6 ACA as lipopolysaccharide binding protein (LBP), complement C4b-binding protein (C4BP), and the thrombin-antithrombin (AT) complex, respectively. Adsorption of each of these cofactor preparations with cardiolipin liposomes led to suppression of ACA reactivity, concomitant with the loss of bands from SDS gels corresponding to sequenced material. Bacterial lipopolysaccharide (which forms high-affinity complexes with LBP) specifically neutralized the cofactor activity of the LBP preparation in a concentration-dependent manner. Bovine serum and plasma, as well as the C4BP preparation, optimally supported the binding of a rabbit anti-C4BP antiserum to immobilized cardiolipin. The binding of a rabbit anti-AT antiserum to solid-phase cardiolipin was sustained by the thrombin-AT preparation and bovine serum, but neither by bovine plasma nor by native AT, thus reproducing the behavior of patient no. 6 ACA. Taking advantage of the restricted recognition by the latter ACA of a cofactor from bovine origin appearing upon clotting, we studied the generation of such activity in human plasma supplemented with bovine AT or bovine prothrombin before clotting. In these conditions, patient no. 6 antibody binding to cardiolipin required the addition of bovine AT, whereas addition of bovine prothrombin alone was ineffective. We therefore concluded that those ACA targeted bovine AT once it has been modified/cleaved by thrombin. These findings underline the wide heterogeneity of ACA and the links that may exist between various coagulation pathways, inflammation and the complement system.

Several bacterial-expressed recombinant fragments encompassing the extracellular part of the beta 3 subunit of the integrin alpha IIb beta 3 were shown to recognize and bind soluble and immobilized forms of fibrinogen. Two of them, designated as rIII-11 (beta 3 274–368) and rIII-13 (beta 3 274–403), did not contain the established RGD-ligand binding sequence. In fact, they interacted, in a Ca(2+)-independent manner, with the C-terminal part of the fibrinogen gamma chain. Both beta 3 fragments blocked the participation of fibrinogen in the induction of platelet aggregation induced by adenosine diphosphate. Fragment rIII-13 was recognized by the anti-beta 3 monoclonal antibody B2A. This antibody, which possesses an epitope exposed on both resting and activated platelets, inhibited fibrinogen binding as well as platelet adhesion and aggregation. In conclusion, the results demonstrated that the 274–368 sequence of the beta 3 subunit of integrin alpha IIb beta 3 constitutes a fibrinogen ligand binding domain, distinct from the RGD-binding site, that is required for both platelet adhesion and aggregation.

Several bacterial-expressed recombinant fragments encompassing the extracellular part of the β3 subunit of the integrin αIIbβ3 were shown to recognize and bind soluble and immobilized forms of fibrinogen. Two of them, designated as rIII-11 (03 274-368) and rlll-13 (β3 274-403), did not contain the established RGD-ligand binding sequence. In fact, they interacted, in a Ca2+-independent manner, with the C-terminal part of the fibrinogen gamma chain. Both β3 fragments blocked the participation of fibrinogen in the induction of platelet aggregation induced by adenosine diphosphate. Fragment rlll-13 was recognized by the anti-β3 monoclonal antibody B2A. This antibody, which possesses an epitope exposed on both resting and activated platelets, inhibited fibrinogen binding as well as platelet adhesion and aggregation. In conclusion, the results demonstrate that the 274-368 sequence of the β3 subunit of integrin αIIbβ3 constitutes a fibrinogen ligand binding domain, distinct from the RGD-binding site, that is required for both platelet adhesion and aggregation.

Rudolph: Boehringer Ingelheim RCV: Employment. Off Label Use: Volasertib is an investigational agent. Albrecht:Boehringer Ingelheim RCV GmbH & Co KG: Employment. Geiselmann:Boehringer Ingelheim RCV GmbH & Co KG: Employment. Impagnatiello:Boehringer Ingelheim RCV GmbH & Co KG: Employment. Garin-Chesa:Boehringer Ingelheim RCV: Employment. Wernitznig:Boehringer-Ingelheim: Employment. Moll:Boehringer-Ingelheim: Employment. Kraut:Boehringer Ingelheim RCV: Employment.

Background:Polo-like kinase 1 (Plk1), a key regulator of cell cycle progression and accurate spindle assembly, is an attractive target for cancer drug discovery. We have previously shown that volasertib (BI 6727), a potent and selective small-molecule inhibitor of Plk, induces a distinct mitotic arrest phenotype in prometaphase (“polo-arrest”) with subsequent apoptosis in a variety of different cancer cell lines, irrespective of their mutational status. When used in vivo, volasertib administered intravenously shows potent anti-tumor activity in xenograft models of human epithelial cancers at well-tolerated doses. The present study was designed to extend the analysis of volasertib to additional preclinical models of human AML, including bone marrow samples from AML patients. Volasertib is the most advanced Plk inhibitor in clinical development and has demonstrated encouraging results in phase II clinical trials. It is currently being investigated in a phase III clinical trial in patients with previously untreated AML, who are ineligible for intensive remission induction therapy.

