bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Autologous Tumor-Specific T-Cell Responses Induced by Liposome Encapsulated Lymphoma Membrane Proteins.
Authors: Neelapu Sattva S. , Gause Barry L. , Harvey Linda , Frye Andrea R. , Horton Jessie , Robb Richard J. , Popescu Mircea C. , Kwak Larry W.
Published: October 2019

The unique antigenic determinants (Idiotype [Id]) of the immunoglobulin expressed on a given B cell malignancy can serve as a tumor-specific antigen for active immunotherapy. We have previously demonstrated that Id vaccines in follicular lymphoma (FL) patients administered in the minimal residual disease (MRD) state are immunogenic and are associated with induction of complete molecular remissions and long-term disease-free survival (Nature Med 5:1171–1177, 1999). This hybridoma-derived vaccine is now being tested in a pivotal Phase III clinical trial. However, the production of Id protein by hybridoma technology for such vaccine formulation is an expensive and laborious process requiring an average of 3 to 6 months to manufacture the vaccine for each patient. To overcome this difficulty, we developed a novel vaccine formulation where we directly extracted the membrane proteins from lymph node biopsy-derived tumor cells and incorporated them into liposomes along with IL-2. Testing in preclinical studies showed this formulation to be as potent as our prototype hybridoma-derived Id protein vaccine. In the present study, 11 previously untreated and/or relapsed FL patients received 5 injections of this novel vaccine formulation subcutaneously and/or intratumorally at approximately monthly intervals. The vaccine was well tolerated and induced only minor local reactions at the sites of injection. T cell responses were evaluated by cytokine induction and IFNg ELISPOT against autologous tumor. Post-vaccine, but not pre-vaccine, peripheral blood mononuclear cells (PBMC) from 6 out of 10 patients that were assessed, recognized autologous tumor cells, as demonstrated by TNFa, GM-CSF and/or IFNg production. Significant production of cytokines was observed only in response to autologous tumor cells, but not normal B cells. The precursor frequency of tumor-reactive T cells was significantly increased in postvaccine PBMC (range 19–115 IFNg spots/100,000 PBMC), compared with prevaccine PBMC (range 2–7 IFNg spots/100,000 PBMC). Anti-MHC Class I and Class II antibodies inhibited cytokine production suggesting that both CD4+ and CD8+ T cells were involved in the anti-tumor immune responses. Vaccination was associated with induction of a sustained complete response in one patient and correlated with the generation of a potent anti-tumor T cell response. The remaining 10 patients progressed after a median duration of 8 months. We conclude that liposomal delivery of lymphoma membrane proteins is safe, induces tumor-specific CD4+ and CD8+ T cell responses, and may serve as a model for vaccine development against other human cancers. The induction of clinical response warrants further testing of this novel formulation in the setting of MRD where the immunosuppressive effects of the tumor are likely to be least.
Mcl-1 Sequesters Pro-Apoptotic BH3-Only Bim To Inhibit Its Collaboration with Bax in Myeloma Cells.
Authors: Gomez-Bougie Patricia , Clement Monique , Juin Philipe , Bataille Regis , Amiot Martine
Published: October 2019

Multiple Myeloma is a fatal malignancy of B-cell origin characterized by the accumulation of plasma cells within the bone marrow. IL-6 is a major myeloma cell survival factor that induces the activation of both, Ras/MAP kinase and JAK/STAT pathways, the latter being involved in cell survival. Thus, IL-6 starvation or IL-6 signaling blockade by AG490 (a JAK 2 inhibitor) trigger apoptosis. In myeloma cells, others and us have shown that Mcl-1 is the major anti-apoptotic protein, since Mcl-1 antisenses induce apoptosis while Bcl-2 or Bcl-xL antisenses have no effect. In the present study, we examined the pro-apoptotic BH3-only molecule Bim which is implicated in apoptosis induced by growth factor deprivation. The three major Bim isoforms (EL, L and S) are expressed in viable human myeloma cell lines and are negatively regulated by IL-6. Blockade of IL-6 signaling induces the up-regulation of Bim isoforms concomitant to the down-regulation of Mcl-1. Of major interest, in viable myeloma cells, Bim is strongly associated to Mcl-1, to Bcl-2 and to a lesser extent to the dynein light chain, suggesting that Bim is always complexed in viable cells. Bim/Mcl-1 endogenous interaction is disrupted upon apoptosis induction, either by IL-6 starvation or AG 490 treatment. Of note, Bim/Bcl-2 complex is not affected under the same conditions. In parallel to disruption of Bim/Mcl-1 interaction and Bim release, Bax activation was observed. Microinjection of a peptide comprising the minimal BH3 domain from Bim triggered apoptosis in myeloma cells, confirming that free Bim might exert a direct pro-apoptotic effect in these cells. Therefore, in myeloma cells Mcl-1 neutralises Bim through endogenous complex formation. Under apoptosis induction, Mcl-1 drastic down-regulation leads to Bim release, which in turn exerts its pro-apoptotic function through Bax activation.
Dysregulation of BMPR2, Arginase II and HMGA2 Expression in Idiopathic Myelofibrosis and Secondary Myelofibrosis.
Authors: Liu Enli , Frost Adaani E. , Popat Uday R. , Prchal Josef T.
Published: October 2019

