bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapamycin -- a Potential Mechanistically Targeted Therapeutic for Juvenile Myelomonocytic Leukemia.
Authors: Liu Y. Lucy , Castleberry Robert P. , Emanuel Peter Dean
Published: April 2020

Juvenile myelomonocytic leukemia (JMML) is a mixed myelodysplastic /myeloproliferative disorder (MDS/MPD) of infancy and early childhood. It is characterized by monocytosis, leukocytosis, elevated fetal hemoglobin, hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF), a low percentage of myeloblasts in the bone marrow, and absence of the Philadelphia chromosome or the BCR/ABL fusion gene. The pathogenesis of JMML has been clearly and definitively linked to dysregulated signal transduction through the RAS signaling pathway. A series of studies conducted over the last decade have shown that mutations or other abnormalities in RAS, NF1, and PTPN11, are potentially responsible for the pathogenesis of JMML in up to 75% of cases. Treatment has been very difficult. There is no effective therapy for JMML. Only allogeneic stem cell transplantation (SCT) can extend survival. However, the relapse rate from allogeneic SCT is inordinately high in JMML (28–55%), with 5-year disease-free survival rates of 25-40%. Rapamycin is a macrolide antibiotic with established clinical applications in organ transplantation. Recent studies have proved that the Mammalian Target of Rapamycin (mTOR) plays an important role in cytokine receptor signaling and induction of apoptosis. Numerous studies have suggested that mTOR functions as a nutritional checkpoint and is connected to energy sensing through AMP-dependent kinase (AMPK) which senses the AMP: ATP ratio in cells. Its function is regulated by the RAS/PI3-kinase pathway. In searching for novel mechanistically-targeted reagents to treat JMML, we conducted an in vitro pilot study with JMML cells. The CFU-GM formation assay was used to test the therapeutic sensitivity of rapamycin to JMML cells. Mononuclear cells (MNCs) from peripheral blood of 9 JMML patients were collected and plated on 0.3% agar medium with rapamycin at a concentration of 1-8nM(0.91-7.28μg/L) and carrier (DMSO). Greater than 50% inhibition of spontaneous CFU-GM growth was observed in all cultures in a dose-dependent fashion, with the exception of one patient sample which had colonies resistant to rapamycin. The effective concentrations in our cultures are equivalent to the safe and tolerable whole blood concentrations achieved in organ transplant patients in clinical settings (5-30μg/L). Our data suggests that rapamycin may be considered as a potentially safe and effective reagent to treat JMML, but that in vitro sensitivity testing might be recommended since one patient sample demonstrated complete resistance to rapamycin in vitro. Further studies are ongoing to explore the mechanism of rapamycin in inhibiting hypersensitivity of JMML cells to GM-CSF.
The Macrophage αMβ2 Integrin αM-Lectin Domain Mediates the Phagocytosis of Chilled Platelets.
Authors: Josefsson Emma C. , Gebhard Harry H. , Stossel Thomas P. , Hartwig John H. , Hoffmeister Karin M.
Published: April 2020

αMβ2 integrin receptors on myeloid cells mediate the adhesion or uptake of diverse ligands, and particular sequences on this integrin’s alpha subunit are responsible for recognition specificity. Inhibition of neutrophil adhesion to platelet monolayers by αM-derived peptides has implicated an αM sequence (I-domain) that also binds iC3b, fibrinogen, ICAM-1 and other ligands. However, αMβ2 also recognizes βN-acetylglucosamine (βGlcNAc) residues on the platelet von Willebrand factor receptor (GPIbα) that are clustered after cooling, suggesting that the αM lectin domain is important for this recognition. The phagocytosis of chilled platelets can be reconstituted when CHO cells are transfected with αMβ2. Removal of the N-T portion of GPIbα, containing the exposed βGlcNAc residues, from the chilled platelet surface using the snake venom mocarhagin inhibits this phagocytosis, as does the addition of 1 mM β-methyl-N-acetyl-D-glucosamine. Replacement of the I domain, or the lectin domain of the αM-chain with the corresponding domain from the αx-chain (p150), reveals that the activity of the αMβ2 integrin toward chilled platelets resides within the lectin-domain, and does not require the I domain. Other evidences for this conclusion include: (1) Sf9 cells expressing αM lectin domain but not β2 bind 3-4 x more chilled platelets than control cells; βGlcNAc inhibits this binding. (2) Soluble recombinant αM lectin domain inhibits the phagocytosis of chilled platelets by αMβ2-expressing THP-1 cells while I-domain substrates (fibrinogen or LPM19c), recombinant I-domain protein, or a synthetic peptide that inhibits neutrophil adhesion to platelet monolayers, are without inhibitory effect. We conclude that chilled platelets are removed from blood by an interaction between βGlcNAc residues on clustered GPIbα and the lectin domain of αM-chain of the αMβ2 integrin. Since the phagocytosis and clearance of chilled platelets by hepatic macrophages has precluded refrigeration of platelets procured for transfusion, a more precise molecular definition of the recognition mechanism could inform strategies for its inhibition, thereby accommodating cold platelet storage.
Molecular Characterization of PS-341 (bortezomib) Resistance: Implications for Overcoming Resistance Using Lysophosphatidic Acid Acyltransferase (LPAAT)-β Inhibitors.
Authors: Hideshima Teru , Chauhan Dharminder , Ishitsuka Kenji , Raje Noopur , Kumar Shaji , Podar Klaus , Mitsiades Constantine , Hideshima Hiromasa , Bonham Lynn , Munshi Nikhil C. , Richardson Paul G. , Singer Jack W. , Anderson Kenneth C.
Published: April 2020