Consolidation using radioimmunotherapy (RIT) is a promising approach for elderly patients with diffuse large B-cell lymphoma (DLBCL) who are ineligible for autologous stem-cell transplantation. RIT using fractionated injections of 90Y-epratuzumab tetraxetan (Immunomedics, Inc.), a radiolabeled humanized anti-CD22 antibody, has been evaluated in relapsed patients with indolent or aggressive non-Hodgkin lymphoma (NHL), providing long-term disease control with manageable hematologic toxicities (Morschhauser et al., J Clin Oncol. 2010;28(23);3709-16). A French phase II trial sponsored by the LYSA group now assessed front-line treatment using fractionated RIT with 90Y-epratuzumab tetraxetan as consolidation therapy after R-CHOP in previously untreated elderly (age >60 years) patients presenting with stage I/II bulky or stage III/IV DLBCL.The trial included 6 courses of R-CHOP delivered q2wks followed by 2 infusions of 90Y-epratuzumab tetraxetan (2 × 15 mCi/m2 [555 MBq/m2], 7 days apart), 8 wks later. Patients were enrolled at time of diagnosis.From October 2008 to December 2010, 75 patients (41 males, 34 females) have been accrued prospectively at 19 French institutions. The median age was 69 (range, 60–79 years); 57 patients (76.0%) were Ann Arbor stage III/IV. Seventy-one of the 75 completed 6 courses of R-CHOP-14 and 61/75 (81.2%) were eligible for RIT. Thus, 14 patients were considered ineligible for RIT because of R-CHOP toxicity (N= 5), progressive disease (PD, N=3), patient refusal (N=3), or concomitant illness (N=3). RIT toxicity consisted of grade 3–4 hematologic toxicity in 51/61 patients (83.6%): grade 3–4 neutropenia in 46 (75.4%), grade 3–4 anemia in 15 (24.6%), and grade 3–4 thrombocytopenia in 47 (77.0%), with a nadir at 42, 48, and 43 days after RIT and a median duration of 18, 5, and 17 days, respectively. Following RIT, RBC and/or platelet transfusions were given to 31 patients (50.8 %). Serious febrile neutropenia was observed in 13 cases (17.3 %) after R-CHOP and in 3 patients (4.9%) following RIT. RIT's severe non-hematologic toxicity consisted of grade 4 gastrointestinal in 1 patient (1.6 %) and grade 4 infection in 3 (4.9%). No patient had mucositis after RIT. In the follow-up, 2 patients (2.6%) developed myelodysplastic syndrome 5 and 20 months after RIT.Using the 1999 International Workshop for Response Criteria for NHL (Cheson 1999), the overall response rate (ORR) after 6 × R-CHOP14 was 94.6% (71/75); 52 patients (69.3%) achieved CR/CRu and 19 (25.3%) had a partial response (PR). Among the 4 remaining patients, one had stable disease and 2 had PD; no assessment was obtained in the other. In an intention-to-treat analysis, CR/CRu rate after 6 × R-CHOP14 followed by RIT was 72.0% (N=54). Seven patients (9.3%) remained in PR and 8 (10.7%) progressed (2 patients previously in PR with PET-positive findings, 3 previously in CRu, including 1 PET-positive, and 3 in PD before RIT and then ineligible for RIT). No response assessment was obtained in the 6 others ineligible for RIT. At a median follow-up of 24 months (range, 1–46), 18 patients experienced lymphoma progression and/or a related death, yielding an estimated 2-year event-free-survival (EFS) of 73.3% (60.7-82.5%) and an estimated 2-year overall survival (OS) of 83.2% (71.4-90.4%). For the 61 patients who received 6 courses of R-CHOP followed by RIT consolidation, ORR was 91.8% (56/61); 50 patients (81.9%) achieved CR/CRu. Eight of 16 patients (50.0%) who had less than a CR/CRu with R-CHOP converted to CR/CRu after RIT. According to a PET analysis (Cheson 2007; N=55), 12 of the 24 patients (50.0%) who were not PET-negative after R-CHOP improved their metabolic response after RIT, resulting in a CR rate of 72.7%. Among these 61 patients, 12 experienced progression and/or a related death, yielding an estimated 2-year EFS of 78.7% (65.1–87.4%) and an estimated 2–year OS of 90.1% (77.7–95.8%).This phase II study clearly shows that fractionated RIT with 90Y-epratuzumab as a consolidation therapy after 6 × R-CHOP-14 is feasible and tolerable in elderly untreated DLBCL patients with advanced disease. RIT markedly improved response status observed after 6 × R-CHOP14. EFS data achieved with R-CHOP plus RIT compare favourably with those achieved with R-CHOP alone in the same patient population.Wegener: Immunomedics: Employment. Goldenberg:Immunomedics: Employment, Equity Ownership.

Abstract 906This icon denotes a clinically relevant abstract

The prognosis of patients (pts) with relapsed or refractory (rel/ref) AML who are considered unlikely to benefit from or tolerate intensive salvage treatment is unfavorable and novel treatment strategies are needed. Repeated cycles of LD-Ara-C are a therapeutic option for palliative treatment; however, the outlook for these pts remains unsatisfactory.Plks are critical in cellular division and mitotic progression and Plk1 is overexpressed in many cancers including AML. Volasertib is a first in class, selective and potent cell cycle kinase inhibitor that induces mitotic arrest and apoptosis by targeting Plk. In phase I/II trials in pts with solid tumors, volasertib demonstrated a favorable safety profile and encouraging antitumor activity. Here, we present updated results from the phase I part of an ongoing phase I/II study of volasertib in combination with LD-Ara-C or as monotherapy in AML pts considered ineligible for intensive salvage treatment.This study follows a two-stage design. The phase I part, reported here, investigates the maximum tolerated dose (MTD) of volasertib as a 1-hr intravenous infusion on days 1 and 15 Q4W as monotherapy or in combination with fixed dose LD-Ara-C 20 mg bid subcutaneously on days 1–10 Q4W in pts with rel/ref AML. Dose escalation follows a 3+3 design with de-escalation. Blood samples for pharmacokinetic (PK) analyses were taken in cycles 1 and 2 and concentrations of volasertib and LD-Ara-C were determined.In the monotherapy arm, increasing volasertib doses (150, 200, 350, 400, 450 mg) were evaluated in 29 pts (median age: 71 yrs [range 26–84]). Drug-related adverse events (AEs) were reported in 8 pts (27.6 %). Most frequent drug-related AEs (>5%) were anemia in 3 pts (10.3%), and thrombocytopenia, epistaxis, and nausea in 2 pts each (6.9%). Grade 3/4 drug-related AEs included thrombocytopenia (2 cases), anemia, diarrhea, mucositis, neutropenia, and pneumonia (1 case each); there was 1 fatal (grade 5) drug-related AE (fungal pneumonia). Of the drug-related AEs, the following were dose-limiting toxicities (DLTs) per protocol: grade 4 pneumonia and fatal fungal pneumonia (n=1, at 150 mg), and grade 3 mucositis (n=1, at 400 mg). Monotherapy dose escalation is ongoing; pts have received volasertib doses of 500 mg without having reached the MTD. Preliminary best response data indicated minor antileukemic activity at low doses (150 and 200 mg); with 4/13 pts achieving no change as best response, mostly of short duration (median number of cycles initiated: 1 [range 1–5]). At higher monotherapy doses (≥350 mg), antileukemic activity was observed with 4/16 pts achieving a complete remission with incomplete blood count recovery (CRi) and 5/16 having temporarily stable blood values as best response.In the combination arm, volasertib doses of 150–400 mg were investigated. The MTD for volasertib in combination with LD-Ara-C was 350 mg (Bug et al ASH 2010). Seven out of 32 pts treated with volasertib + LD-Ara-C achieved a complete remission (CR or CRi). In responding patients, a median number of 6 treatment cycles was initiated (range 3–13) and a preliminary analysis revealed a median overall survival of 551 days (range 165–595).PK analysis showed that volasertib is a moderate clearance drug with multi-compartmental PK behavior with a large volume of distribution (>4000 L) and a long terminal half-life (∼111 hrs). No drug interaction after co-administration of LD-Ara-C was observed.The phase I part of the study determined the MTD of volasertib in combination with LD-Ara-C to be 350 mg; the MTD of volasertib monotherapy has not yet been determined. Volasertib was well tolerated in this heavily pretreated AML pt population at doses above the recommended phase II volasertib dose used in pts with solid tumors. Most of the reported higher grade drug-related AEs were due to the myelosuppressive effect of volasertib. Preliminary results from the phase I trial show antileukemic activity of volasertib as monotherapy and in combination with LD-Ara-C. These results indicate Plk to be a potential new target for AML treatment and warrant proceeding with further clinical investigation of volasertib in AML pts.Bug: Novartis Pharma GmbH: Consultancy, Honoraria; Celgene GmbH: Consultancy, Honoraria. Off Label Use: Volasertib is an investigational agent. Müller-Tidow:Boehringer Ingelheim: Research Funding. Krug:Boehringer Ingelheim: Research Funding. Voss:Boehringer Ingelheim: Employment. Taube:Boehringer Ingelheim: Employment. Fritsch:Boehringer Ingelheim: Employment. Garin-Chesa:Boehringer Ingelheim: Employment. Ottmann:Boehringer Ingelheim: Consultancy. Döhner:Celgene, Clavis: Membership on an entity's Board of Directors or advisory committees.

