bluuebunny/crossref_metadata_2025 huggingface dataset:
Induction, Selection and Expansion of Acute Myeloid Leukemia Reactive Autologous T Cells for Adoptive Immunotherapy.
Authors: Zhong Rui-kun , Ball Edward D.
Published: October 2019

To generate highly AML reactive autologous CTL, the optimal protocol of growth factor combination and sequence of administration were screened and compared in an AML-DC culture system aimed to induce AML cell DC differentiation, activate and expand naïve T cells against AML; and simultaneously activate and expand possible rare existing AML reactive memory T cells. Primary AML peripheral blood or marrow cells containing more than 90% AML blasts were cultured in 96 well plates. Every single well was regarded and treated as an independent culture. Various growth factor combinations of GM-CSF, IL2, IL-3, IL-4, IL7, IL12, CD40L, TNF-α, anti-CTLA-4 mAb were compared for efficiency of AML cell DC differentiation induction and autologous T cell activation in 7 day culture. Significantly enhanced CD80/CD86 expression and total cell number (include both AML-DC and T cells, see attached figure) in the culture was observed when GM-CSF/IL-4 combined with IL-12. The T cells in each well were than expanded with high dose IL-2 (6000 u/ml) and CTL activity against autologous AML were examined by both 51Cr release assay and culture supernatant IFN-γ concentration assay. Multiple wells of AML cell culture from the same patient with the same cell number and culture condition exhibited substantial functional variation demonstrated by MHC-restricted autologous AML cell killing (3 - 70% specific lysis) and IFN-γ secretion (25 - 2994 pg/ml), indicating that the T cells randomly put in the independent wells vary greatly in efficiency of AML antigen recognition and activation. IL-12 alone exerted significant enhanced IFN-γ secretion in 4 of 5 patients. IL-12 combined with GM-CSF/IL-2/IL-4/IL-7 enhanced IFN-γ secretion compared with IL-12 alone in 3 of 5 patients. CTLA-4 blockade further significantly enhanced T cell activity in 5 of 5 patients. Highly active CTL lines were selected from high IFN-γ secretion wells and rapidly expanded using OKT-3/CD28 coated, mitomycin-c treated autologous AML cells. After expansion, the highly active lines still maintained high autologous AML cell killing compared to the low active lines. A therapeutic quantity of AML reactive autologous T cells (20.2±30.1)x109, (n=5) can be obtained after 6 weeks of culture from (6.4±2.9)x107 AML PBMC. This study demonstrated AML cells cultured in IL-12 or IL-12 combined with GM-CSF/IL-2/IL-4/IL-7/anti-CTLA4 mAb activated the potential of the patient immune system and may maximize the possibility of AML reactive CTL generation for adoptive immunotherapy.

Figure Figure
Prolonged Remissions with Autologous and Allogeneic Stem Cell Transplantation for Burkitt’s Lymphoma: Long Term Follow-Up at a Single Institution.
Authors: Gada Purvi , Defor Todd , Weisdorf Daniel J. , Miller Jeffrey S. , Orchard Paul J. , MacMillan Margaret L. , Arora Mukta , Tomblyn Marcie , Burns Linda J.
Published: October 2019