PS-341 (bortezomib, Velcade™) is a promising novel agent for treatment of advanced multiple myeloma (MM); however, 65% of patients with relapsed refractory disease in a phase II study did not respond. Lysophosphatidic acid (LPA) is a phospholipid which mediates tumor cell migration and invasion. Recent studies have shown that inhibition of LPAAT-β inhibits both Ras/Raf/Erk and PI3K/Akt signaling cascades. We have previously shown that lysophosphatidic acid acyltransferase (LPAAT)-β inhibitor CT-32615 triggers caspase-dependent apoptosis, and can overcome resistance to conventional therapeutics (ie, dexamethasone, doxorubicin, melphalan) in MM cells. In this study, we determined whether CT-32615 could also overcome resistance to PS-341. We first characterized molecular mechanisms of resistance to PS-341 in DHL-4 lymphoma cells. DHL-4 cells express low levels of caspase-3 and caspase-8; furthermore, no cleavage in caspase-8, caspase-9, caspase-3, poly ADP-ribose polymerase (PARP), or DNA fragmentation factor (DFF) 45 is triggered by PS-341 treatment. We have previously shown that PS-341 treatment triggers phosphorylation of c-Jun NH2-terminal kinase (JNK), which subsequently induces caspase-dependent apoptosis; conversely, JNK inhibition blocks PS-341-induced apoptosis. Here we show that PS-341 does not induce phosphorylation of SEK-1, JNK, and c-Jun in DHL-4 cells, suggesting that it does not trigger a stress response. Importantly, CT-32615 inhibits growth of DHL-4 cells in a time- and dose-dependent fashion: a transient G2/M cell cycle arrest induced by CT-32615 is mediated via downregulation of cdc25c and cdc2. CT-32615 triggers swelling and cell membrane destruction in DHL-4 cells, without caspase/PARP cleavage or TUNEL-positivity, suggesting a necrotic response. Our studies therefore demonstrate that LPAAT-β inhibitor CT-32615 triggers necrosis even in PS-341-resistant DHL-4 cells, providing the framework for its evaluation to overcome clinical PS-341 resistance and improve patient outcome.
A Phase II Trial of Doxil, Vincristine and Reduced Schedule Dexamethasone (DVd) for Patients with Relapsed or Refractory Multiple Myeloma.
Authors: Masci Paul , Karam Mary A. , Platt Luba , Andresen Steven , Lichtin Alan , McGowan Bridget , Hussein Mohamad
Published: April 2020

Patients with newly diagnosed multiple myeloma (MM) typically have responses to initial cytotoxic or steroid based therapy. Disease relapse occurs in all patients. As high as 90% of patients with relapsed or refractory disease will have over-expression of the multi-drug resistance (MDR) gene. Pharmacokinetic data suggest that prolonged exposure to high concentrations of doxorubicin can overcome MDR. Pegylated liposomal doxorubicin can theoretically achieve this goal as the angiogenic activity of the MM bone marrow is significantly increased. We proceeded with a phase II trial to evaluate the response rate of patients with relapsed or refractory MM (R/R-MM) to the DVd regimen. Eligible patients had clinically active R/R-MM following at least one prior cytotoxic based treatment regimen. Patients received intravenous (IV) pegylated liposomal doxorubicin 40 mg/m2 day 1, vincristine 2 mg day 1 and oral or IV dexamethasone 40 mg daily days 1–4. Cycles were repeated every 28 days for a minimum of 6 cycles and 2 cycles after best response. Myeloma parameters were measured at the start of each cycle. SWOG criteria were used to determine response. Thirty-five patients (21 male and 14 female) with R/R-MM clinically active disease were enrolled. Median age was 59 years (range 43–87). Patients received a median of 2 (range 1–4) prior cytotoxic based treatments. All patients received at least one cycle of treatment (median=5; range 1–12) and were evaluable for response. Ten (29%) patients responded to therapy; 5 partial responses (PR > 50%) and 5 responses (R > 75%) were observed after a median of 2 cycles (range 1–9). Median progression free survival of responding patients (PR + R) was 4.5 mos. (range 0.67–44.8). Patients achieving R had a median progression free survival of 32.5 mos. (3.0–44.8). Thirteen (37%) patients had stable disease (SD) for a median of 1.4 mos. (range 0.8–9.9). Twelve (34%) patients had progressive disease after a median of 1 cycle (range 1–5). The most common toxicities were hematologic; there were four occurrences of febrile neutropenia. Three patients experienced grade 3 constipation and one grade 3 palmar-plantar erythrodysethesia was observed. This study suggests that in patients with R/R-MM, DVd alone yields response rates similar to bortezomib with patients achieving an R experiencing a durable plateau phase. Ongoing studies of DVd in combination with thalidomide or CC-5013 in patients with R/R-MM have resulted in higher and better quality response rates (comparable to autologous SCT) translating to a durable progression free survival. We would not recommend the DVd regimen in patients with R/R-MM without the addition of an immune modulator such as thalidomide.
Dysregulated CD4+CD25+ T- Regulatory Cells and TLRs in Myeloma.
Authors: Prabhala Rao , Neri Paola , Tassone Pierfrancesco , Shammas Masood A. , Bae Jooeun , Cumming Virginia M. , Allam Charles K. , Daley John F. , Desarneud Frank , Chauhan Dharminder , Blanchard Elizabeth , Anderson Kenneth , Munshi Nikhil C.
Published: April 2020