Abstract 1549

Radioimmunotherapy (RIT) is under study as a consolidation treatment after chemotherapy induction in follicular lymphoma patients. This approach also appears interesting in diffuse large B-cell lymphoma (DLBCL) patients >60 years, who are not candidates for bone marrow transplantation. 90Y-epratuzumab tetraxetan (Immunomedics, Inc.) is a radiolabeled humanized anti-CD22 antibody that has been used for a fractionated RIT, showing high rates of durable complete responses with manageable hematologic toxicity in previously-treated indolent and aggressive non-Hodgkin lymphoma (NHL) patients (Morschhauser et al., J Clin Oncol. 2010;28(23);3709-16). A French phase II trial sponsored by the GOELAMS group is ongoing, assessing fractionated RIT using 90Y-epratuzumab tetraxetan as a consolidation therapy after first-line chemotherapy in disseminated DLBCL patients >60 years. The protocol has been designed to include 75 patients; 64 patients have been already enrolled. We report the initial results, in particular safety data, on the first 29 available patients.From October 2008 to November 2009, 29 untreated DLCBL patients >60 years were studied in several French institutions with an initial course of six cycles of R-CHOP14 followed 8 weeks later by two weekly infusions of 90Y-epratuzumab tetraxetan (15 mCi/m2 [555 MBq/m2]) 7 days apart. Hematologic and non-hematologic toxicities were evaluated using NCI-CTC v.3.0. Treatment responses were classified according to the 1999 International Workshop for Response Criteria for NHL.Twenty-six patients underwent the entire course of R-CHOP and 23 received the 2 weekly RIT injections. Following R-CHOP, grade 3–4 neutropenia was observed in 20 patients (68.9%) and grade 3–4 thrombocytopenia in 4 (13.7%). During RIT infusions, 4 patients showed transient change of pulse or blood pressure, with 2 attributed to vasovagal reactions. RIT toxicity included grade 3–4 hematologic toxicity in 18 of 23 patients (78.3%); the most common grade > 3 toxicities were neutropenia (N=18, 78.3%) and thrombocytopenia (N=17, 73.9%). Serious febrile neutropenia was observed in 4 cases (13.8%) after R-CHOP and in 2 patients (8.7%) following RIT. Compared to R-CHOP, RIT non-hematologic toxicity was uncommon; moderate or severe gastrointestinal toxicity was observed in 10 patients (34.5%) after R-CHOP and in 2 (8.7%) following RIT; moderate or severe infection in 9 patients (31.0%) after R-CHOP and in 1 (4.3%) after RIT; and moderate or severe mucositis in 10 (34.4%) patients following R-CHOP, while no patient had mucositis after RIT. Following RIT, red cells and/or platelets transfusions were given to 12 patients (52,2%). Following R-CHOP, 10 of the 25 patients (40.0%) achieved a complete response (CR) or unconfirmed CR (CRu), 13 patients (52.0%) had a partial response (PR) and 2 patients (8.0%) had a stable disease. Six weeks after RIT, 13 patients (56.5%) achieved a CR or CRu, 9 patients (39.1%) had PRs, and 1 patient (4.3%) had progressive disease. Four of 13 patients (30.7%) who achieved less than a CR or CRu with R-CHOP improved their remission status 6 weeks after RIT.These preliminary results indicate the feasibility and safety of fractionated RIT with 90Y-epratuzumab as a consolidation therapy for elderly DLBCL patients. Additional data will be presented at the time of the communication.Off Label Use: monoclonal antibody epratuzumab labeled with yttrium 90 in phase II clinical trial. Wegener:Immunomedics, Inc.: Employment, shareholders. Goldenberg:Immunomedics, Inc.: Employment, shareholders.