Idiopathic myelofibrosis (IMF) is a fatal clonal myeloproliferative disorder, characterized by variable pancytopenia, splenomegaly, leukoerythroblastic blood smear, marrow fibrosis (MF), and myeloid metaplasia. Like all myeloproliferative disorders, it is due to an acquired somatic mutation of a single hematopoietic progenitor. MF has also been reported in systemic lupus and metastatic malignancies involving bone marrow. We have recently demonstrated the occurrence of MF in 85% patients with pulmonary hypertension (PH), which (unlike IMF) is associated with polyclonal hematopoiesis and normal number of circulating CD34+ cells. The pathophysiological mechanisms resulting in IMF and secondary MF (SMF) in PH remain still unclear. To date, 52 heterozygous germline mutations in the gene (BMPR2) encoding the bone morphogenetic protein type II receptor (BMPR-II) have been found to characterize many cases of familial (Lane, 2000) and sporadic primary PH (PPH) (Thomson, 2000). Recently, increased activity of arginase in pulmonary tissue of patients with PH was found. This may contribute to the pathogenesis of pulmonary hypertension by decreasing the substrate for NO synthesis. We examined BMPR2 and Arginase II mRNA levels by quantitative real-time RT-PCR in mononuclear cells (MNC), platelets (PLT) and granulocytes (GNC) from IMF patients and PH patients with secondary MF. We show for the first time that the mRNA level of BMPR2 is increased in PLT and GNC of IMF compared with normal controls. Arginase II mRNA was significantly increased in GNC of IMF as well as PH patients with SMF. Rearrangement and over expression of high-mobility group AT-hook2 (HMGA2) gene in myeloid progenitors in two patients with IMF was recently reported. This gene is known to play a major role in fetal growth and development, and is expressed in trace amount in adult lung, kidney, and synovia. We found significantly increased HMGA2 mRNA expression in MNC of IMF patients. In summary, the biomarkers studied here can be abnormal in both IMF and PH with secondary MF and may not be useful to discriminate between the two types of MF. Dysregulation of BMPR2, Arginase II and HMGA2 expression may play a pathophysiological role in MF of both IMF and PH.

Expression of BMPR2 , Arginase II and HMGA2 in Blood Cells of IMF and SMF

Gene Cells Control (ΔCT) Idiopathic MF (ΔCT) Secondary MF (ΔCT) *The P value represents a significant difference of gene expression in IMF or SMF compared with normal controls. A lower ΔCT indicates a higher gene expression. BMPR2 PLT 18.5±1.2 16.7±1.4 (p=0.038) * 18.4±2.2 (p=0.881) GNC 19.2±1.0 18.3±1.3 (p=0.009) * 19.1±1.2 (p=0.893) MNC 18.3±1.2 19.2±1.8 (p=0.059) 19.0±2.7 (p=0.554) Arginase II GNC 24.5±0.6 21.9±1.7 (p=0.000) * 22.6±1.2 (p=0.004) * MNC 24.4±3.5 24.4±3.9 (p=0.965) 22.5±2.4 (p=0.222) HMGA2 MNC 32.4±1.6 29.3±1.7 (p=0.000) * 30.8±1.8 (p=0.085)
BMS-354825 Is a SRC/ABL Inhibitor with High Nanomolar Activity Against the Kit D816v Mutation, Which Drives Systemic Mastocytosis and Is Imatinib-Resistant.
Authors: Shah Neil P. , Lee Francis Y. , Sawyers Charles L. , Akin Cem
Published: October 2019

The vast majority of systemic mastocytosis cases are associated with a somatic KIT oncoprotein point mutation which substitutes a valine for aspartic acid (D816V), resulting in KIT receptor auto-phosphorylation in a ligand-independent manner. Previous reports have demonstrated that this mutation is inherently imatinib-resistant. Although interferon-alpha has some activity against aggressive systemic mastocytosis, major responses are uncommon, and the drug is associated with significant toxicity. To date, there remains no effective therapy for systemic mastocytosis. We recently described BMS-354825, a novel orally bioavailable SRC/ABL inhibitor that has activity against multiple imatinib-resistant BCR-ABL isoforms in vitro (Shah et al, Science 305:399, 2004). BMS-354825 is presently undergoing evaluation in a phase I clinical trial of imatinib-resistant CML patients, and is showing signs of clinical efficacy. Pharmacokinetic analysis suggests that high nanomolar concentrations of the compound can be safely achieved in humans (see Sawyers et al, Talpaz et al, abstracts submitted for this meeting). To determine if this compound warrants study in other human hematologic conditions, we tested BMS-354825 for activity against human mastocytosis cell lines HMC-1560 and HMC-1560,816, carrying an activating c-kit mutation in juxtamembrane domain (codon 560) with or without a second mutation in tyrosine kinase domain (codon 816), respectively. While 1 um imatinib failed to inhibit the growth of HMC-1560,816 cells carrying the tyrosine kinase domain c-kit mutation, BMS-354825 led to an almost complete growth inhibition at the same concentration, with an IC50 of 0.1–1 uM. In addition, growth of HMC-1560 cells carrying the juxtamembrane c-kit mutation alone was more effectively inhibited by BMS-354825 as compared to imatinib (IC50 of <0.01 vs 0.01–0.1 micromolars respectively). Significantly, detection of phospho-KIT by Western blot analysis was significantly reduced in the presence of BMS-354825 at nanomolar concentrations. An ex vivo assessment of D816V-harboring mast cell sensitivity using a flow cytometric method in systemic mastocytosis bone marrow samples is ongoing. Our findings suggest that studies to evaluate BMS-354825 for the treatment of systemic mastocytosis are warranted. Additionally, the compound may harbor activity in other disease settings that contain activating KIT mutations.
Sepsis-Induced Coagulation in the Baboon Lung Is Associated with Decreased Endothelial Tissue Factor Pathway Inhibitor.
Authors: Tang Haiwang , Ivanciu Lacramioara , Hamm Todd , Taylor Fletcher B. , Lupu Cristina , Lupu Florea
Published: October 2019