Although current vaccine strategies have achieved anti-myeloma immune responses, meaningful clinical responses have not been observed. This may be related with the significant immune dysfunction observed in myeloma that may interfere with the development of effective anti-myeloma immune responses. The initiation of immune response is controlled by CD4+CD25+ T regulatory (Treg) cells, which can suppress anti-tumor immune responses. This property of Treg cells to modulate anti-tumor immune responses may function as a barrier to cancer immunotherapy. In order to overcome such suppression, signaling through Toll-like receptors (TLRs) can induce adjuvant effect by increasing local production of chemokines, and pro-inflammatory cytokines, and by enhancing antigen-presentation by APCs. Here, we have evaluated the role of Treg cells and the effects of TLRs in myeloma. We observed significant increase in CD4+CD25+ Treg cells in MM patient samples compared to normal donors (23±4% vs 6±3%). Proliferation of T cells depleted of Treg cells with anti-CD3 antibody was significantly lower in MGUS (n=9, SI=12±2) and MM (n=9, SI=28±8) compared with normal donors (n=9, SI=74±9, p<0.01). As expected addition of Treg cells was able to suppress T cell proliferation in normal donors (↓31%), however, their effects were less suppressive in MGUS (↓26%), and in fact, they increased T cell proliferation in MM (↑29%). Additionally, LPS which functions through Toll-like receptor 4 (TLR4), is able to overcome suppression of T cell proliferation by Treg cells in normal controls (110%), however, it had significantly limited influence in MGUS (49%) and MM (24%). Expression profile of myeloma cells (n=15) showed up-regulation of TLR4 (7.4 folds) and its related gene MD2 (2.5 folds) in all the MM cells tested compared to normal plasma cells indicating a possible modulation of immune responses by myeloma cells through TLR4 or its soluble agent. This is the first demonstration of dysregulated Treg cells and TLRs in myeloma. Understanding of the molecular basis of Treg cell dysfunction is underway with a view to eventually target these mechanisms to improve immuno-therapeutic strategies in myeloma.
Human Lymphoid and Myeloid Cell Development in NOD/LtSz-scid IL2rγnull Mice Engrafted with Mobilized Human Hematopoietic Stem Cells.
Authors: Shultz Leonard , Lyons Bonnie L. , Burzenski Lisa M. , Gott Bruce , Chen X. , Chaleff Stanley , Kotb Malak , Gillies Stephen D. , Greiner Dale L. , Handgretinger Rupert
Published: April 2020

We have developed, characterized, and validated a new genetic stock of IL-2r common γ (gamma) chain deficient NOD/LtSz-scid (NOD-scid IL2rγnull) mice that support high levels of human hematopoietic stem cell (HSC) engraftment and multilineage differentiation. Histology, flow cytometry, and functional assays document a severe depletion of lymphocytes and NK cells in NOD-scid IL2rγnull mice. These mice survive beyond 16 months of age and untreated as well as sub-lethally irradiated NOD-scid IL2rγnull mice are resistant to the development of lymphomas and are “non-leaky” throughout life. Intravenous injection of sub-lethally irradiated NOD-scid IL2rγnull mice with 7 x 105 human mobilized CD34+ stem cells leads to high levels of multilineage engraftment. At 10 weeks after engraftment, percentages of human hematopoietic CD45+ cells are six-fold higher in the bone marrow of NOD-scid IL2rγnull mice as compared to NOD-scid controls. Human CD45+ cells include immature and mature B cells, NK cells, myeloid cells, plasmacytoid dendritic cells and HSCs. Spleens from engrafted NOD-scid IL2rγnull mice contain high percentages of immature and mature B cells but low percentages of T cells. Treatment with human Fc-IL7 fusion protein leads to a high percentage of human CD4+CD8+ immature thymocytes and high percentages of CD4+CD8− and CD4−CD8+ mature human T cells in the spleen and blood. Validation of de novo human T cell development was carried out by quantifying T cell receptor excision circles in thymocytes and by analyses of TCRβ repertoire diversity. Human T cell function was evidenced by proliferative responses to PHA and streptococcal superantigen. NOD-scid IL2rγnull mice engrafted with human HSC generate differentiated functional human T and B cells and provide an in vivo model of multilineage human hematopoietic cell engraftment.
Bendamustine/Mitoxantrone/Rituximab (BMR): A Very Effective and Well Tolerated Immuno-Chemotherapy for Relapsed and Refractory Indolent Lymphomas - Results of a Multicentre Phase-II-Study of the German Low Grade Lymphoma Study Group (GLSG).
Authors: Weide Rudolf , Koeppler Hubert , Heymanns Jochen , Thomalla Joerg , Hess Georg , Gamm Heinhold , Aldaoud Ali , Schmitz Stephan , Haak Ursula , Plewe Dirk , Burkhard Oswald , Gaede Bernd , Hinrichs Fokke , Pflueger Karl-Heinz , Dreyling Martin , Unterhalt Michael , Hiddemann Wolfgang
Published: April 2020

In a previous phase-I-study we could demonstrate that the combination of the two chemotherapeutic agents bendamustine and mitoxantrone in combination with the lymphocyte-specific antibody rituximab (BMR) is a highly effective regimen in the treatment of relapsed or refractory indolent lymphomas and CLL (Leukemia and Lymphoma 2002, 43(2):327–331). Based on these data we have conducted a multicenter phase-II-study to further evaluate the efficacy and toxicity of BMR. BMR treatment schedule consisted of Bendamustine 90mg/m2 (days 1+2), Mitoxantrone 10mg/m2 (day 1) and Rituximab 375mg/m2 (day 8). Treatment was repeated on day 29 for a total of 4 cycles. Between 04/03 and 07/04, 39 patients (pts) with symptomatic stage III/IV indolent lymphomas were treated with BMR (18 follicular, 11 mantle cell, 3 marginal zone, 6 immunocytoma and 1 patient with hairy cell leukemia). All pts were treated on an outpatient base. Median age was 67 years (40 – 83) and their performance score ranged from 0–2. Median number of previous treatment regimens was 1 (1–8). 16 pts (41%) had rituximab pretreatment. Currently, 25 pts are evaluable: A reversible grade 3/4 hematotoxicity occurred in 19 pts (76%), no treatment related death was reported. The overall response rate was 92% with 36% complete remissions and 56% partial remissions. Updated results on progression-free and overall survival rates will be presented. We conclude that BMR is a very effective and well tolerated immuno-chemotherapy for relapsed or refractory indolent lymphomas.
Blockade of p110delta Isoform Activity of Phosphoinositide 3-Kinase Inhibits Blast Cell Proliferation in Acute Myeloblastic Leukemia.
Authors: Bouscary D. , Bardet V. , Sujobert P. , Cornillet-Lefebvre P. , Hayflick J. S. , Prie N. , Verdier F. , Vanhaesebroeck B. , Muller O. , Pesce F. , Ifrah N. , Berger M. Hunault , Bertho C. , Guillerm G. , Villemagne B. , Audhui B. , Solary E. , Witz F. , Harousseau J. L. , Himberlin C. , Lamy T. , Lioure B. , Bene M. C. , Cahn J. Y. , Dreyfus F. , Mayeux P. , Lacombe C.
Published: April 2020