Abstract 2875

Despite its often indolent clinical course, follicular lymphoma (FL) is a heterogeneous disease. Current criteria for early identification of patients with a poor prognosis are suboptimal - the FLIPI and F2 index are insufficient prognostic markers for individual patients and the limitations of post-treatment conventional response criteria have long been acknowledged. FL shows increased FDG uptake, but unlike DLBCL, minimal data exist about the role of PET-CT in response assessment. We have used the prospective conventional response assessment and 42 month patient follow-up in the PRIMA (Primary Rituximab and Maintenance, Salles et al., ASCO 2010, Abstr#8004) study as a platform for analysis of the utility of PET-CT in FL.The PRIMA database was interrogated and investigators surveyed to identify PET-CT scans performed during staging and induction response assessment. Single modality PET-only scans were not eligible for inclusion. Local PET interpretation (positive + or negative -) was used to explore associations with patient outcomes. The primary endpoint was PFS from PRIMA registration.277 PET-CT scans on 160 patients from 40 centres were identified. Baseline patient characteristics did not differ from the overall PRIMA patient population. Positive PET-CT scans were recorded in 119/120 (99%) at diagnosis, 11/33 (33%) interim restaging scans and 32/124 (26%) post induction treatment (R-CHOP or R-CVP).There was significant correlation between PET-CT result and conventional response assessment at the end of immunochemotherapy (p<0.0005). The incidence of post-treatment PET+ increased across the categories of lesser conventional responses, occurring in 8% (4/50) CR, 31% (12/39) CRu, 41% (11/37) PR, 67% (2/3) SD, and 80% (4/5) PD. While 73/91 (80%) of PET- patients were in CR/CRu, given the very high overall response rate on study, 16/33 (48%) of the PET+ population were also in CR/CRu.With a median follow-up of 42 months, a significantly inferior actuarial 3yr PFS was observed in post-treatment PET+ vs. PET- patients (Figure 1): 32% (95% CI 17–48%) vs. 74% (95% CI 63–82%) (log rank p<0.0001, HR 3.5, 95% CI 2.0–6.1), median PFS 19 months (13-35) vs. not reached, (52-NR).Using proportional hazard regression analysis, both conventional response (overall p=0.0002) and PET+ status (HR 2.8 p=0.0007) were significant predictors of inferior PFS. However, the predictive power of conventional response assessment was limited to non-responders: SD/PD vs. CR/CRu (HR 6.5, p<0.0001), and SD/PD vs. PR (HR 5.2, p=0.0009). Comparison of PR vs. CR/CRu was not different (HR 1.2, p=0.5). While PET+ status had a significant negative impact on PFS in both the CR/CRu (HR 2.6, p=0.015) and PR (HR 4.3 p=0.018) patient groups, there was no difference in outcome between CR/CRu vs. PR patients within the PET+ (HR 1.5 p=0.42) and PET- (HR 1.0 p=0.98) subgroups.When only patients randomised for the maintenance element of the PRIMA study were considered, post-treatment PET+ (15/59) remained predictive of 3yr PFS (27 vs. 69%, HR 3.1, p=0.005) in the observation arm, but post-treatment PET+ (9/47) was not significantly associated with an adverse outcome in patients receiving rituximab maintenance (3-year PFS 56 vs. 81%, HR 2.2, p= 0.18).In a multivariate Cox model including responding patients the following factors were negative predictors of PFS: post-treatment PET+ (HR 3.1 p<0.0014); R-CVP induction therapy (HR 2.8, p<0.014); and baseline β2M ≥3 (HR 2.6 p<0.0042), while conventional PR and FLIPI were not.This PRIMA sub-study demonstrates that post-treatment PET-CT is a powerful predictor of PFS that complements conventional response evaluation after first line immunochemotherapy for FL. Patients who are PET- can expect a prolonged PFS whether in conventional CR or PR, but for those remaining PET+, with a median 19 month PFS, the disease cannot be characterized as indolent. Future clinical trials should evaluate an FDG PET-CT response adapted approach focused on improving outcomes for this group.Seymour: Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Shpilberg:Roche: Consultancy, Honoraria.

Abstract 855

Treatment strategies of acute myeloid leukaemia (AML) largely depend on their cytogenetic status. However this stratification remains insufficient for almost half of the patients, requiring subsequent molecular investigations. In our study, we used mass spectrometry-based proteomic approaches to characterize de novo-AML. Samples (blood mononuclear cells collected and frozen before any treatment) were retrospectively selected from two independent sets of newly diagnosed AML patients, included in prospective clinical trials of the GOELAMS (Groupe d'Etude Ouest-Est des Leucémies aigues). We showed that protein signature of leukemic cells defined 2 groups of patients that displayed significant variation of overall and disease free survival (Fig-1A). This proteomic classification refined cytogenetic classes. Combination of proteomic and cytogenetic allowed a new stratification highly correlated with outcome. In particular, AML with intermediate and high risk cytogenetic could be respectively subdivided according to their protein profiles in two subgroups with significantly different survival (Fig-1B). Interestingly in both type 2 and type 3 cytogenetic groups, a good proteomic profile identified subsets of patients with significantly increased probability of survival, suggesting that the weigh of a good proteomic risk might predominate over a bad predictive cytogenetic. Among proteins expressed by leukemic cells, we isolated a 10800 Da marker that retained the highest discriminative value between alive and deceased patients. The median intensity of the 10800 peak was 94.3 ± 97.7 (7.5-368.7) in living patients compared to 214.2 ± 163.7 (9.0-693.4) in dead patients (p= 0.009). Among normal cytogenetic patients, intensity levels of the marker was also significantly different between dead (217.4 ± 155.7 range 10.4-612.8) and alive patients (26.0 ± 73.1, range 6-168.1; p= 0.008) (Fig-1C). Using a thresholds of 100 (calculated by ROC curves) we were able to correctly identify 82% of patients who died (patients greater than 100) and 70% of patients who stayed alive (patients lower than 100) with a specificity of 65% and 82% respectively. These data were confirmed in a second independent set of patients. 10800 Da marker was identified by MS peptide sequencing as S100A8 (also designated MRP8 or calgranulin A), a cytosolic protein of mature granulocytes. Western blot analysis confirmed its expression mainly in AML patients with the worst prognostic but not in all AML patients, neither in some leukemic and lymphoma cell lines, arguing for a selective deregulation associated with poor prognosis. These results show that expression of S100A8 in leukemic cells at diagnosis might be considered as a predictor of low survival.No relevant conflicts of interest to declare.

Febrile neutropenia (FN) is the most frequent complication of chemotherapy-induced neutropenia. Only 30% of the episodes of FN are microbiologically documented by routine procedures, mainly blood cultures (BC). In order to improve the diagnostic yield of BC in this setting, we prospectively assessed new procedures of BC in FN, by implementing a BC automate in the ward and combining molecular and rapid identification methods directly applied on the blood culture bottle when detected as positive.This is a prospective study, run in the Hematology department of Henri Mondor Hospital. Adult patients were eligible at their first episode of FN if they were neutropenic (PMN<0.5 ×109/L), febrile (temperature ≥ 38°3 or twice ≥ 38° within 8h), and had a central venous catheter (CVC). For each episode we collected: 2 standard BC sampled through CVC and one through peripheral punction. These BC were incubated in the laboratory and processed according to the routine procedure for identification (morphologic, culturing and biochemical characters including API, Biomérieux) (routine process); 1 study BC (2 aerobic and 2 anaerobic bottles) sampled through the CVC and immediately incubated in the ward (BacT/Alert3D®), then processed directly with Vitek2® (Biomérieux) and Genotype® (Hain Lifescience) for identification (study process). We compared the routine process with the study process on : (1) the rate of BC positivity; (2) the time elapsed between blood sample and BC positivity; (3) the time elapsed between blood sampling and final pathogen identification. Pairwise analyses were performed using the McNemar test to compare documentation rates of both techniques, and the Wilcoxon signed-rank test to compare delays.120 consecutive episodes of febrile neutropenia were included from Feb 2008 to Mar 2009: 97 (80%) acute leukemia/myelodysplasia, 26 (17%) lymphoid malignancies and 4 (3%) others. The median age was 50y (20-75). Eighteen (15%) episodes were at the initial phase of allogeneic stem cell transplantation (SCT), and 16 (13%) at the initial phase of autologous SCT. 34 episodes (28%) were microbiologically documented by routine BC, 33 (27%) by study BC, without significant difference between the 2 methods (McNemar's chi2=0.20, p=0.65). Fifty bacteria were identified from BC: 24 (48%) streptococci sp., 14(28%) staphylococci, 9 (18%) gram-negative bacteria, and 3 (6%) others. No fungi was identified. In the microbiologically documented episodes, the time elapsed between incubation and positivity in BactAlert was significantly shorter for the study BC (median rank: 740 min [662-947]) than for the routine BC (median rank: 800 min [726-1079]) (p=0.0003). The time needed for final identification was significantly shorter in the Vitek2®+Genotype® study BC (respectively 1.5 days [CI 1.2-2.7] and 1.2 [CI 1.0-2.9) days]) than in the routine BC (median rank: 3.0 days [CI 2.2-4.4]), (respectively p=0.018 and p=0.047).Immediate incubation combined with the use of rapid method for early identification does not improve the rate of positivity of blood cultures in FN episodes, when compared to BC routine process. However, it reduced the time to positivity about 60 minutes and time to identification about 1.5 days. This could allow an earlier adaptation of antibiotics for the pathogens resistant to the initial antibiotic therapy. Further studies are needed to evaluate the clinical impact of these results.No relevant conflicts of interest to declare.