Burkitts Lymphoma (BL) is a highly aggressive form of non-Hodgkins lymphoma (NHL) that accounts for 50% of childhood cases of NHL, yet is rare in adults. B symptoms, advanced stage and extranodal disease are risk factors previously associated with poor survival. Hematopoietic stem cell transplantation (HCT) is often used because of either incomplete or short duration of remission with standard therapy yet little published data for HCT and BL exists. We evaluated the comparative safety and efficacy of a cyclophosphamide/total body irradiation-containing myeloablative conditioning regimen followed by either an autologous HCT (autoHCT) or allogeneic related donor HCT (alloHCT) in 38 patients who received transplants between October 1975 and June 2004. Twenty-five patients (median age 16 years [range, 4–65]) underwent an autoHCT; 13 patients (median age 13 [range, 4–62]) received an alloHCT. The median number of conventional chemotherapy regimens prior to transplant was 2 (range 1–4); the median duration of first complete remission (CR) was 0.4 years (range, 0–8.8). The majority of patients were in a complete remission (CR) at transplant (auto HCT - 16 [64%] [40% CR1]), alloHCT - 9 [69%] [23% CR1]). Patient demographics, disease characteristics at diagnosis, at relapse and at transplant were comparable between the two groups except for a greater incidence of high risk factors, including B symptoms, advanced stage at diagnosis, and extranodal (bone marrow and central nervous system) disease in the alloHCT group. The median follow up is 7 years (range 1–12) and 24 years (range 2–27) for the autoHCT and alloHCT groups, respectively. Post-transplant, 71% of auto-HCT and 75% of the alloHCT recipients obtained a CR. The 1-year treatment related mortality (TRM) was comparable in the two groups: 8% and 15% for the autoHCT and alloHCT groups, respectively (p=NS). Ten-year progression free survival (PFS) was 21% (95% CI, 4–38%) and overall survival (OS) 23% (95% CI, 5–41%) after autoHCT compared to 31% (95% CI, 6–66%) and 31% (95% CI, 6–66%) for alloHCT (p=NS). Six patients in autoHCT group and 3 in alloHCT survive disease free between 1 and 27 years; 5 survive beyond 10 years and 3 beyond 15 years from HCT. Donor choice did not significantly alter PFS. Two factors were predictive of superior PFS: fewer chemotherapy regimens prior to transplantation (1 vs ≥ 2) and CR (vs relapsed/persistent disease) at time of transplant. Patients with high risk factors more commonly underwent alloHCT, yet outcomes were comparable to autoHCT, suggesting that a powerful and durable graft versus lymphoma effect exists. These results demonstrate that prolonged remissions can be obtained with either auto or alloHCT, especially for high risk patients in CR. New approaches for patients in relapse are needed to improve these outcomes.
The Role of the Protein Kinase C (PKC) Family in Glucocorticoid-Induced Apoptosis of Human Leukemic Cells.
Authors: Komak Spogmai , Miller Aaron L. , Johnson Betty H. , Thompson E. Brad
Published: October 2019

Glucocorticoids (GC’s) are among the most important clinical agents for the treatment of human leukemias. Cells exist, however, that have a functional glucocorticoid (GC) receptor, but are resistance to GC-induced apoptosis—suggesting that major signal transduction pathways (ie PKC family) may influence cellular response to GC. In order to determine whether glucocorticoid-induced apoptosis is mediated by members of the PKC family, we first screened the CEM acute T-cell lymphoblastic leukemic cell line for the presence or absence of all known PKC isoforms. We used two clonal cell lines that were derived from the same parent, CEM-CCRF, but which subsequently developed a pronounced difference in their sensitivity to glucocorticoids. One of these cell lines, CEM C7-14 is sensitive to dexamethasone-induced apoptosis, while the second cell line, CEM C1-15, is resistant. Of the 12 known PKC isoforms, 5 were detected at the basal level, and furthermore that specific members (PKC iota) was up-regulated during resistance, while PKC theta was up-regulated during apoptosis. Selectively inhibiting the entire PKC family caused increased cellular susceptibility to GC-induced apoptosis. Selectively targeting specific isoforms (PKC iota) caused resistant cells to become sensitive to apoptosis. Using immunoflouresence imaging, we showed the selective translocation of PKC’s iota, theta, and alpha after treatment with glucocorticoids. In addition, we showed specific phosphorylation of the isoforms iota, theta, and alpha in cells undergoing DEX-induced apoptosis. Two-dimensional proteomic analysis indicated a large number of proteins in CEM cells that show significant change after DEX treatment. We successfully identified several proteins which were regulated both after treatment with DEX alone, and after treatment with DEX + the PKC specific inhibitor Safingol. These proteins may represent the potential cross talk between the glucocorticoid and PKC apoptosis pathways. Taken together, these studies show that the PKC family is directly involved in glucocorticoid-induced apoptosis.
Disseminated Intravascular Coagulation Associated with Acute Hemoglobinemia or Hemoglobinuria Following Rho(D) Immune Globulin Intravenous Administration for Immune Thrombocytopenic Purpura.
Authors: Gaines Ann R.
Published: October 2019