AMLs constitute an heterogeneous group of hematopoietic stem cell disorders of rather poor prognosis. Abnormal activation of signaling pathways including STAT, MAPK and NF-KB have been described in AMLs and could constitute new targets for therapy. In this study, we focused on the PI3K/AKT and mTOR/P70S6K activity in primary blast cells purified from the bone marrow of patients with primary (n = 54) or secondary (n =10) AMLs. Patients with de novo AMLs were issued from the AML2001 French Multicenter Trial directed by the GOELAMS. The FAB classification was the following: M0 (n = 8), M1 (n = 10) M2 (n = 21), M4 (n = 10), M4eo (n = 2), M5 (n = 11), unknown AML (n = 2). In 58% of these patients, a constitutive activation of PI3K/AKT was detected in blast cells by western blot analysis showing AKT and FOXO3a phosphorylation, and confirmed by immunofluorescence confocal microscopy and flow cytometry analysis in CD34+ blast cells. Constitutive GAB1/2 phosphorylation was detected in all PI3K+ patients. However, we did not find any correlation between PI3K activation and FLT3 gene mutations. PTEN and SHIP-1 expression was normal in all tested PI3K+ patients. Proliferation (3H thymidine incorporation) was significantly increased in PI3K+ samples compared to PI3K- samples (p=0.001). The mTOR/P70S6K pathway was activated and the mTOR inhibitor rapamycin selectively reduced proliferation of PI3K+ samples. We determined the expression of the alpha, beta and delta isoforms of the catalytic sub-unit of PI3K (p110) in leukemic cells and found that p110 delta was the only consistently expressed isoform. In 8 PI3K+ samples tested, IC87114 (ICOS Corporation), a specific p110delta inhibitor compound used at 10μM totally suppressed AKT and FOXO3a phosphorylation. IC87114 inhibited proliferation of PI3K+ leukemic cells whereas it did not induce apoptosis. IC87114 did not significantly impaired the proliferation of PI3K- blast cells. Interestingly, IC87114 did not inhibit the proliferation or clonogenicity of CD34+ cord blood cells cultured in SCF, FLT3L and TPO used as controls.

Overall, our results report, a/ a constitutive activation of PI3K in 58% of AML, b/ no correlation between FLT3 gene mutations and PI3K positivity, c/ no implication of PTEN or SHIP-1 phosphatases in PI3K activation, d/ a major role of the p110 delta isoform of PI3K in leukemic cell proliferation, e/ a potential therapeutic value of inhibiting mTOR and P110 delta activity.
EB10, an Anti-FLT3 Monoclonal Antibody, Prolongs Survival and Reduces NOD/SCID Engraftment of AML Cell Lines and Primary Blasts.
Authors: Piloto Obdulio , Levis Mark , Li Li , Nguyen Bao , Kim Kyu-Tae , Huso David , Li Yiwen , Li Hongli , Wang Mei-Nai , Witte Larry , Hicklin Daniel J. , Small Donald
Published: April 2020

The FLT3 receptor is a potential target in AML due to its role in leukemogenesis and its high degree of expression on blasts from approximately 90% of acute myeloid leukemia (AML) patients. In addition, mutant forms of FLT3, including internal tandem duplications (ITD) in the juxtamembrane region and point mutations in the kinase domain, constitutively activate FLT3 signaling. ITD mutations in particular are also associated with poor prognosis. A number of small molecule tyrosine kinase inhibitors (TKI) against FLT3 are currently in clinical trials and have shown some clinical activity. However, TKIs have various limitations, including their lack of specificity, which may produce toxicities, and can select for drug resistant cells. In an attempt to overcome some of these limitations and to generate new agents which might cooperate in targeting FLT3, we generated a fully humanized phage display monoclonal antibody (EB10). This antibody is capable of inhibiting both ligand-activated wild-type and, to a lesser degree, ligand-independent mutant FLT3 signaling. When EB10 is used to treat cells expressing activated FLT3, inhibition of downstream pathways including STAT5, AKT and MAPK are also frequently seen. EB10 treatment of cells expressing FLT3 in the presence of NK cells leads to antibody-dependent cell-mediated cytotoxicity (ADCC). EB10 treatment of NOD/SCID mice injected with FLT3 expressing AML cell lines or with primary AML blasts significantly prolongs survival and/or reduces engraftment of leukemic cells. EB10 proved efficacious in vivo against cells even when in vitro EB10 treatment did not significantly reduce FLT3 signaling. This indicates that ADCC may be the primary mechanism mediating cytotoxicity as opposed to direct FLT3 inhibition. In contrast to the effects on AML cell lines and primary samples, EB10 treatment did not significantly reduce NOD/SCID engraftment of normal human CD34+ hematopoietic stem cells. Anti-FLT3 antibodies, like EB10, may be a promising therapeutic agent that can specifically target malignant cells with limited toxicities against normal hematopoietic stem cells and should be considered for clinical trials.
Reduced Plasma Fibrinolytic Potential Is a Risk Factor for Venous Thrombosis.
Authors: Lisman Ton , de Groot Philip G. , Adelmeijer Jelle , Meijers Joost C.M. , Rosendaal Frits R.
Published: April 2020