Activation of tissue factor (TF)-dependent coagulation is an early event in the pathogenesis of sepsis, responsible for microvascular thrombosis and consequent organ injury. We hypothesized that sepsis-induced TF procoagulant activity may be paralleled by a decreased expression or function of its natural inhibitor, tissue factor pathway inhibitor (TFPI). To test this hypothesis we used a non-human primate sepsis model in which adult baboons were infused with live E. coli at 109 CFU/kg (sublethal dose), 1010 CFU/kg (lethal dose) or saline (control group) and the animals were sacrified after 2, 8, or 24 hours. Lung tissue was snap frozen for protein and mRNA extraction or fixed and processed for light and electron microscopy. Tissue cryosections were immunostained using double/multiple fluorescence labeling approaches for TF, TFPI, factor VII/FVIIa and fibrin, in conjunction with cell markers for endothelial cells (CD31 or von Willebrand factor), leukocytes (PSGL-1), macrophages (CD68), PMN (myeloperoxidase) and platelets (gpIIb/IIIa). Large amounts of TF were detected in leukocytes, endothelial cells and platelet-rich microthrombi, starting from 2 hours and throughout the examined period. Concomitantly, confocal and electron microscopy analysis revealed increased leukocyte infiltration, platelet aggregates and fibrin deposition in the intravascular and interstitial compartments. In addition, TF induction was documented by semiquantitative RT-PCR, ELISA, western blot and factor Xa activation assays. Whereas TFPI mRNA showed only a modest increase, tissue-associated TFPI protein was found considerably decreased, especially during the first eight hours post E. coli infusion. Moreover, TFPI inhibitory activity of lung extracts from septic animals was 6–8 fold lower comparing to controls. TF activity measurements in the presence of inhibitory anti TFPI antibodies showed that only a very small fraction of endogenous TF was inhibited by tissue-associated TFPI, suggesting that most of the active TFPI available in the vascular compartment was depleted. The decrease of TFPI inhibitory potency cannot be exclusively explained by its proteolytic degradation, as we did not find significant amounts of truncated TFPI on western blots. In conclusion, our studies demonstrate that the exposure to septic and inflammatory stimuli lead to a decrease of TFPI-dependent endothelial anticoagulant potential, simultaneous with a strong TF-dependent procoagulant response. Activation of TF-dependent coagulation pathway not adequately countered by TFPI may have important roles in the pathogenesis of sepsis-associated disseminated intravascular coagulation. Strategies aimed to restore the physiological anticoagulant function of TFPI may help preventing sepsis-induced multiple organ dysfunction syndrome and death.
Allogeneic Hematopoietic Cell Transplantation after Fludarabine and 2 Gy Total Body Irradiation for Relapsed and Refractory Mantle Cell Lymphoma.
Authors: Maris Michael B. , Sandmaier Brenda M. , Storer Barry E. , Chauncey Thomas , Stuart Monic Jain , Maziarz Richard T. , Agura Edward , Langston Amelia A. , Pulsipher Michael , Storb Rainer , Maloney David G.
Published: October 2019

We carried out HLA-matched related (n=16) and unrelated (n=17) hematopoietic cell transplantation (HCT) in 33 patients with relapsed and refractory mantle cell lymphoma after nonmyeloablative conditioning with fludarabine and 2 Gy total body irradiation. Postgrafting immunosuppression consisted of cyclosporine and mycophenolate mofetil. Fourteen patients had failed high-dose autologous HCT. The median time from disease diagnosis to HCT was 2.8 (range 0.4 to 9.5) years. The median number of prior treatment regimens was 4 (range 1 to 10). Recipients of unrelated grafts were more heavily pretreated than those of related grafts (median treatment regimens 5 versus 3, respectively). Forty-two percent (10/17 unrelated recipients versus 4/16 sibling recipients) of patients had failed autologous HCT. Thirty-nine percent of patients were refractory to salvage chemotherapy or planned autologous HCT. Forty-one percent of the unrelated recipients had moderate to large disease burdens compared to 25% of the related recipients. The median follow-up after HCT for surviving patients was 24.6 (range 2.7 to 41.7) months. Thirty-one of the 33 patients had stable engraftment, while 2 patients experienced non-fatal graft rejections. The incidences of acute grades II, III and IV, and chronic GVHD were 27%, 17%, 13%, and 64%, respectively. The overall response rate in the 20 patients with measurable disease at the time of HCT was 85% [n=17; 75% complete remissions (CR) and 10% partial remissions (PR)], while 3 patients had progressive disease. Only 1 of the 17 patients who responded and none of the 13 transplanted in CR had disease relapse with a median follow-up of 24.6 months. Relapse and non-relapse mortalities were 9% and 24%, respectively, at 2 years. The Kaplan-Meier probabilities of overall and disease-free survivals at 2 years were 65% and 60%, respectively. On univariate analysis, patients less than 55 years old had worse non-relapse mortality compared to patients who were older than 55 years (p=0.003), b2 microglobulin level greater than the median of 1.85 predicted worse survival (p=0.03), and preceding chemotherapy regimens > 4 predicted a higher risk of relapse. In the time-dependent analysis of the relationship of disease response with GVHD, there was no association between disease response and acute grades I-IV acute GVHD (p=0.36), but a trend was observed with chronic extensive GVHD (p=0.09). Allogeneic HCT after nonmyeloablative conditioning is a promising salvage strategy for patients with relapsed and refractory mantle cell lymphoma. The high response and low relapse rates with this approach suggest that mantle cell lymphoma is susceptible to graft-versus-tumor responses.
The Inhibitor of VEGF-Receptors Tyrosine Kinase PTK-787 Blocks the Invasive Potential of Adult T-Cell Leukemia Cells through the Endothelium.
Authors: Bazarbachi Ali , Harb Roa , Khoury Hilda , Haddad Lara , Kfoury Youmna , Atweh Lamia , Hamadeh Eva , Hermine Olivier , de The Hugues , El-Sabban Marwan E.
Published: October 2019