The Food and Drug Administration (FDA) licensed Rho(D) immune globulin intravenous (anti-D IGIV) in March 1995 for treatment of immune thrombocytopenic purpura (ITP). The presumed mechanism of action of anti-D IGIV is extravascular hemolysis of anti-D-sensitized RBCs by splenic macrophages. Although seemingly inconsistent with this mechanism of action, acute hemoglobinemia and hemoglobinuria have been reported and are listed in the professional package insert for anti-D IGIV as possible adverse events. Through November 30, 2004, FDA received 6 reports of disseminated intravascular coagulation (DIC) associated with “acute hemolysis” (or similar terms) following anti-D IGIV treatment for ITP (Gaines AR. Disseminated intravascular coagulation associated with acute hemoglobinemia or hemoglobinuria following Rho(D) immune globulin intravenous administration for immune thrombocytopenic purpura. Blood.2005, 106(5):in press). All patients were clinically stable and were initially discharged home following anti-D IGIV administration. All were subsequently hospitalized for signs and symptoms of acute hemolysis or DIC. The mean decrease between pretreatment and nadir posttreatment hemoglobin levels was 5.8 g/dL (range: 3.0 to 9.6 g/dL); 4 patients received 2 to > 5 units of packed RBCs. Four patients whose baseline serum creatinine levels were within normal limits developed renal insufficiency; 2 of those patients underwent dialysis. The pediatric patient was subsequently discharged from the hospital without sequelae. However, all 5 adult patients remained hospitalized and died 3 to 10 days after anti-D IGIV administration. Attending or consulting physicians assessed that acute hemolysis and/or DIC caused or contributed to each death. Data from previously reported cases further suggested that previous uneventful administration of anti-D IGIV does not preclude the development of acute hemolysis upon subsequent administration of anti-D IGIV. These cases suggested that patients treated with anti-D IGIV for ITP who experience acute hemoglobinemia or hemoglobinuria be monitored for the development of DIC. To increase our knowledge about the seemingly rare but potentially serious complication of DIC following anti-D IGIV treatment for ITP, physicians and other health care professionals are encouraged to submit serious adverse event reports to the anti-D IGIV manufacturer or to FDA. Contact information for reporting adverse events to the manufacturer of any FDA-approved product is generally available in the professional package insert or on manufacturer- or distributor-sponsored web sites. Alternatively, adverse events can be reported directly to FDA through its adverse event reporting system, MedWatch, by Internet at http://www.fda.gov/medwatch, by telephone at 1-800-FDA-1088; by fax at 1-800-FDA-0178; or by mail at MedWatch, HF-2, 5600 Fishers Lane, Rockville, MD 20852-9787.
Stimulation of Platelet Production in Healthy Volunteers by a Novel Pegylated Peptide-Based Thrombopoietin (TPO) Receptor Agonist.
Authors: Cerneus Dirk , Brown K. , Harris R. , End D. , Molloy C. , Yurkow E. , Koblish H. , Franks C. , Moolenaar M. , Burggraaf K.
Published: October 2019