The role of the fibrinolytic system in the development of deep vein thrombosis (DVT) is unclear. We determined the plasma fibrinolytic potential of patients enrolled in the Leiden Thrombophilia Study (LETS), a population-based case-control study on risk factors for DVT. Plasma fibrinolytic potential was determined in 421 patients and 469 controls who were not using oral anticoagulants at the time of the blood draw by means of a tissue factor- and tissue-type plasminogen activator (tPA)-induced clot lysis assay. This assay represents an overall measure for plasma fibrinolytic potential, as it was previously shown to be dependent on plasma levels of plasminogen, α2-antiplasmin, plasminogen activator inhibitor type 1, and thrombin activatable fibrinolysis inhibitor. Using clot lysis times above the 70th, 80th, 90th, 95th, and 99th percentile of the values found in control subjects as cut-off levels, we found a dose-dependent increase in risk for DVT in patients with hypofibrinolysis (odds ratio’s [95 % CI] of 1.4 [1.1–1.8], 1.6 [1.2–2.1], 1.9 [1.3–2.9], 2.1 [1.3–3.6], 2.2 [0.7–7.5], respectively). This indicates a two-fold increased risk of DVT in subjects with clot lysis times above the 90th percentile. Using the 90th percentile, we showed that the risk was not affected by age or sex (odds ratio [95% CI] 2.0 [1.3–3.0]), and after correction for all possible confounders (age, sex, and levels of procoagulant proteins which were shown to associate with clot lysis times in the control population) the risk estimate was marginally reduced (odds ratio [95% CI] 1.6 [1.0–2.5]). Taken together, these results indicate that plasma hypofibrinolysis constitutes a risk factor for venous thrombosis. Whether this hypofibrinolytic state is determined by genetic or acquired factors, or a combination of these, and which proteins are involved is at present unknown.
Economic Evaluation of Bortezomib (VELCADE) for Relapsed and Refractory Multiple Myeloma.
Authors: Bagust Adrian , Haycox Alan R. , Boland Angela , Chu Patrick , Morgan Gareth J. , Mujica Mota Ruben E. , Dubois Dominique , Dhawan Ravinder
Published: April 2020

Bortezomib (VELCADE), a new proteasome inhibitor, was recently licensed by the FDA for treating patients with relapsed and refractory multiple myeloma (MM), and has now also been approved for similar use in Europe. A model was developed to evaluate and compare the costs and health benefits of bortezomib, as a third-line treatment for patients with relapsed and refractory MM, relative to best supportive care in the UK. A two-part mathematical model of survival was built and calibrated using individual patient data from the SUMMIT trial (n=202 ; NEJM2003;348:2609-17) , a multi-center, pivotal phase II trial of bortezomib in 202 patients with relapsed and refractory MM. Patients in SUMMIT were 18 years of age or older, with a life expectancy of more than three months and a Karnofsky score >=60. In the first part of the survival model, the time to disease progression for patients on bortezomib (n=202) or best supportive care (n=38) was estimated. In the second part, the time from disease progression till death was calculated, based on the sub-sample of patients for whom relevant data were available (n=79). Health-related utility was assessed for the effects of the drug and also the natural course of the disease on quality of life, yielding different utility weights pre- and post-disease progression. Those values were derived by mapping individual patient reports of quality of life in SUMMIT (EORTC-QLQ30 and MY24, FACIT-Fatigue and FACT/GOG-Neurotoxicity subscales) onto the 5 scales of the EQ-5D instrument. Resource use data from SUMMIT were used to estimate costs from the perspective of the UK NHS for bortezomib administration, hospital care, medications and diagnostic tests and surgical procedures on an individual patient basis. Results: By delaying the rate at which disease progresses, bortezomib produced survival gains relative to best supportive care that range between 7.75 to 12.09 months of life depending on the method of survival estimation. Additional total direct costs associated with the novel agent were £17290 or £24121 if the additional costs incurred during the extended period of survival are included. The incremental cost-effectiveness ratio for bortezomib was in the range £17161- £33539 per life year gained and £26714 - £51,666 per quality-adjusted life year gained. Conclusions: These results suggest that bortezmib is a cost-effective third-line treatment option for patients with RRMM in the UK. This study provides a methodological framework for undertaking preliminary economic evaluations of new drugs for the treatment of cancer with the aim of informing purchasing decisions based on limited outcome data.
Non-Hematopoietic Bone Marrow Cells Can Be Mobilized and Differentiate into Cardiomyocytes after Myocardial Infarction: Possible Contribution of Mesenchymal Stem Cells.
Authors: Kawada Hiroshi , Fujita Jun , Kinjo Kentaro , Matsuzaki Yumi , Tsuma Mitsuyo , Miyatake Hiroko , Muguruma Yukari , Okano Hideyuki , Hotta Tomomitsu , Fukuda Keiichi , Ando Kiyoshi
Published: April 2020