Extravasation of tumor cells through the endothelial barrier is a critical step in cancer metastasis. HTLV-I associated adult T-cell leukemia/lymphoma (ATL) is an aggressive disease characterized by multiple organ invasion. We have shown that ATL-derived cells induce angiogenesis and communicate with endothelial cells through gap junctions. This results in induction of endothelial-derived metalloproteinases, sub-endothelial basement membrane degradation followed by endothelial cell retraction allowing neoplastic lymphocytes extravasation. In this study we have used the specific tyrosine kinase inhibitor of VEGF receptors PTK787, functional blocking antibodies against VEGF and the gap junction communication inhibitor18-a-G to dissect the respective role of VEGF signaling pathway and cell-to-cell communication in endothelial cell retraction and tumor cell extravasation. PTK787 has mild growth inhibitory and cytotoxic effects on both endothelial and tumor cells at a concentration of 50μM and significantly inhibited cell proliferation at 100μM. Hence, 20μM PTK787 which did not exhibit any cytotoxic or antiproliferative effects on both cell types was used in experiments that last 24 hours or longer (in vitro tube formation assay and invasion assay). Endothelial cells monolayers incubated with either ATL derived cells or their cell-free conditioned medium for 48 hours induced in vitro tube formation in Matrigel angiogenesis assay. This was inhibited by both anti-VEGF antibody and 18-a-G and to a greater extent and in a dose dependent manner by PTK787. Importantly, PTK787, and to a lesser extent anti-VEGF antibody and 18-alpha-G significantly attenuated the ability of ATL-derived cells to traverse endothelial cell monolayers in double compartment invasion assays. Signaling cascade that is activated upon VEGF stimulation of endothelial cells was investigated in confluent endothelial cells cocultured with ATL derived cells or cell-free conditioned medium in the absence or presence of PTK787 or anti-VEGF antibody, using phospho-specific antibodies against FAK Tyr 861, FAK Tyr 397, Src, ERK, PLCgamma, and Akt/PKB, known to be signal mediators in the Ras-Raf-MEKK-ERK signaling cascade. The addition of ATL derived cells or cell-free conditioned medium resulted in a transient activation of the above signal mediators with a peak increase at 15 minutes and gradual return to baseline after 60 minutes. This activation was down regulated in the presence of anti-VEGF antibody and to a greater extent in the presence of PTK787. This data establishes a novel role of VEGF signaling in tumor cell invasion and supports the potential use of PTK787 in the treatment of ATL and other hematological malignancies with angiogenic and invasive properties.
Tumor Necrosis Factor-α-Converting Enzyme (ADAM17) Mediates GPIbα Shedding from Platelets In Vitro and In Vivo.
Authors: Bergmeier Wolfgang , Piffath Crystal L. , Cheng Guiying , Dole Vandana S. , Zhang Yuhua , von Andrian Ulrich H. , Wagner Denisa D.
Published: October 2019

Interaction of the platelet receptor GPIb-V-IX with von Willebrand factor exposed at a site of vascular injury is an essential step in the initiation of a hemostatic plug. Proteolytic cleavage (shedding) of the GPIbα subunit was first described more than 25 years ago, the protease mediating this event as well as its physiological function, however, have not been elucidated. We have recently reported that shedding of GPIbα and platelet clearance induced by platelet storage or mitochondrial injury involves a platelet-derived metalloproteinase(s). Here we show that GPIbα shedding in response to mitochondrial injury or physiological activation is inhibited in platelets obtained from chimeric mice, which express inactive tumor necrosis factor-α converting enzyme (TACEΔZn/ΔZn) in blood cells only. Shedding was also inhibited in mouse and human platelets in the presence of two potent TACE inhibitors, TAPI and TMI-1. Our data further suggest that TACE is important in the regulation of GPIbα expression in vivo, as we observed an ~ 90% reduction in soluble GPIbα (glycocalicin) in plasma of TACEΔZn/ΔZn chimeras as well as significantly increased levels of GPIbα on circulating platelets. In contrast, shedding of P-selectin from activated platelets was not affected by the mutation in TACE. Mitochondrial damage in TACEΔZn/ΔZn platelets did not result in reduced posttransfusion recovery or impaired hemostatic function of such platelets in mice, indicating that TACE not only mediates GPIbα shedding but that it may also be crucial for the development of cellular changes leading to platelet clearance. In conclusion, our data demonstrate that TACE is expressed in platelets and that it is the key enzyme mediating shedding of GPIbα. Inhibition of TACE during platelet storage could be a powerful means to improve the effectiveness of platelet transfusions.
Akt-1 Regulates Angiogenesis in Skin.
Authors: Byzova Tatiana , Chen Juhua , Somanath Payaningal R.
Published: October 2019

The major mechanism to adapt to ischemic conditions is the development of neovascularization, i.e. angiogenesis, a process driven by members of VEGF family of growth factors. Phosphoinositide 3-kinase/Akt pathway is a critical component of the signaling network that regulates endothelial cell function related to angiogenesis. VEGF treatment of endothelial cells results in rapid phosphorylation of Akt. Our studies demonstrated that Akt kinase activity is necessary for VEGF-induced and integrin-mediated endothelial cell adhesion and migration. Moreover, cell transfection with a constitutive active form of Akt (myr-Akt) leads to increased function of integrin receptors. Using Akt-1 null mice we found that Akt-1 controls VEGF-induced and integrin-dependent endothelial cell responses in vitro. Impaired endothelial cell migration and adhesion to extracellular matrix and a reduced rate of cell proliferation were observed in Akt-1 (−/−) endothelial cells compared to WT. There are three Akt isoforms with different tissue distribution, however, it appears that Akt-1 is a predominant isoform in skin and in skin microvasculature. This observation prompted us to perform series of in vivo experiments designed to assess the angiogenic response in skin in the absence of Akt-1. Angiogenesis assay using matrigel plugs revealed that the weight and hemoglobin content of matrigel plugs is about two fold higher in Akt (−/−) mice compared to WT mice. Tumor angiogenesis also appears to be enhanced in Akt(−/−) mice, resulting in the significantly lower degree of tumor necrosis. Blood vessels in Akt (−/−) mice appear to be smaller in diameter and have reduced laminin content. Our analysis revealed significant changes in blood vessel wall matrix composition of Akt (−/−) mice as compared to WT animals. These changes resulted in increased vascular permeability in skin of Akt (−/−) mice. Akt-1 is known to target multiple cellular processes including adhesive properties, cell survival, transcription and translation. It appears that the phenotype of Akt-1 (−/−) mice depends on the equilibrium between pro-angiogenic and anti-angiogenic roles of Akt-1 and reveals a central role for Akt-1 in the regulation of matrix production and maturation of blood vessels.
Time To Complete Remission Is Not a Significant Prognostic Factor in AML: A Report on 1,959 Consecutive Patients Registered to 6 Studies of the Eastern Cooperative Oncology Group (ECOG).
Authors: Rowe Jacob M. , Kim Haesook T. , Cassileth Peter A. , Litzow Mark R. , Wiernik Peter H. , Tallman Martin S.
Published: October 2019