A novel, pegylated, peptide-based thrombopoietin receptor agonist (peg-TPOmp) was shown to possess in vitro and in vivo thrombopoietic activity. In cell-based assays, peg-TPOmp was active at picomolar concentrations. In vivo, peg-TPOmp increased platelet production dose-dependently in rats (ED50 single i.v. dose ~ 100 μg/kg), dogs and mice. A phase I study was conducted in healthy male volunteers to investigate the tolerability, PD and PK of peg-TPOmp. Forty volunteers were randomized to receive peg-TPOmp or placebo as a single i.v. bolus injection in a ratio of 6:2. The peg-TPOmp dose range explored was 0.375, 0.75, 1.5, 2.25 or 3 μg/kg. PK analysis indicated dose-related kinetics of peg-TPOmp, although at doses of 0.75 μg/kg or lower, plasma concentrations were generally below the LOQ of 6.25 ng/mL. Mean Cmax values ranged from 11 ng/mL for 0.75 μg/kg to 62 ng/mL at 3.0 μg/kg. The mean terminal half-life ranged from approx. 18 to 36 hours. Platelet counts increased dose-dependently reaching peak levels at Day 10–12, and counts returned to baseline within 3–4 weeks. Mean peak platelet levels ranged from 315 x109/L at 0.375 μg/kg to 685 x 109/L at 3 μg/kg. Mean increase of peak platelet counts from baseline ranged from 1.4-fold at 0.375 μg/kg to 3.2-fold at 3.0 μg/kg. Endogenous TPO levels dose-dependently increased, reaching peak levels at 3 days post-dose, possibly due to a reduced rate of clearance. No significant changes were observed in blood levels of IL-6, IL-11 and EPO levels. Platelet function, assessed as collagen-induced platelet aggregation in whole blood, was not different between the treatments. None of the subjects experienced serious adverse events or dose-limiting toxicities. The most frequently observed adverse events included mild headache and fatigue and occurred both after active treatment and placebo. No antibodies against peg-TPOmp were detected. Based on the safety, PK and PD data, peg-TPOmp shows promise as an agent to treat thrombocytopenic disorders.
Erythroid Kruppel-Like Factor Regulates E2F4 and the G1 Cdk Inhibitor, p18.
Authors: Perkins Andrew C. , Keys Janelle R. , Hodge Denise J. , Tallack Michael R.
Published: October 2019

Erythroid Kruppel-Like Factor (EKLF) is a zinc finger transcription factor which is essential for β-globin gene expression. Knockout mice die from anemia at E15, but restoration of globin chain imbalance does not rescue anemia or increase survival. Cell lines derived from EKLF null mice undergo proliferation arrest upon reactivation of a conditional EKLF-ER fusion protein, suggesting a role in cell cycle control. A transcriptional profiling experiment comparing the global gene expression in EKLF null and wild type fetal liver identified many differentially expressed genes, a number of which function in G1 and at the G1/S checkpoint of the cell cycle. The Cyclin dependent kinase (Cdk) inhibitor, p18, and the S phase transcription factor E2F4 were both found to be significantly down regulated in EKLF null mice and this result was confirmed by real-time PCR. Interestingly, E2F4 knockout mice have a similar phenotype to EKLF knockout mice. Bioinformatic searches of the p18 and E2F4 genes shows that each contains phylogenetically conserved CACC box motifs capable of binding EKLF within longer regions of conservation in promoter and intron regions. The p18 gene contains two conserved CACCC sites upstream of the start of transcription, which are required for EKLF dependent promoter activity in luciferase reporter assays. The transcription factor E2F4 contains a conserved EKLF-binding CACC site within an intron that is closely associated with two conserved GATA1 binding sites. We show by a chromatin immunoprecipitation (ChIP) assays that the E2F4 intron and p18 promoter are occupied by EKLF in vivo. Together, these results suggest that EKLF is likely to directly regulate expression of key cell cycle genes in vivo to drive the switch from proliferation to differentiation of erythrocytes. The loss of EKLF is likely to result in aberrant proliferation and predisposition to leukemia.
Cytoplasmic Tail of Tissue Factor(TF): Role in TF Trafficking to Microparticles.
Authors: Zakarija Anaadriana , Hobbs Jennifer , Chalasani Pavani , Lieberman Elliot , Morrissey James H. , Soff Gerald A.
Published: October 2019

Introduction: Tissue factor (TF) is a transmembrane protein that is primarily extracellular. It has a 21 amino acid cytoplasmic tail, which is not essential for coagulation but may have a role in signal transduction. We studied the activity of deletion variants of TF, transfected into a pancreatic cancer line (MiaPaCa-2), which lacks TF RNA or protein.

Methods: TF variants: lctTF (lacking cytoplasmic tail), sTF(soluble, lacking transmembrane and cytoplasmic domains), wtTF (wild type) were transfected into MiaPaCa-2 cells. Microparticles were prepared from serum-free conditioned media(CM) by ultracentrifugation at 100,000g. Presence of TF antigen was detected by immunoblot using a monoclonal antibody to TF, 10H10. Tissue factor activity (TFA) was assessed by recalcification time.