Abstract 276

Abstract 4309

Telomeres consist of repetitive DNA sequences and specific proteins, creating a specialized structure called the telosome. Unraveling the specific telomerase and telosome changes in leukemia is thought for providing new knowledge about oncogenesis, together with useful clinical markers and specific therapeutic targets. Here we measured telomerase activity (TA) by a quantitative TRAP assay in 57 human acute leukemia samples including 20 acute lymphoblastic leukemias (ALL) and 37 acute myeloid leukemias (AML). AML, TALL (n=7) and BALL (n=13) displayed significantly higher levels of TA than their normal counterparts [normal bone marrow CD34+ cells deriving from donors (BMCs, 17 samples) for AML, B-lymphocytes (n=20) for BALL and T-lymphocytes (n=24) for TALL; p<0.005 for all]. hTERT encodes telomerase reverse transcriptase which is the rate-limiting factor for TA. In BALL and TALL, hTERT expression was respectively 53 (p=0.0005) and 12 (p=0.05) times higher than in control cells. In contrast AML blasts displayed 4.5 times lower hTERT expression levels than BMCs (p=0.0008). hTERT transcripts include the full-length functional hTERT mRNA and non-functional sequences. AML blasts displayed a global decrease of all hTERT transcripts (p<0.0006) without any shift in the splicing pattern. c-Myc is a transcriptional activator of hTERT; we evidenced a positive correlation between c-Myc and hTERT transcription in BMCs (p=0.005) but not in myeloblasts. WT1, which represses hTERT transcription ex vivo in certain cell lines, can act as a transcriptional regulator or repressor depending on the cellular context. WT1 was significantly overexpressed in all leukemic subtypes (p<0.05 for all). In AML, WT1 expression negatively correlated hTERT expression (p=0.022) whereas both transcripts were positively correlated in BALL (p=0.029). We investigated the transcriptional control of hTERT by comparing the hTERT promoter occupancy between AML blasts and BMCs. Nuclear extracts deriving from 9 AML and 3 normal BMC samples were incubated with the hTERT core promoter (hCP). The protein complexes interacting with hCP were purified and components were identified by mass spectrometry. The specific AML hCP peptidome included 19 proteins of which 4, 5, 9, and 1 are involved in transcriptional regulation (including 3 factors possessing negative transcriptional effects), chromatin remodeling (including the DEK protein), mRNA maturation, and DNA methylation [DNA methyl transferase 1 (DNMT1)], respectively. With the exception of DNMT1, which is involved in hTERT transcriptional repression via CpG methylation, these factors have not been previously found negatively correlated with hTERT transcription. DEK is a chromatin remodeling protein frequently overexpressed in AML. Using CHIP, RNAi, and transfection experiments we validated and characterized the transcriptional repression of hTERT by DEK. To investigate the paradoxical association of hTERT transcriptional repression with elevated TA in AML, we measured the transcription of 36 genes involved in the post-translational regulation of hTERT and/or encoding shelterin and non-shelterin telosome genes. AML blasts overexpressed AKT1 (p=0.01) and underexpressed Ku70 (p=0.039). AKT1 activates hTERT through phosphorylation while Ku70 may act as a negative regulator of hTERT by reducing the ability of telomerase to access the exposed 3′ overhang. One can propose that these gene deregulations help compensate hTERT transcriptional repression towards an increased TA in AML. In B and TALL, telomeric gene deregulation involved shelterin and non-shelterin genes with a transcriptome pattern consistent with telomere deprotection. This is the first report of a hTERT transcriptional repression in de novo AML resulting from a global repression of all hTERT transcripts, depending on WT1 expression and involving an acquired defect in the transcriptional control of hTERT by c-Myc. DEK and DNMT1 participate to this process in vivo; our proteomic survey evidenced additional promising candidates. hTERT transcriptional repression is known to promote telomere dysfunctions and thereby genetic instability. We propose that it helps initiate the AML phenotype, which is subsequently stabilized by additional deregulations including AKT1 and Ku70-dependent hTERT post-transcriptional activation. Finally these results support important telosome modifications in acute leukemias with specific features depending on the disease phenotype.

Background: BI 2536 is a potent and selective inhibitor of Plk-1, which plays a crucial role in the regulation of mitosis. Inhibition of Plk-1 leads to mitotic arrest and apoptosis. Therefore, Plk-1 inhibitors are currently in clinical testing as anti-proliferative agents in cancer patients. BI 2536, the first specific Plk-1 inhibitor in clinical testing, demonstrated strong anti-proliferative effects on AML cell lines in vitro and in in vivo models. To investigate target inhibition in the malignant cell, bone marrow samples from patients who participated in a Phase I/II study of BI 2536 single-agent therapy in elderly patients with relapsed or refractory AML were analyzed.