Recent studies have suggested that bone marrow (BM) cells can contribute to regeneration processes in various tissues. Cardiomyocytes derived from BM cells have been observed after myocardial infarction (MI), and BM-derived cells mobilized by cytokines were capable of regenerating the myocardial tissue, leading to an improvement in survival or cardiac function after MI. However, the responsible BM cells have not been fully identified. The most likely candidates for the BM-derived stem cells with the ability to regenerate myocardial tissue are hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC). The aim of this study was to determine the precise origin of the BM cells mobilized by cytokines to repair infarcted myocardium. Here, we used two independent clonal studies; we transplanted genetically marked single HSC or clonal MSC into lethally-irradiated recipient mice, induced MI, treated the mice with G-CSF, and analyzed the cardiac tissues. First, we transplanted single CD34−c-kit+Sca-1+lineage− side population (CD34−KSL-SP) cells or whole BM cells from mice ubiquitously expressing enhanced green fluorescent protein (EGFP) into lethally-irradiated mice, induced myocardial infarction (MI), and treated the animals with G-CSF to mobilize stem cells to the damaged myocardium. At 8 weeks post-MI, from 100 specimens we counted only 3 EGFP+actinin+ cells in myocardium of CD34−KSL-SP cells-transplanted mice, but more than 5,000 EGFP+actinin+ cells in whole BM cell-transplanted mice, suggesting that most of EGFP+actinin+ cells derived from non-hematopoietic BM cells. These results suggested that the major population of cells mobilized from the BM and active in the regeneration of cardiomyocytes was non-hematopoietic in origin. Next, clonally purified mesenchymal stem cells, CMG cells, transfected with a pMLC2v-EGFP plasmid encoding EGFP driven by the myosin light chain promoter were transplanted directly into BM of lethally-irradiated mice, MI was induced, and they were treated with G-CSF. As a result, a number of EGFP+actinin+ cells were observed in the ischemic myocardium, indicating that CMG cells had been mobilized and differentiated into cardiomyocytes. Together, these results suggested that the vast majority of BM-derived cardiomyocytes are of mesenchymal origin.
Thrombotic Events in Adults with Acute Lymphoblastic Leukemia.
Authors: Cserti Christine M. , Brandwein Joseph , Lipton Jeffrey H. , McLeod Anne G. , Messner Hans , Minden Mark D. , Schimmer Aaron D. , Schuh Andre C. , Seki Jack T. , Yeo Erik L. , Yi Qi-Long , Wells Richard A.
Published: April 2020

A strong link between asparaginase-containing (ase +) chemotherapy regimens and venous thrombotic events (VTE) has been established for pediatric acute lymphoblastic leukemia (ALL), but this association has not been studied in adult ALL. We recently adopted as our first-line therapy for adult ALL a treatment protocol (DFCI 00–01) that employs weekly high-dose ase throughout a 27 week intensification phase. We reviewed the records of 92 adult patients consecutively accrued at Princess Margaret Hospital (from Jan 2000 to Dec 2003) for venous thrombotic events (VTE). Median age at diagnosis of ALL was 41±17 y, or 19±3 y for those who presented after a history of pediatric ALL (n=6), and 42±16 y for adults who presented with de novo ALL (n=86). Thirty-five (37%) were female, 59 (63%) male. Twenty-four (26%) were Philadelphia-chromosome positive; 71% of these received regimens containing imatinib. At least 1 symptomatic, radiologically confirmed VTE was seen in 19 patients (21%), with 3 (3%) diagnosed prior to any antileukemic therapy. Overall survival (OS) versus thrombosis-free survival (TFS) is illustrated in the Kaplan-Meier curve below. Median time to VTE was 94±92 d [95% CI]. Average age of patients with VTE was 44 ±15y; no events were observed in patients with a prior pediatric history of ALL. The sites of VTE were: 10/22 (46%) in the lower extremities, 5/22 (23%) propagating from central venous access catheters (CVAC), 4/22 (18%) as pulmonary emboli (2 in isolation), and 3/22 (14%) CNS (2 cerebral venous sinus and 1 retinal venous thrombus). The first antileukemic regimen administered was DFCI 00–01 ± imatinib in 79/92 (86%), HyperCVAD ± imatinib in 7 (8%), an unclassified ase+ regimen in 1 (1%), or an unclassified asparaginase-excluding (ase−) regimen in 5 (5%). A significant difference in the rate of ase-dependent VTE was observed when all regimens (1° or subsequent) were counted, with 18 VTE during 103 ase+ regimens (17.5%), versus only 1 VTE in 32 ase− regimens (3.1%), [p=0.04]. Median time to VTE in DFCI 00-01was 109±98d, most often occurring during the intensification phase. The odds ratio for VTE with any T-cell subtype of ALL compared to any B-cell subtype was 4.6 [p=0.0043].

Conclusions : A high, significant VTE rate (~18%) was observed in adults with ALL undergoing ase+ antileukemic therapy, nearly 6X the rate observed either prior to treatment or during ase− regimens (~3%). Ase is thus strongly associated with VTE, warranting improved surveillance and antithrombotic prophylaxis. Risk factors (underlying thrombophilias, leukemic subtypes) similarly deserve further study.

Figure Figure
Survivin-Driven Therapy for Leukemia.
Authors: Caldas Hugo , Altura Rachel A.
Published: April 2020

Survivin, a member of the inhibitor of apoptosis (IAP) family of proteins, is widely expressed in transformed cell lines and in many different primary cancer cells, including both hematopoietic and non-hematopoietic malignancies. It is not expressed in many non-malignant adult tissues, but is essential for fetal development, as demonstrated by conventional gene-targeting experiments in mice that show embryonic lethality at day 4–6 of development. In adult cancers, including lymphoma and many epithelial carcinomas (colon, breast, gastric) the expression level of survivin, as assayed by immunohistochemical analysis and RT-PCR, correlates with overall survival.