Results: Immunoblot confirmed the presence of TF in the cell lysates of MiaPaCa-2 cells transfected with wtTF or lctTF. In the sTF MiaPaCa-2 cells, TF was not detected in the cell lysates, but was present in CM. TF was present in CM of lctTF-transfected cells, but only trace antigen was observed in CM of wtTF cells. TF was microparticle-associated in the CM collected from lctTF cells, but not in the sTF cells. The TF protein made by sTF cells was not functional in the CM as demonstrated by no difference in TFA as compared to untransfected MiaPaCa-2 cells (>350 sec vs 351 sec). The CM from lctTF MiaPaCa-2 cells shortened clotting time by over 50% (159 sec vs 351 sec), while wtTF MiaPaCa-2 cells did not (349 sec vs 351 sec). The microparticles from lctTF had the most TFA when compared to wtTF or untransfected cells (107 sec vs 194 sec vs 436 sec). In the CM from cells with lctTF, TFA was exclusively in the microparticle section and absent in the supernatant (107 sec vs 440 sec).

Conclusions: Wild type TF remains primarily cell associated. In contrast, a higher fraction of the lctTF was released into the CM and remained microparticle associated. TF protein that lacks the transmembrane portion is soluble but not microparticle-associated and does not have procoagulant activity. Lack of the cytoplasmic tail results in increased generation of functional, microparticle-associated TF compared with the wild-type protein. These data suggest the cytoplasmic tail may influence the trafficking of functional TF to the microparticle fraction.
Novel Synthetic Histone Deacetylase Inhibitor (SK-7041) Potently Inhibits Proliferation in Acute Myeloid Leukemia Cell Lines.
Authors: Kim Young-Ju , Guo Susan , Park Ju Won , Bae Eun Kyung , Ahn Kwang-Sung , Kim Inho , Lee Jong-Seok , Lee Young Yiul , Park Seonyang , Kim Byoung Kook , Yoon Sung-Soo
Published: October 2019

Histone acetyltransferase (HAT) and histone deacetylase (HDAC) activities determine the acetylation status of histones, and have the ability to regulate gene expression through chromatin remodeling. Aberrant histone acetylation is known to play a key role in leukemogenesis. To improve the treatment outcome in AML patients, we examined the antiproliferative activity of SK-7041, a novel hybrid synthetic HDACI, in acute myelogenous leukemia (AML) cell lines (KG-1,HL-60 and HEL). This HDACI preferentially inhibited the enzymatic activities of HDAC1 and HDAC2, compared to other HDAC isotypes, indicating that class I HDAC is the major target of SK-7041. SK-7041 effectively induced the time-dependent hyperacetylation of histones, H3 and H4 in AML cell lines. We found that this compound exhibited potent antiproliferative activity against AML cell lines in vitro. The growth inhibitory effect of SK-7041 was related to the induction of apoptosis in AML cell lines. SK-7041(4uM) induced potent cell death, triggering apoptosis in over 60% of cells in 48h by FACS analysis. After 12h of treatment by western analysis, apoptotic cells were increased through activation of caspase-7,-9,-3 and cleaved-PARP (caspase-8 was not affected). Cyclin D1 expression was decreased, and CDK4 expression level was not affected by SK-7041. The level of p21 expression was gradually increased in KG-1 and HL-60 cell lines, whereas treated HEL cell lines showed no change. For further investigation of the genes affected by SK-7041, 20Kb oligonucleotide microarray analysis was performed. The expression of candidate genes, which we selected in chip analysis, was confirmed using real-time PCR. Fifteen genes (RGL1, FYN, CARD9, ABCA7, TNFRSF6B, CASP9, ENPP2, etc.) were consistently up-regulated and twelve genes (PTPN7, CD34, INSIG1, IL16, LHX6, TRIB3, BID, PDCD4, etc.) were down-regulated more than 4-fold by the SK-7041 compound. We suggest that the efficacy of SK-7041 was mainly mediated by the inhibition of proliferation and induction of apoptosis. Taken together, our data suggested that novel HDACI SK-7041 treatment is effective in AML cell lines, and that the further study is warranted.
Mesenteric Castleman’s Disease with Systemic Symptoms in Children.
Authors: Kim Sun Young , Sul Ji Young , Kim Jin Man , Chung Nak Gyun , Cho Bin , Jeong Dae Chul , Kim Hack Ki
Published: October 2019