Background: BI 2536 is a potent and selective inhibitor of Plk-1, which plays a crucial role in the regulation of mitosis. Inhibition of Plk-1 leads to mitotic arrest and apoptosis. Therefore, Plk-1 inhibitors are currently in clinical testing as anti-proliferative agents in cancer patients. BI 2536, the first specific Plk-1 inhibitor in clinical testing, demonstrated strong anti-proliferative effects on AML cell lines in vitro and in in vivo models. To investigate target inhibition in the malignant cell, bone marrow samples from patients who participated in a Phase I/II study of BI 2536 single-agent therapy in elderly patients with relapsed or refractory AML were analyzed. Methods: Pharmacodynamic analyses including immunocytochemistry (ICC) and flow cytometry (FACS) of bone marrow (BM) were performed to examine cell morphology, phosphorylation of histone H3 (phospho-H3), and induction of apoptosis. Samples were acquired before and 24 h after the first administration of BI 2536. FACS analysis included propidium iodide (PI)-FACS to determine the percentage of cells residing in various cell cycle stages (G0/1-, S- and G2/M-phases) and Annexin V-Cy5-staining for apoptosis. Immunocytochemistry included staining for phosphorylated histone H3 and TUNEL assay for apoptosis. Results: At the time of analysis, data from 28 patients treated at doses in the range of 50 to 400 mg BI 2536 were available. BM taken after BI 2536 administration showed an increase of phospho-H3 positive cells as compared to baseline prior to treatment. There was a trend toward a positive correlation between phospho-H3 increase and increase in dosage of BI 2536 (p=0.16) when treatment at low doses (50 to 60 mg) of BI 2536 were compared to treatment at higher doses (100 to 400 mg). Furthermore, trends were observed that an increase of phospho-H3 is positively correlated with both a higher percentage of cells in G2/M and an increase in apoptotic cells. The typical morphology of cells in mitotic arrest could be demonstrated by ICC. Interestingly, when patients with progressive disease after one cycle (PD) were compared to patients with disease stabilization or response (nonPD), a statistically significant positive correlation between PD and increase in phospho-H3 compared to baseline was found (p=0.03). Also, there was a clear trend for an increase in the percentage of cells in G2/M phase and apoptosis in patients with PD compared to patients with nonPD. Preliminary evaluation of other disease characteristics including karyotype (normal vs complex), secondary AML, complete response in previous treatments, baseline value for blasts in the bone marrow or in the peripheral blood, did not reveal any signs of correlation with the clinical response to BI 2536 treatment. Conclusion: BI 2536 treatment increases the number of cells in G2/M phase (as detected by FACS analysis and phospho-H3 staining) and the number of apoptotic cells within 24 h after administration. In line with the clinical observation of rapid blast reduction both in the BM and the peripheral blood, these findings indicate that BI 2536 induces mitotic arrest and apoptosis of the malignant target cells in AML patients. The biological meaning of the correlation between the BM findings and the clinical response to BI 2536 is unclear, but if substantiated by more data, these results may suggest a predictive value of BM examinations for the response to BI 2536.

Background: In acute leukemia (AL) patients, mortality rates of the most common IFI (i.e., aspergillosis and candidiasis) are well illustrated in the literature from the IFI diagnosis, as well as the rate of fungal relapse during subsequent periods at risk, such as neutropenic phases, or transplant. However, few data are available about the impact of IFI on the subsequent chemotherapy schedule and the indirect impact of IFI on the relapse-free and overall survival. Clinicians are usually reluctant to give the full chemotherapy doses on time, due to the risk of life-threatening fungal relapse during the subsequent courses. Even with secondary prophylaxis, they often delay or decrease the doses of chemotherapy. This may impact on the leukemia outcome. The aim of this case-control study was to assess the potential impact of proven or probable IFI onset on the application of the chemotherapy schedule in AL patients, and its consequences on the leukemia outcome, by comparing patients with and without IFI in a single institution. Delays and changes in chemotherapy doses and drug choices were evaluated and compared to the planned schedule in the protocol. Methods: All consecutive AL patients with a first episode of proven or probable IFI according to the EORTC-MSG criteria between 2000–2006 were reviewed. All patients have been treated in, or according to, clinical research protocols where timing and doses of chemotherapy were predefined. Patients who were planned for allogeneic transplant were excluded as those who were at their last consolidation course when they got IFI or in a palliative phase of the leukemia. Any delay, dose decrease, or dose change were defined as any difference compared to the planned schedule. We planned to include 3 control patients for each case, selected among AL patients without IFI, and matched for age, sex, type of AL, chemotherapy protocol, and year of treatment. 27 case and 76 control patients were finally included. The event-free survival (EFS) was defined as survival without evidence of relapse or progression, or death of any cause. Results: The mean age of the 27 case patients (26 myeloid and 1 lymphoblastic AL) was 52 y (± 13), the M/F ratio was 14/13 IFI (7 candidiasis, 19 aspergillosis, 1 zygomycosis) was proven for 10 patients (37%), and probable for 17 (63%). Twenty (71%) of these IFI occurred during the first induction phase. All patients were treated for their IFI with ≥ 1 antifungal, and 4 of them had a surgical resection of the main fungal lesion(s). These 27 patients were compared to 76 controls (73 myeloid and 3 lymphoblastic AL) without IFI. A delay of the next course of chemotherapy according to the planned protocol was significantly more frequent in the IFI group (16/27, 59%) than in the control group (16/76, 21%) (p=.001). Similarly, the dose or choice of the drugs was modified more frequently in the IFI group (7/27, 26%) than in the control group (6/76, 8%) (p=.037). Only 9 (33%) patients got their next chemotherapy course without any modification in time, dose, or choice of drug, vs. 19/76 (75%) in the control group (p<.0001). The EFS of the IFI group was lower than that of the control group, although this difference was not significant. Conclusion: In this single-institution case-control study, the occurrence of IFI significantly modified the application of chemotherapy courses, both on timing of the courses, and dose and choice of the drugs when compared to patients without IFI. Although the difference was not significant, there was a tendency for a lower EFS in the IFI group when compared to the control group.−

Background: In acute leukemia (AL) patients, mortality rates of the most common IFI (i.e., aspergillosis and candidiasis) are well illustrated in the literature from the IFI diagnosis, as well as the rate of fungal relapse during subsequent periods at risk, such as neutropenic phases, or transplant. However, few data are available about the impact of IFI on the subsequent chemotherapy schedule and the indirect impact of IFI on the relapse-free and overall survival. Clinicians are usually reluctant to give the full chemotherapy doses on time, due to the risk of life-threatening fungal relapse during the subsequent courses. Even with secondary prophylaxis, they often delay or decrease the doses of chemotherapy. This may impact on the leukemia outcome. The aim of this case-control study was to assess the potential impact of proven or probable IFI onset on the application of the chemotherapy schedule in AL patients, and its consequences on the leukemia outcome, by comparing patients with and without IFI in a single institution. Delays and changes in chemotherapy doses and drug choices were evaluated and compared to the planned schedule in the protocol.