Prognostic factors for AML at diagnosis include cytogenetics, FLT3 ITD, age, MDR status and white cell count at presentation. Several groups (Blood2000; 95:72; BJH1999; 107:69; Haematologica2004; 89:528) have reported that time to achievement of complete remission (CR) is an independent post-induction prognostic factor in AML. Furthermore, some investigators have classified patients who do not achieve CR with one cycle as primary induction failure. Between 1983 and 1993 1,959 patients were registered to 6 consecutive ECOG studies (E3483, PC486, E1490, E2491/INT0129, E3489 and E3993). These studies were for patients age 18–55 years (E3483, PC486 and E3489); age 56–70 years (E1490); age >56 years (E3993) and with no age limit (E2491/ INT0129). Of these 1,959 patients, 1261 achieved CR (64%) and were available for analysis. All patients received standard induction therapy, consisting of daunorubicin 60 mg/m2 (E3483, PC486 and E1490), or 45 mg/m2 (E2491/ INT0129) or idarubicin 12 mg/m2 (E3489), or a randomization between daunorubicin 45 mg/m2, idarubicin 12 mg/m2 or mitoxantrone 12 mg/m2 (E3993) - all for 3 days - together with cytarabine 100–200 mg/m2 by continuous infusion for 7 days. It should be noted that in the North American Intergroup APL study (E2491/ INT0129) patients who received ATRA in induction are excluded from this analysis. All patients were to receive a second cycle of identical induction therapy if the day 10–14 bone marrow demonstrated unequivocal residual leukemia. Identical study-specific post-remission therapy, including consolidation therapy and/or allogeneic or autologous bone marrow transplantation, was given to all patients who went into CR - irrespective if this was achieved after 1 or 2 cycles. This retrospective analysis compared patients who achieved CR after 1 cycle versus 2 cycles of induction therapy. The data were obtained from the ECOG database that recorded the number of days that patients received induction therapy (≤8 days vs >8 days). The table presents the data for disease-free survival (DFS) and overall survival (OS). These data are based on the Cox model, adjusting for other variables.

Total Failed Median DFS/OS (mo) 5-year DFS/OS p-value DFS 1 cycle 406 321 10.2 24.4% 2 cycles 855 629 11.9 28.6% 0.07 OS 1 cycle 406 287 23.7 36.1% 2 cycles 855 583 24.1 35.7% 0.7

In this large cohort of patients, by multivariate analysis, age, WBC at diagnosis were significant factors for DFS and OS. However, the number of cycles given for induction or the time to achieve a CR in AML patients could not be demonstrated to have any independent prognostic significance. These data suggest that post-remission strategies in AML should not be influenced by the number of cycles required to achieve CR.
A Phase 2 Evaluation of Single Agent Clofarabine as First Line Treatment for Older Patients with AML Who Are Not Considered Fit for Intensive Chemotherapy.
Authors: Burnett Alan K. , Russell Nigel , Kell Jonathan W. , Milligan Donald , Culligan Dominic
Published: October 2019

Patients over 70 years represent a significant proportion of patients with AML. They respond poorly to intensive chemotherapy. Although 50% of patients can achieve CR, only 8% are alive at 2 years (MRC Database). In addition significant numbers of these patients either do not wish to undergo, or are not considered fit for intensive chemotherapy. This represents a significant unmet medical need. Nucleoside analogues (Ara-C) provide an active group of agents in AML, Fludarabine however is ineffective at tolerable doses. Clofarabine has been modified by introducing a Fluorine in the 2′ position which confers reduced susceptibility to cleavage of the glycosidic linkage and also reduced toxicity caused by halogenated nucleobases. As such the drug has potential use as an oral formulation. Previous clinical and in vitro studies have established a dose schedule at 40mg/m2 days 1–5. This is associated with CRs in relapse/refractory AML, but with grade ¾ liver toxicity in 25% of patients.

Because of these characteristics we undertook a non-randomised study using a dose of 30mg/m2 days 1-5 IV, which could be repeated for up to 4 courses at a minimum of 28 days apart. The target patient group were patients >70 years, or patients 60–70 with a cardiac history, who were not considered fit for conventional chemotherapy.

Twenty-eight patients entered the study: 16 males, 12 females of a median age 71 years (range 60 – 79). The FAB distribution was, M0=1;M1=9; M2=8; M4=2; M5=4; M6=3; Unknown=1. The cytogenetic risk group (MRC criteria) were 26 standard and 2 unfavourable risk. All patients received course 1. Five patients died before response could be assessed from causes which were not thought to be associated with the drug. One patient is too early to assess response. Five did not enter complete remission although there was a reduction in bm blasts from 58 to 18%, 55 to 20% and 30 to 15% in 3 cases. Sixteen patients (59%) achieved complete remission after course 1. Grade 3 toxicity was seen in 3 patients, but this recovered in all cases in a few days. Patients who did not enter CR did not show haematological recovery after course 1 and were considered treatment failures. Of the 16 patients who entered CR, haematological recovery to ANC 1.0 x 109/l took 28 days (range 21–42) Platelets to 50x109/l took 25 days (range 21–38).