Castleman’s disease (CD) is a lymphoproliferative disorder of unknown origin and rarely occurs in children. It has been further classified into two major subgroups: solitary or localized and multicentric CD. Clinically, hyaline-vascular type is rarely associated with systemic symptoms, but the plasma cell type is frequently associated with the constitutional symptoms of fever, malaise, night sweat and the abnormal laboratory markers. We encountered a case of the hyaline-vascular type CD located in the mesentery with systemic symptoms. She admitted suffering from prolonged fever, anemia, poor weight gain, dyspnea, and abdominal distension. Labaroatory data showed that microcytic hypochromic anemia, polyclonal gammaglobulinemia, hyperfibrinogenemia, elevated C-reactive protein, and increased IL-6 level. Computed tomography confirmed that the mass was located in the mesentery with multiple lymphadenopathies. The clinical and biochemical abnormal findings improved after surgical resection of the involved lymph node. Currently she has no signs of recurrence at 12 months postoperatively.
High Catalase Activity: Another Possible Pathway for Imatinib-Resistant CML Patients.
Authors: Luque Diogo G. , Ferreira Ana Carolina S. , Vasconcelos Flavia C. , Maia Raquel C. , Kwee Jolie K.
Published: October 2019

Imatinib (imatinib mesylate: Novartis Pharmaceuticals, Basel, Switzerland) is a well-known selective inhibitor of BCR-ABL tyrosine kinase activity, a hallmark enzyme in the pathogenesis of chronic myeloid leukemia (CML). Resistance to imatinib in CML patients has been associated with different and complex types of mechanisms ranging from nonspecific multidrug resistance protein, preventing the drug to reach its target, to BCR-ABL kinase domain point mutations, reducing the affinity of the enzyme to Imatinib. More recently, it was described that modulation of reduced glutathione (GSH) intracellular levels restores sensitivity to Imatinib in Imatinib-resistant cell line. This suggests that induction of hydrogen peroxide (H2O2), a well-known apoptotic reactive oxygen species (ROS), might be another Imatinib anti-leukemic mechanism action. Additionally, it is known that catalase activity is enhanced through phosphorilation by the c-abl tyrosine kinase (TK). In a preliminary work, our group found normal levels of GSH but high levels of antioxidant enzyme catalase in CML patients. The aim of this work is to evaluate the possible role of catalase as another pathway to Imatinib resistance. White blood cells from blood donors, CML patients and patients with reactional leukocytosis caused by infectious bacterian diseases were obtained using Ficoll-Hypaque. Catalase activity was measured by monitoring decomposition of 10mM H2O2 at 240nm according to the method described by Aebi. The viability of eritroleukemia cell lineage K562 previously treated or not with 100μM 3-aminotriazole (catalase inhibitor) was evaluated by MTT method with Imatinib 1, 5 and 25μM for 24 and 48 hours. Imatinib induced intracellular ROS generation was monitored by flow cytometry with 40μM 2′, 7′-dichlorofluorescein diacetate (DCFH-DA), a well-known ROS capturing reagent. Catalase activity was increased up to 30x in CML patients but not in reactional leukocytosis. When BCR-ABL positive cell line K562 was treated with Imatinib (50μM), catalase activity levels decreased in 38% compared with untreated cells. Also, we found that 3-aminotriazole can overcome the resistance to Imatinib in K562 cells resulting in 25% enhancement of dead cells. The protection of catalase against Imatinib-ROS generation was confirmed by DCFH-DA/FACs in cells incubated with PEG-catalase (monomethoxypolyethylene glycol covalent attached to catalase). Taken together, these results suggest that catalase might be another attractive target for overcoming resistance to Imatinib in Imatinib-resistant CML cells with high catalase activity.
A Prospective Analysis of Adverse Events in a Phase 2 Trial of Imatinib in the Treatment of Polycythemia Vera.
Authors: Jones C. Michael , Dickinson Tina M.
Published: October 2019