Telomeres consist of repetitive DNA sequences and specific proteins, creating a specialized structure called the telosome. Unraveling the specific telomerase and telosome changes in leukemia is thought for providing new knowledge about oncogenesis, together with useful clinical markers and specific therapeutic targets. Here we measured telomerase activity (TA) by a quantitative TRAP assay in 57 human acute leukemia samples including 20 acute lymphoblastic leukemias (ALL) and 37 acute myeloid leukemias (AML). AML, TALL (n=7) and BALL (n=13) displayed significantly higher levels of TA than their normal counterparts [normal bone marrow CD34+ cells deriving from donors (BMCs, 17 samples) for AML, B-lymphocytes (n=20) for BALL and T-lymphocytes (n=24) for TALL; p<0.005 for all]. hTERT encodes telomerase reverse transcriptase which is the rate-limiting factor for TA. In BALL and TALL, hTERT expression was respectively 53 (p=0.0005) and 12 (p=0.05) times higher than in control cells. In contrast AML blasts displayed 4.5 times lower hTERT expression levels than BMCs (p=0.0008). hTERT transcripts include the full-length functional hTERT mRNA and non-functional sequences. AML blasts displayed a global decrease of all hTERT transcripts (p<0.0006) without any shift in the splicing pattern. c-Myc is a transcriptional activator of hTERT; we evidenced a positive correlation between c-Myc and hTERT transcription in BMCs (p=0.005) but not in myeloblasts. WT1, which represses hTERT transcription ex vivo in certain cell lines, can act as a transcriptional regulator or repressor depending on the cellular context. WT1 was significantly overexpressed in all leukemic subtypes (p<0.05 for all). In AML, WT1 expression negatively correlated hTERT expression (p=0.022) whereas both transcripts were positively correlated in BALL (p=0.029). We investigated the transcriptional control of hTERT by comparing the hTERT promoter occupancy between AML blasts and BMCs. Nuclear extracts deriving from 9 AML and 3 normal BMC samples were incubated with the hTERT core promoter (hCP). The protein complexes interacting with hCP were purified and components were identified by mass spectrometry. The specific AML hCP peptidome included 19 proteins of which 4, 5, 9, and 1 are involved in transcriptional regulation (including 3 factors possessing negative transcriptional effects), chromatin remodeling (including the DEK protein), mRNA maturation, and DNA methylation [DNA methyl transferase 1 (DNMT1)], respectively. With the exception of DNMT1, which is involved in hTERT transcriptional repression via CpG methylation, these factors have not been previously found negatively correlated with hTERT transcription. DEK is a chromatin remodeling protein frequently overexpressed in AML. Using CHIP, RNAi, and transfection experiments we validated and characterized the transcriptional repression of hTERT by DEK. To investigate the paradoxical association of hTERT transcriptional repression with elevated TA in AML, we measured the transcription of 36 genes involved in the post-translational regulation of hTERT and/or encoding shelterin and non-shelterin telosome genes. AML blasts overexpressed AKT1 (p=0.01) and underexpressed Ku70 (p=0.039). AKT1 activates hTERT through phosphorylation while Ku70 may act as a negative regulator of hTERT by reducing the ability of telomerase to access the exposed 3′ overhang. One can propose that these gene deregulations help compensate hTERT transcriptional repression towards an increased TA in AML. In B and TALL, telomeric gene deregulation involved shelterin and non-shelterin genes with a transcriptome pattern consistent with telomere deprotection. This is the first report of a hTERT transcriptional repression in de novo AML resulting from a global repression of all hTERT transcripts, depending on WT1 expression and involving an acquired defect in the transcriptional control of hTERT by c-Myc. DEK and DNMT1 participate to this process in vivo; our proteomic survey evidenced additional promising candidates. hTERT transcriptional repression is known to promote telomere dysfunctions and thereby genetic instability. We propose that it helps initiate the AML phenotype, which is subsequently stabilized by additional deregulations including AKT1 and Ku70-dependent hTERT post-transcriptional activation. Finally these results support important telosome modifications in acute leukemias with specific features depending on the disease phenotype.

Invasive fungal infection (IFI) occurs in 2–12% of acute leukemia patients, with different prevalences according to centers, and phases of treatment. The mortality rates of the main IFI (i.e., aspergillosis and candidiasis) are well illustrated in the literature, as well as the risk of fungal relapse during subsequent periods at risk, such as a new neutropenic phase, or transplant. However, few data are available about the impact of IFI on the subsequent chemotherapy schedule. Clinicians are usually reluctant to give the full chemotherapy doses on time, due to the risk of life-threatening fungal relapse during the subsequent courses. Even with secondary prophylaxis which is now widely given, they usually delay or decrease the doses of chemotherapy. This may impact on the leukemia outcome. Objective: The aim of this single-institution retrospective study was to look at the impact of proven or probable IFI onset on the application of the chemotherapy schedule as planned in the initial strategy in patients with acute leukemia. Delays, and changes in chemotherapy doses and drug choices were evaluated and compared to the planned schedule in the protocole. Methods: All consecutive acute leukemia patients with a first episode of proven or probable IFI according to the EORTC-MSG criteria between 2000–2006 were reviewed. All patients have been treated in, or according to, clinical research protocols where timing and doses of chemotherapy were predefined. Patients who were planned for allogeneic transplant were excluded as those who were at their last consolidation course when they got IFI. Any delay, dose decrease or dose change were defined as any difference compared to the planned schedule. Results: 28 patients (7 candidiasis, 20 aspergillosis, 1 zygomycosis) were included (M/F: 15/13; mean age: 54y), including 27 acute myeloid leukemia and one acute lymphoblastic leukemia. Eleven (39%) were proven, 17 (61%) were probable. Twenty (71%) of these IFI occurred during the first induction phase. All patients were treated for their IFI with ≥ 1 antifungal, and 4 of them had a surgical resection of the main fungal lesion (s). Seventeen of 28 (60%) had their next course delayed (median delay: 14 days) when compared to the planned protocole. The doses of the antineoplastic drug(s) were modified in 7 (25%) patients. Only 9 (32%) patients got their next chemotherapy course without any modification in time, dose, or choice of drug. Although the number of patients in our study does not allow definite conclusions, these changes should have an impact on leukemia-free and overall survivals. Conclusion: Due to the risk of fungal relapse during a subsequent period at risk, IFI has a practical impact on the dates, doses, and choices of chemotherapy in acute leukemia in more than two thirds of the patients, due to subjective decisions of the clinicians. Effective antifungal strategies are needed to avoid the onset of IFI in acute leukemia patients.