Conclusions: Clofarabine is an active agent when used alone as first line treatment. It is well tolerated but is associated with cytopenia of an average 28 days. At the dose chosen for this study grade ¾ liver toxicity was uncommon. The drug is a candidate for evaluation in a randomised trial and/or in combination schedules, and lower doses should be explored in older patients. This study was partially supported by an unrestricted grant from Bioenvison Inc.
Intravenous Voriconazole (IV-VORI) Prevents Invasive Fungal Infections (IFI) in Patients (pts) with Acute Myelogenous Leukemia (AML) and High-Risk Myelodysplastic Syndrome (MDS).
Authors: Mattiuzzi Gloria N. , Alvarado Gladys , Kantarjian Hagop M. , Verstovsek Srdan , Cabanillas Maria , Faderl Stefan , Lim Joann T. , Giles Francis , Beran Miloslav , O’Brien Susan , Estey Elihu H.
Published: October 2019

Background: IFI is the most frequent cause of mortality in pts with AML and MDS undergoing chemotherapy. None of the antifungal prophylactic regimens used since 1992 at MD Anderson Cancer Center appeared to be significantly superior in the prevention of IFI.

Study Aims: To compare the efficacy and safety of IV-VORI versus IV-ITRA as antifungal prophylaxis in AML and MDS pts receiving chemotherapy

Patients and Methods: Pts older than 18 years old receiving induction or salvage chemotherapy, without documentation of prior IFI were eligible. Pts were randomized on day 1 of chemotherapy to receive IV-VORI 400 mg q12 h x 2 days followed by 300 MG IV twice per day or IV-ITRA 200 mg q12 h x 2 days followed by 200 mg IV once per day. Prophylaxis continue until recovery from neutropenia, developed possible/proven IFI, complete remission, declared resistant or up to 35 days for induction pts and up to 42 days for salvage pts.

Results: 114 pts were evaluable (49 on IV-ITRA, 65 on IV-VORI). Baseline characteristics were similar in both groups. 102 were induction pts and 12 were on first salvage. Median time on prophylaxis was 21 (induction) and 17 days (salvage) for both groups. 45% pts on IV-ITRA and 48% pts on IV-VORI completed prophylaxis without modification (p=ns). Two pts on IV-ITRA developed IFI (1 disseminated C. glabrata and 1 disseminated Fusarium) as oppose to none on the IV-VORI arm (p=0.101). No significant differences were seen in the number of pts that required empiric antifungal therapy due to persistent fever or possible fungal pneumonia (14% on IV-VORI, 18% on IV-ITRA). 15/49 pts on IV-VORI (23%) versus 4/49 (8%) on IV-ITRA discontinued prophylaxis due to side effects (p=0.036). Reversible increase in the liver function tests (9 on IV-VORI, 4 on IV-ITRA) and hallucinations (5 on IV-VORI) were the most frequent adverse events. Response to induction chemotherapy, overall induction mortality and survival were similar in both groups.

Conclusions: 1) IV-VORI appears to be more efficacious than IV-ITRA in preventing IFI in AML and MDS pts receiving chemotherapy. More pts are needed to confirm this finding. 2) IV-VORI tends to be more toxic than IV-ITRA. The usage of high dose IV-VORI may explain the incidence of side effects.
Long Term Follow-Up Shows No Clinical Benefit for CD34+ Selection of Autologous Transplants Following Myeloablative Therapy in Patients with Newly Diagnosed Myeloma : An EBMT Phase III Randomized Study.
Authors: Bourhis Jean-Henri , Bouko Yasmina , Koscielny Serge , Greinix Hildegard , Derigs Gunter , Salles Gilles , Feremans Walter , Bakkus Marleen , Apperley Jane , Samson Diana , Bjorkstrand Bo , Niederwieser Dietger , Gahrton Gosta , Pico Jose-Luis , Goldschmidt Hartmut
Published: October 2019

CD34+ selection of peripheral blood progenitor cells (PBPC) was used in Multiple Myeloma (MM) in an attempt to reduce tumor cell contamination and relapse rate. From May 1995 to November 1999, 127 patients from 17 EBMT centers with newly diagnosed advanced MM were entered into a phase III trial. Responders to 3 cycles of VAD were randomized to receive either CD34+ selected (arm A) or unselected PBPC (arm B) following myeloablative conditioning treatment. PBPC were harvested following mobilization with cyclophosphamide 4g/m² and G-CSF (Filgrastim, AMGEN, Europe). Conditioning regimen in both arms was TBI and high dose melphalan 140 mg/m². CD34+ cell selection was performed in arm A using the CellPro Ceprate-SC device. The analysis concerns the 111 patients who were transplanted (56 arm A and 55 arm B). The median follow-up at the time of the analysis was 65 months. CD34+ selection resulted in a median purity of 87% and a yield of 50%. Molecular analysis showed a median tumor cell depletion of 2.0 log (range 0.3–85.5). The median number of CD34+ cells reinfused/kg was 5.8 x 106 (1.42–50.4) in arm A and 7.4x106 (1.85–99.2) in arm B. The median time to neutrophil engraftment (ANC >0.5x109/l) was 10 days (range 8–14) in arm A and 10 days (range 8–21) in arm B. The median time to platelet engraftment (plts>20 x 109/l) was 11 days (range 5–26) in arm A and 9 days (range 5–42) in arm B (p=0.005). One patient died before platelets engraftment in arm A. Twelve patients experienced at least one episode of serious infection (total episodes =17) before day 100 in arm A compared to 3 patients (total episodes = 6) in arm B (p=0.02). For 3 patients in arm A, these infections were fatal (including the patient who died before time to platelet engraftment). There was no significant difference in CR rate at 1 year as defined by EBMT/IBMTR/ABMTR criteria (27% in arm A and 20% in arm B). The 5 year overall survival (OS) was 51% in arm A and 45% in arm B (p=0.80). The 5 year event free survival was 20% in arm A and 18% in arm B (p=0.45). The increased incidence of severe infections in the CD34+ selected arm in the early post transplant may be due to impaired immune reconstitution. Thus, no long term clinical benefit is obtained by CD34+ selection despite significant tumor cell reduction.
Addition of Bortezomib (Velcade™) to High Dose Melphalan (Vel-Mel) as an Effective Conditioning Regimen with Autologous Stem Cell Support in Multiple Myeloma (MM).
Authors: Hollmig Klaus , Stover Julie , Talamo Giampalamo , Zangari Maurizio , Thertulien Raymond , van Rhee Frits , Fassas Athanasios , Anaissie Elias , Tricot Guido , Barlogie Bart
Published: October 2019