Imatinib has recently been demonstrated to be an effective treatment in Polycythemia Vera. Initial response rates are high, approaching 80%. Patients enrolled in our trial, Novartis CST 1571B (US 144), were asked to complete a questionnaire identifying side effects of imatinib known from previous trials in chronic myeloid leukemia and GIST. Questionnaires were completed weekly for the first month, biweekly for the second month, and monthly for the remainder of the study. Mean time on study is now 1.1 years. Target enrollment was 20 patients. Mean age was 54 years; male to female ratio was 1.3: 1. Fourteen patients were Caucasian and 2 African American. Twelve patients were naïve to treatment and 4 patients were previously treated with hydroxyurea. Two dose escalations were allowed sequentially to 600mg and 800mg daily based on response using the Polycythemia Vera Study Group Criteria. Toxicity was graded according to the NCI Common Toxicity Criteria, version 2.0. Patient reported toxicities are expressed as the total number of events reported during the 120 week study period. Diarrhea was the most common reported toxicity at 53 events. Periorbital edema (33), pruritis (39), fatigue (29), arthralgias (20), headache (18), nausea (19), reflux (38), and dry mouth (15) were the next most commonly reported events on study. Other toxicities reported less commonly were chest pain (3), depression (4), cough (5), insomnia (6), weight gain (3), taste changes (7), and depigmentation (3). Most toxicities were mild at grade 1 and resolved spontaneously within 30 to 40 weeks from the time of treatment initiation. The exceptions to this were diarrhea, periorbital edema, and reflux esophagitis, which persisted in a number of patients throughout the study. The severity of gastrointestinal side effects was dose dependent. Previous studies of imatinib induced toxicity in patients with chronic myeloid leukemia have reported cutaneous manifestations as the most common event. Our study demonstrates the toxicities of imatinib in patients with polycythemia vera are generally mild and self limited, although resolution may take up to 35 weeks. In contrast with studies of imatinib and chronic myeloid leukemia, gastrointestinal toxicity was more common and tended to persist, but did not result in patient withdrawal from study. These differences from our findings may be the result of several factors: the use of a patient reported adverse events scale, small trial size, or differences in drug metabolism in a related myeloproliferative disorder.
A Photodynamic Pathway to Apoptosis Induced by Hypericin in Human Myeloma ARH-77 Cell Line: Possible Relevance to Photodynamic Therapy.
Authors: Avcu Ferit , Ural Ali U. , Baran Yusuf , Sobaci Gungor , Deveci Mustafa , Cetin Turker , Pekel Aysel , Sarper Meral , Elci Pinar , Gunduz Ufuk
Published: October 2019

Hypericin, a photosensitizing plant pigment from Hypericum perforatum, is a protein tyrosine kinase inhibitor that has been exploited in models for anti-tumor and anti-viral activity. In this study, we investigated the effects of hypericin on the human myeloma cell line ARH-77 as a model to determine whether hypericin-induced cell death is available. The cells were incubated with hypericin at concentrations ranging from 0.001 to 10 microg/ml in RPMI at 37°C in 5% CO2 atmosphere for 4 h. Then, the cells were irradiated at 532 nm (fluence=24 J/cm2) using a dye laser pumped by an argon laser (Orion). After 72 h exposure, the IC50 of hypericin was 0.005 microg/ml as determined by the MTT assay. Hypericin exerted phototoxic effect on ARH-77 cells, while it did not produce toxic effect in the absence of irradiation. After 72 h exposure to 0.005 microg/ml photoactive hypericin, apoptosis was assessed by morphological changes, DNA fragmentation and flow cytometric analysis using Annexin V and propidium iodide staining. Most of the cells were accumulated in the late stage of apoptosis and these cells were brightly stained and fragmented nuclei and cytoplasmic blebbing were observed. A decrease in the number of apoptotic cells was detected when protein kinase C was inhibited by addition of staurosporine to photoactive hypericin induced ARH-77 cells. From these results, we demonstrated that exposing myeloma cell line ARH-77 to photoactive hypericin inhibits cell growth in a dose dependent manner, induces apoptotic cell death by protein kinase C activation, and provides a rationale for potential applications in vivo.
Normal Bone Marrow Stromal Cells Inhibit the Growth of Myeloma Cells While Myeloma Bone Marrow Stromal Cells Improve It.
Authors: Zhuang Junling , Wu Yongji , Wang Xuan , Sun Wanling
Published: October 2019