Recently, a naturally occurring regulatory T cell population (Tregs) has been identified and shown to actively suppress immune responses of self-reactive T cells. The molecular basis for the function of Tregs remains unclear. We are interested in identifying molecules specifically expressed in human Tregs that are candidates for mediating suppression by comparative proteomic and transcriptomic analysis of human CD4+/CD25+ and CD4+/CD25- T cells using two-dimensional electrophoresis (2-DE) followed by electrospray tandem MS on the Micromass Q-Tof (HPLC-MS/MS) and high-density cDNA microarray technology, respectively. Thus far, the most revealing results of the 2-DE analysis are the detection of an upregulation in the expression of a protein kinase inhibitor and FUSE binding protein (FBP), a regulator of c-Myc expression. The gene expression pattern of the Tregs was overall very similar compared to CD4+/CD25- T cells despite their major functional differences. We have identified several sets of genes differentially expressed in the Tregs. Of special interest are adhesion molecules and chemokine receptors that could reveal distinct trafficking patterns. We are in the process of validating and screening at the protein level several of these candidate genes that could lead to the identification of specific molecular events in the Tregs. The upregulation of the PKC inhibitor is especially interesting, as the reduction of IL-2 production may explain the hypoproliferative status of Tregs. The increase in FBP and a resulting increase in c-Myc transcription may promote the survival of Tregs, thereby contributing to the prevention of autoimmunity.

Recently, a naturally occurring regulatory T cell population (Tregs) has been identified and shown to actively suppress immune responses of self-reactive T cells. The molecular basis for the function of Tregs remains unclear. We are interested in identifying molecules specifically expressed in human Tregs that are candidates for mediating suppression by comparative proteomic and transcriptomic analysis of human CD4+/CD25+ and CD4+/CD25- T cells using two-dimensional electrophoresis (2-DE) followed by electrospray tandem MS on the Micromass Q-Tof (HPLC-MS/MS) and high-density cDNA microarray technology, respectively. Thus far, the most revealing results of the 2-DE analysis are the detection of an upregulation in the expression of a protein kinase inhibitor and FUSE binding protein (FBP), a regulator of c-Myc expression. The gene expression pattern of the Tregs was overall very similar compared to CD4+/CD25- T cells despite their major functional differences. We have identified several sets of genes differentially expressed in the Tregs. Of special interest are adhesion molecules and chemokine receptors that could reveal distinct trafficking patterns. We are in the process of validating and screening at the protein level several of these candidate genes that could lead to the identification of specific molecular events in the Tregs. The upregulation of the PKC inhibitor is especially interesting, as the reduction of IL-2 production may explain the hypoproliferative status of Tregs. The increase in FBP and a resulting increase in c-Myc transcription may promote the survival of Tregs, thereby contributing to the prevention of autoimmunity.

Invasive fungal infection (IFI) occurs in 2–12% of acute leukemia patients, with different prevalences according to centers, and phases of treatment. The mortality rates of the main IFI (i.e., aspergillosis and candidiasis) are well illustrated in the literature, as well as the risk of fungal relapse during subsequent periods at risk, such as a new neutropenic phase, or transplant. However, few data are available about the impact of IFI on the subsequent chemotherapy schedule. Clinicians are usually reluctant to give the full chemotherapy doses on time, due to the risk of life-threatening fungal relapse during the subsequent courses. Even with secondary prophylaxis which is now widely given, they usually delay or decrease the doses of chemotherapy. This may impact on the leukemia outcome.

The tumor suppressor INK4B(p15) gene is silenced by CpG island hypermethylation in a majority of acute myeloid leukemias (AML). This silencing can be reversed by the treatment with hypomethylating agents, and these agents are currently being tested for therapeutic intervention. So far, it was not investigated whether or not the INK4B is hypermethylated in all cytogenetic subtypes of AML. Our experiments, which compare levels of INK4B methylation in AML with inv(16), t(8:21) and t(15;17) reveal a strikingly low level of methylation in all leukemias with inv(16). This contrasts with significant levels of DNA methylation in a high proportion of the AML from the other two groups. Surprisingly, even though there is a lack of INK4B methylation in samples from patients with inv(16), expression of the gene is very low when compared to that of PBL from healthy individuals or HL60 cells. Subsequent experiments uncovered a novel mechanism to explain the low level of INK4B expression in the inv(16) AMLs. Overexpression of the aberrant chromosome 16-associated gene CBFb-MYH11 in U937 cells results in failure to induce INK4B in response to vitamin D3. Furthermore, CBFb-SMMHC, encoded by CBFb-MYH11 directly represses transcription from an INK4B promoter in a reporter assay. Electromobility shift assays in the AML-derived cell line ME-1 and U937 cells expressing the fusion gene demonstrate that the repression is due to a change in the composition of the complexes recognizing the binding sites for the transcription regulator CBF. In conclusion, we have found that methylation is not the only way to induce silencing of the tumor suppressor p15INK4B in AML. In inv(16)-containing AML, loss of gene expression is accomplished by the direct transcriptional repressor activity of CBFβ-SMMHC.

The tumor suppressor INK4B(p15) gene is silenced by CpG island hypermethylation in a majority of acute myeloid leukemias (AML). This silencing can be reversed by the treatment with hypomethylating agents, and these agents are currently being tested for therapeutic intervention. So far, it was not investigated whether or not the INK4Bis hypermethylated in all cytogenetic subtypes of AML. Our experiments, which compare levels of INK4Bmethylation in AML with inv(16), t(8:21) and t(15;17) reveal a strikingly low level of methylation in all leukemias with inv(16). This contrasts with significant levels of DNA methylation in a high proportion of the AML from the other two groups. Surprisingly, even though there is a lack of INK4B methylation in samples from patients with inv(16), expression of the gene is very low when compared to that of PBL from healthy individuals or HL60 cells. Subsequent experiments uncovered a novel mechanism to explain the low level of INK4Bexpression in the inv(16) AMLs. Overexpression of the aberrant chromosome 16-associated gene CBFb-MYH11in U937 cells results in failure to induce INK4Bin response to vitamin D3. Furthermore, CBFb-SMMHC, encoded by CBFb-MYH11directly represses transcription from an INK4Bpromoter in a reporter assay. Electromobility shift assays in the AML-derived cell line ME-1 and U937 cells expressing the fusion gene demonstrate that the repression is due to a change in the composition of the complexes recognizing the binding sites for the transcription regulator CBF. In conclusion, we have found that methylation is not the only way to induce silencing of the tumor suppressor p15INK4Bin AML. In inv(16)-containing AML, loss of gene expression is accomplished by the direct transcriptional repressor activity of CBFβ-SMMHC.