Velcade has shown promising activity alone and, more recently, in combination with other anti-myeloma agents (dexamethasone, thalidomide, doxil, standard dose melphalan) for relapsed or refractory multiple myeloma. We have now explored the efficacy and safety of Velcade™ 1.0 or 1.3mg/m2 administered on days -4 and -1 prior to Melphalan, which was given in two fractionated doses for a total of 100–250 mg/m2 on days -4 and -1 or as single dosing on day -1 supported on day 0 by PBSC infusion. Follow-up is now 1–5 months; detailed follow-up data will be available at presentation. Of 37 patients currently evaluable for toxicity, 27 are also evaluable for response. Patient characteristics are outlined in Table 1, depicting a high risk population.

The treatment was administered in the out-patient setting in 37 patients, 27 of whom were entirely managed through ambulatory care. Hospital admission was required for pneumonia in 4, sepsis in 3, nausea and vomiting in 2 and ileus in 1 patient. In 37 evaluable patients, hematopoetic recovery depended upon the timing of PBSC collection (prior to the first, second or the Vel-Mel transplant). Neutrophil recovery to > 1,000/μL occurred at a median of 13 days and platelet recovery to 50,000/μl at a median of 17 days. Non-hematologic toxicities included ≥ grade III mucositis in 5, diarrhea in 11, febrile neutropenia in 5, pneumonia/sepsis in 14, and fatigue in 22 patients. No fatal complications were seen.

Of 27 evaluable patients, partial response (≥ 75% of serum M protein reduction, ≥ 75% of urinary M excretion) was obtained in 9 (39%) including 6 (26%) who showed a complete disappearance of serum M and urine M. Bone marrow follow-up examinations were available in 17 patients and revealed a median reduction in plasmacytosis of 75% including 2 patients achieving a normal bone marrow plasmocytosis

Our results are promising and demonstrate that Vel can be safely added to Mel at doses as high as 250 mg/m2 in fractionated dosing with a high response rate also in high-risk, advanced disease. This approach will be formally evaluated in a randomized trial comparing tandem transplants with Mel 200 mg/m2 alone versus added Vel 1.0 mg/m2 on days 1 and 4 followed on each day by Mel 140 (total 280 mg/m2).

Velcade 1.0 mg/m² Velcade 1.3 mg/m² Parameter Mel 250 Mel 220–240 Mel 200 Mel 100–150 Mel 250 Mel 200 Mel 100–150 N 4 4 12 10 4 2 1 % AGE ≥ 65 0 25 33 30 0 0 100 %Abn Cytogen 75 0 8 30 50 50 0 % Prior Autotx 75 25 33 40 75 0 100 % Prior VTD 50 25 25 30 75 50 100
Gender as a Prognostic Factor in CLL. Biological Pointers to the Improved Outcome of Women.
Authors: Catovsky Daniel , Matutes Estella , Morilla Alison , Burford Anna , Brito-Babapulle Vasantha , Del-Giudice Ilaria , Orchard Jennifer , Oscier David G. , Halsey Jim , Richards Sue
Published: October 2019

Gender is not widely regarded as a prognostic indicator in CLL. However, the combined data from three MRC randomised trials, CLL1, 2 and 3, and two observational studies for patients with Binet stage A, CLL2A and 3A, over a period of 20 years (1978-1998) totalling 2370 patients, showed a significant survival advantage for women (2p<0.0001). Cox regression analysis of the three randomised trials showed that gender, age, stage and response to therapy were independent prognostic variables. The response to treatment for women was also better than for men receiving the same therapies. The LRF CLL4 randomised trial, which started in 1999 shows the same trend. Preliminary results in 444 patients Binet stages A progressive, B and C showed the same CR/Nod.PR for both sexes (43.5%) but a higher PR rate in women (45%) vs men (36.5%) and a lower proportion of women non-responders to first line therapy (11.5% vs 20%). A number of laboratory investigations in CLL4, which included FISH analysis with five probes, VH mutational status, CD38 and ZAP-70 expression by flow cytometry, showed differences between the sexes, which were significant for 17p and 11q deletions combined and CD38, always in favour of women, as shown in Tables 1 and 2.

The clinical and laboratory results suggest that CLL is biologically more benign in women. Women have a lower incidence of CLL, an overall higher incidence of stage A (41.7%) than men (27.3%) in CLL 1, 2, 2A, 3 and 3A and respond better to treatment in all the trials. These differences may be underlined by a higher proportion of 13q del as sole abnormality, a lower proportion of 17p (p53 locus) and 11q deletions and lower levels of CD38. Data on VH mutations and ZAP-70 point in the same direction but the number of cases studied is still small. An additional factor that may play a role in the better outcome for women relates to the effect of oestrogen derivatives which are known to target selectively superoxide dismutase and induce cell kill (Huang et al, Nature407, 390, 2000).

Table 1: FISH analysis by gender (Dohner hierarchical model)

Abnormality 17p del 11q del Trisomy 12 13q del Others * = Combined p value < 0.05 (Chi-Square test) Men (286) 12% 19% 10% 34% 25% Women (94) 7% 13% 11% 44% 25% p value * * 0.052

Table 2: Other biological markers

CD38 negative (<30%) VH mutated ZAP-70 negative Men 173/335 (52% ) 51/149 (34%) 94/192 (49%) Women 68/100 (68%) 23/52 (44%) 42/68 (62 %) p value <0.005 NS 0.07
IV Busulfan (Bu) with Fludarabine (Flu) or Cyclophosphamide (Cy) - Comparing Ablative Conditioning Regimens for Allogeneic Transplantation in AML/MDS.
Authors: de Lima Marcos , Couriel Daniel , Shahjahan Munir , Alamo Jorge , Thall Peter F. , Russell James , Anderlini Paolo , Giralt Sergio , Shpall Elizabeth J. , Jones Roy B. , Champlin Richard E. , Andersson Borje S.
Published: October 2019

As part of our evaluation of Flu as immunosuppressive (and DNA damage repair inhibiting) agent, and as an alternative to Cy in combination with IV Bu, preparing AML/MDS patients for allogeneic transplant (AlloSCT), we postulated that the IV Bu-Flu regimen would yield improved safety, yet increased antileukemic efficacy. We now compared two consecutive series of patients receiving pretransplant conditioning therapy with either IV Bu-Flu or IV BuCy2.