We have designed a novel gene therapy approach that takes advantage of the high expression levels of survivin in malignant cells, in which the survivin promoter is used to drive the expression of a suicide gene to kill cancer cells by programmed cell death. Our system is based on perforin-independent granzyme B cytotoxicity and therefore does not require pro-drug activation. We designated it SAGA, for survivin and granzyme apoptosis. We used Jurkat cells as an in vitro model for T-cell leukemia, and 697Bcl2 cells as a model for pre-B Bcl2-expressing leukemia, to show this approach is more efficient in killing leukemic cells than conventional chemotherapy. Jurkat cells responded to both vincristine therapy and SAGA whereas 697Bcl2 were unaffected by vincristine, but responded to SAGA. Cell growth curves of Jurkat cells and 697Bcl2 cells are shown in Figure 1. Our approach not only inhibits cell growth, but also induces apoptosis. We detected apoptotic events by Annexin V staining and by changes in mitochondrial potential, as early as 12 hours post-treatment. Rates of early apoptotic events are shown in Table 1. In addition to these events, we also documented DNA fragmentation and caspase-3 activation in treated cells. Cytotoxicity was clearly visible by microscopic analysis 24 hours post-treatment (Figure 2). Our results strongly suggest that survivin-driven suicide gene therapy effectively enhances cell death of leukemic blasts derived from two common sub-types of ALL, one of which expresses the potent anti-apoptotic inhibitor, Bcl-2, known to be clinically more resistant to standard therapy. Experiments evaluating the in vivo effects of SAGA in mouse leukemia models are currently underway.

Cell death at 24 hours

Early Apoptosis Necrosis control vincristine SAGA control vincristine SAGA T-ALL 2% 46% 41% 2% 18% 19% B-ALL 1% 2% 29% 0.4% 1% 30%

Figure Figure Figure Figure
Non-Total Body Irradiation (TBI) Containing Preparative Regimen in Alternative Donor Bone Marrow Transplantation (BMT) for Severe Aplastic Anemia (SAA).
Authors: Lee Je-Hwan , Choi Seong-Jun , Lee Jung-Hee , Lee Young-Shin , Seol Miee , Lee Jung-Shin , Kim M. Wookun , Lee Kyoo Hyung
Published: April 2020

Most preparative regimens in alternative donor BMT for SAA have included TBI to overcome high incidence of graft rejection, which is the major problem in this setting. With routine use of irradiated leukocyte-poor blood products, however, the incidence of graft rejection due to pre-transplant allo-sensitization has decreased. Recently, efforts to reduce dose intensity of the preparative regimen for alternative donor transplants have been made with promising results. Using non-TBI containing preparative regimens, 13 patients with SAA were transplanted from alternative donors in our center and we report on the results of BMT in these patients. Median age of the patients, 6 males and 7 females, was 22 years (range, 15-34). Twelve donors were unrelated volunteers and one was an HLA one-locus mismatched sibling. Median time from diagnosis of SAA to BMT was 304 days (range, 49–5,403). Nine patients had received one cycle of immunosuppressive treatment with ATG (± cyclosporine) before BMT, while 4 had not. All patients except one had been transfused before BMT. Preparative regimens consisted of cyclophosphamide (50 mg/kg x 4) plus ATG (Atgam® 30 mg/kg x 3) in 9 patients, cyclophosphamide (50 mg/kg x 4) plus fludarabine (30 mg/m2 x 3) in 2 patients, and cyclophosphamide (50 mg/kg x 2) plus fludarabine (30 mg/m2 x 5) plus ATG (Atgam® 30 mg/kg x 3 or Thymoglobuline® 3 mg/kg x 3) in 2 patients. All patients received non-T-cell depleted bone marrow graft form the donor. Cyclosporine plus methotrexate were given for GVHD prophylaxis. All patients engrafted on a median of day 21 (range, 15–27) with complete donor chimerism. Grade III-IV acute GVHD developed in 3 (23%) of 13 patients and extensive chronic GVHD in 4 (31%) of 12 evaluable patients. With a median follow-up duration of 1,138 days (range, 118–1,553), 10 patients are alive with durable engraftment showing 75% of survival rate. Causes of death were chronic GVHD in 2 and CNS bleeding in 1. In conclusion, non-TBI containing preparative regimen could ensure durable engraftment in alternative donor BMT for SAA and showed promising results.
Bypassing Cellular Senescence by Ezh2.
Authors: Kamminga Leonie M. , Bystrykh Leonid , Houwer Sita , Douma Jose , Weersing Ellen , Dontje Bert , de Haan Gerald
Published: April 2020

In this study mouse embryonic fibroblasts (MEFs) and hematopoietic stem cells (HSCs) were used to identify genes involved in the process of cellular aging. MEFs are able to undergo a distinct number of population doublings, after which they enter a state of irreversible growth arrest termed replicative senescence. Previous studies of others and us have shown that aging is regulated by an intrinsic genetic program. In order to identify candidate genes involved in senescence we assessed gene expression profiles of proliferating and senescent MEFs. Interestingly, a significant number of genes that was higher expressed in proliferating vs. senescent MEFs, are involved in epigenetic regulation of gene transcription by modulating histone methylation and deacetylation. The top candidate gene was Enhancer of zeste homolog 2 (Ezh2), a Polycomb group protein (PcG), involved in histone methylation and deacetylation and known to function as a negative regulator of gene transcription. It has been reported that Ezh2 is essential during development, since Ezh2-deficient mice die early during embryonic development. Ezh2 expression was rapidly downregulated during senescence in primary MEFs, but was readily detectable in spontaneously transformed cells. Retroviral overexpression of Ezh2 in primary MEFs resulted in bypassing the senescence program. In addition, overexpression of Ezh2 in spontaneously transformed MEFs increased proliferation rates. We found that Ezh2 is highly expressed in purified Lin−Sca-1+c-kit+ (LSK) HSCs. In agreement with MEF studies, terminal differentiation of stem cells in vitro resulted in rapid downregulation of Ezh2 expression. Furthermore, overexpression of Ezh2 in purified HSCs results in increased proliferation rate in vitro. Moreover, HSCs transduced with Ezh2 were enriched for CFU-GM activity. Ongoing studies will address whether overexpression of Ezh2 in stem cells will affect their self-renewal potential in vivo. In an extensive genetic screen we identified genes that correlate with Ezh2 expression in stem cells, potential targets or partners of Ezh2, among others Top2a, MKi67, Pcna, and Eed. These data were deposited in an on-line resource (www.WebQTL.org), to uncover genetic networks that are involved in regulating the process of aging.
Methylation of the Tumor Suppressor Gene PTPRO (Receptor-Type Protein Tyrosine Phosphatase) Is Associated with Expression of Important Apoptosis-Related Proteins in Chronic Lymphocytic Leukemia (CLL).
Authors: Motiwala T. , Ghoshal K. , Majumder S. , Kutay H. , Neuberg D. S. , Kitada S. , Reed J. C. , Dewald G. W. , Flinn I. W. , Tallman M. A. , Liu T. , Plass C. , Lucas D. M. , Byrd J. C. , Jacob S. T. , Grever M. R.
Published: April 2020