Methods: Flu 40 mg/m2 IV was given once daily, each dose followed immediately by Bu 130 mg/m2 IV over 3 hr (days −6 till −3) (n=128 patients), and in the IV BuCy2 the Bu was given at 0.8 mg/kg over 2 hr every 6 hr for 16 doses (days −7 till −4), followed by Cy 60 mg/kg on days −3 and −2 (n=73 patients). Patients with an unrelated (MUD) or mismatched donor received ATG on days −3 to −1. Graft-versus-host disease (GVHD) prophylaxis was based on tacrolimus and mini-methotrexate.

Patients: Median age was 46 (19–66) (IV Bu-Flu group) and 39 (13–64) (IV BuCy2 group) years, respectively. At transplant 45% and 46% patients were in CR1–3, the remainder had active disease. Cytogenetic risk categories were equivalent for the two groups, with poor or intermediate risk in 88 % in the IV Bu-Flu, and in 82 % of the BuCy2group. Donors were HLA-identical siblings (SIBS) in 53 %, and 73%, MUDs in 39% and 15 %, others in 8 % and 12% respectively. Median follow-up for patients still alive (August 2004) was 15 mos (3–37; n=78) for the IV Bu-Flu group, and 45 mos (24–76; n=27)) for the IV BuCy2 patients.

Results: Both regimens were well tolerated; 100-day treatment-related mortality (TRM) was for SIB/MUD 2.7 / 2% after IV Bu-Flu, and 7 / 9% in the IV BuCy2 group, respectively. Overall, one-year TRM was for 7% after IV Bu-Flu and 18% after IV BuCy2. The overall aGvHD rates were 42% and 45%. 2-year overall survival (OS) were 50% for the IV Bu-Flu group, 42% after IV BuCy2. For patients transplanted in CR the 2-yr OS and event-free survival were 69% and 68 % after IV Bu-Flu, and 48% and 41% in the IV BuCy2.

We conclude, that IV Bu-Flu is a safe and highly effective, reduced toxicity myeloablative conditioning regimen for AML/MDS. Its antileukemic efficacy appears to be at least equal to what is experienced with the IV BuCy2 regimen. Thus, our data demonstrate the IV Bu-Flu regimen to be a new standard for myeloablative conditioning therapy for alloSCT in AML/MDS.

Figure Figure
Impact of Acute GVHD on Immune Reconstitution after Cord Blood Transplantation in Adult.
Authors: Nagamura-Inoue Tokiko , Takahashi Satoshi , Ooi Jun , Tomonari Akira , Iseki Toru , Uchimaru Kaoru , Nagamura Fumitaka , Ohno Nobuhiro , Takasugi Kashiya , Futami Munechika , Uchiyama Michihiro , Soda Yasushi , Konuma Taka-aki , Nakayama Shin , Shioya Mika , Watanabe Nobukazu , Asano Shigetaka , Tojo Arinobu , Takahashi Tsuneo A.
Published: October 2019

BACKGROUND: Immune reconstitution following unrelated cord blood transplantation (UCBT) in adult patients is of great concern because of immaturity of cord blood immunological cells.

STUDY DESIGN AND METHODS: Twenty-six adult patients (15 to 58 year-old) with hematological malignancies, who underwent UCBT and sustained engraftment were enrolled in this study. Infused number of immunological cells in thawed CB units including T cells (CD3+), B cells (CD19+), NK cells (CD3-CD56+), monocytes (CD14+) and also CD34 + cells was analysed using bead-contained TRUCOUNT tube (BD, CA). Dead cells after thawing were excluded by gating out with 7AAD dye. Immune reconstitution was analysed every 30 days by 120 days after CBT. Four-colour FACS Caliber and TRUCOUNT tube were utilized to calculate the absolute number of immune cells concentration in blood after UCBT. We put strict volume of 50μl fresh unmanipulated blood in each TRUCOUNT tube.

RESULTS: Thawed-transplanted NC 2.3±x107/kg, CD34 was 0.72±0.3x105/kg (4.1x106 total), T cells; 3.1±1.6x106/kg with CD4/8 ratio of 3.2±2.0, B cells; 1.2±0.5x106/kg, NK cells; 1.0±0.5x106/kg and monocytes; 1.6±0.6x106/kg. There were no correlations between infused CD34+ cells number and T, B, NK and monocytes numbers. Monocytes increased in blood rapidly after CBT at 30 days, then, declined to the normal value. NK cells was recovered in the early after CBT and then did not so change in number from 30 to 120 days after CBT, while T cells increased time dependent manner, and B cells appeared late but influenced by acute GVHD grade. Within 120 days after CBT, T cells showed also CD4+dominant in most cases with relatively high CD25+CD4+ regulatory T (rT) cells compared to normal control. The patients with grade II to IV aGVHD showed significantly higher number of rT cells on 30 days (P<0.05) compared to those with grade 0–I aGVHD. On day 30, the number of rT cells showed 7.7±5.9/μl in grade 0–I aGVHD and 19.4±13.3/ μl in grade II–IV. The patients with grade II to IV aGVHD showed significant delayed recovery of B cells on 90 days after CBT compared to those with 0–I aGVHD (P<0.001).

CONCLUSION: aGVHD in adult patients may influence on the number of regulatory T cells in the early period after UCBT and delayed recovery of B cells.