We previously demonstrated that apoptosis-inhibiting proteins (e.g., Bcl-2, Mcl-1, and XIAP) are important in predicting response to chemotherapy and antibody therapy for CLL. Additionally, genetic abnormalities and epigenetic control of gene expression have become increasingly important in understanding the prognosis and potential therapeutic opportunities in this disease. Greater than 80% of patients with CLL have genetic abnormalities detectable by FISH. Important subsets include those with a less favorable prognosis (deletions of 17p, 11q), an intermediate prognosis (trisomy 12), or a favorable outcome (deletion of 13q). PTPRO, encoding a receptor-type tyrosine phosphatase, is a specific tumor suppressor gene located in chromosomal region 12p12.3. PTPRO has been highly characterized as inhibiting anchorage-independent tumor growth in human lung carcinomas. This gene has been found to be silenced by promoter methylation, and can be re-expressed by hypomethylating agents in human lung tumor models. Thus, we examined the methylation profile of the promoter region of PTPRO in primary leukemic cells from patients with CLL, and in B cells obtained from normal volunteers. Analysis of the PTPRO CpG island by methylation-specific PCR and combined bisulfite restriction analysis (COBRA) demonstrated that this region was methylated in 91% of samples from 46 patients, whereas it was unmethylated in control B cells from normal volunteers. Specific quantitative determinations of important proteins related to apoptosis in the leukemic cells from 20 of these patients showed that, in the group with methylation of the PTPRO promoter, there was a significant elevation of Bcl-2 (p=0.079), Mcl-1 (p=0.004), XIAP (p=0.048), Bcl-2/BAX ratio (p=0.0024), and Mcl-1/BAX ratio (p=0.0017). There was no apparent relationship with FISH category or immunoglobulin VH mutational status. Thus, PTPRO promoter methylation analysis may identify a subset of CLL patients less likely to respond to therapy, and provide a potential reversible target for new therapeutic approaches to this disease. Finally, analysis of methylation of CLL samples by restriction landmark genome scanning also revealed methylation of genes for three additional protein tyrosine phosphatases: PTPRN2, PTPRZ2, and PTPN11. Increases in methylation of two of these (PTPRN2 and PTPRZ2) in CLL samples relative to control B cells was confirmed by COBRA. These results provide additional potential molecular targets for CLL therapy.
Thrombocytosis Increases the Risk of Chemotherapy-Associated Thrombosis.
Authors: Khorana Alok A. , Francis Charles W. , Culakova Eva , Lyman Gary H.
Published: April 2020

Background: Venous thromboembolism (VTE) is a frequent and significant complication of cancer and of chemotherapy, with an estimated risk of 0.04%/month (0.5%/year). Patients with thrombocytopenia are widely perceived to be at low-risk for VTE. Conversely, thrombocytosis may contribute to risk of VTE, but this has not been well-studied. We conducted a prospective analysis to determine the association of platelet counts with the incidence of symptomatic VTE in cancer patients receiving chemotherapy.

Methods: The Awareness of Neutropenia in Cancer (ANC) Study Group is currently enrolling patients with breast, lung, colon, ovary and other cancers, at 137 sites nationwide, with an accrual goal of 4,500 patients. Patients are registered at the time of initiation of a new chemotherapy regimen, and followed for 4 cycles. The association of VTE with platelet counts and other clinical variables was studied in univariate analysis and in a multiple logistic regression model.

Results: A total of 2,151 patients treated with at least one cycle of chemotherapy were available for analysis. Of these, 18.1% had platelet counts <200,000 and 21.5% had counts ≥350,000 prior to initiating chemotherapy. Symptomatic VTE occurred in 46 patients, for an incidence of 2.14% over a median follow-up of 2.5 months (0.85%/month). The incidence of VTE by quartiles of pre-chemotherapy platelet counts is shown in the figure. VTE occurred in 4.34% (1.73%/month) of patients with platelet counts of ≥ 350,000 prior to initiation of chemotherapy. This was significantly greater than 1.8% (0.72%/month) incidence in those with baseline counts of <200,000 (p=0.035). Similarly, the mean platelet count prior to chemotherapy was higher for patients who developed VTE than for those who did not (351,000 versus 288,000, p=0.007). Platelet counts during chemotherapy were also associated with risk for VTE. The mean platelet count at nadir during the cycle VTE occurred was 202,000. This was significantly greater than the mean nadir count of 157,000 for corresponding cycles in patients who did not develop VTE (p=0.007). Other clinical variables associated with development of VTE included primary site of cancer, ECOG performance status ≥ 2, and baseline use of erythropoietin or colony-stimulating factors. A baseline platelet count of ≥ 350,000 continued to be significantly associated with VTE after adjusting for other risk factors in a multivariate analysis (odds ratio 3.00, 95% CI 1.61–5.55, p=0.0005).

Conclusion: This is the first report describing an independent association of platelet counts with symptomatic VTE in cancer patients receiving chemotherapy. Patients with platelet counts of ≥ 350,000 prior to initiation of chemotherapy are at a three-fold greater risk for developing VTE. The role of platelets in the pathogenesis of chemotherapy-associated thrombosis warrants further study. Platelet counts should be taken into account when defining high-risk patients for thromboprophylaxis.

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