Objective: Observe the effect of normal and myeloma bone marrow stromal cells (BMSC) to the growth of myeloma cells. Evaluate the function of adhesion molecules in the mutual action of BMSC and myeloma cells.

Methods: Immunomagnetic beads and anti-CD138 antibody were used to separate myeloma cells from patients’ bone marrow of multiple myeloma (MM). U266 and freshly purified myeloma cells respectively co-cultured with normal BMSC and myeloma BMSC. Single U266 or fresh myeloma cells were cultured as control group. Anti-CD11a, anti-CD29, anti-CD44 and anti-CD49d antibodies were respectively added in the co-cultured system and single myeloma cells. After culture for 72 hours, cell cycle of myeloma cells and the ratio of apoptotic cells were tested by flowcytometry. The level of interleukin 6 (IL-6) in the supernate was measured.

Results: Normal BMSC inhibited the growth of myeloma cells while myeloma BMSC improved it. As to U266, S+G2% of myeloma cells in normal BMSC group and myeloma BMSC group were 55.5±4.0% and 65.4±4.5%(p<0.05)respectively. The ratio of apoptotic cells were 3.92±1.83% and 0.86±0.58%(p<0.05)respectively(figure1 from left to right: U266, U266+normal BMSC, U266+myeloma BMSC, red: apoptotic cells). The level of IL-6 in the supernate were 1236±146.2pg/ml and 1661±161.5pg/ml(p<0.05)respectively. After co-cultured with normal BMSC and myeloma BMSC, S+G2% of freshly separated myeloma cells were 5.2±4.1% and 15.9±5.5%(p<0.05)respectively. The ratio of apoptotic cells were 18.7±7.8% and 8±3.9%(p<0.05) respectively (figure2 from left to right: fresh myeloma cells, fresh myeloma cells+normal BMSC, fresh myeloma cells+myeloma BMSC, red: apoptotic cells). The level of IL-6 in the supernate were 1621.2±121.1pg/ml and 2151.1±170.4pg/ml(p<0.05)respectively. The function of four adhesion molecules was different in the co-culture system. Anti-CD11a hardly affected the growth of myeloma cells. But anti-CD29 and anti-CD44 showed their significant inhibition to the growth of myeloma cells, including the decrease of supernatant IL-6 level, percentage of phase S+G2 and the increase of apoptotic cells (fresh myeloma cells). Anti-CD49d indicated partial inhibition, but no significant change.

Conclusion: We firstly proved that normal BMSC inhibited the growth of myeloma cells. Anti-CD29 and anti-CD44 can block the adhesion of myeloma cells and BMSC, resulting in the growth inhibition of myeloma cells.

Figure Figure
Whole Body MRI Versus Skeletal Survey in Multple Myeloma.
Authors: McHugh Johnny , Johnston Ciaran , Duke Deirdre , Thornton Patrick , Eustace Steve , O’Gorman Peter
Published: October 2019

BACKGROUND: Bone involvement in myeloma is conventionally assessed by radiographic skeletal survey (plain x-rays of spine, skull, chest, pelvis and long bones). However this may not pick up bony involvement in all patients who may then present with serious complications of myeloma bone disease such as spinal cord compression. Whole body MRI may be better than skeletal survey at evaluating myeloma bone involvement.

AIMS: To compare the evaluation of myeloma bone involvement by conventional radiographic skeletal survey (RSS) with whole body magnetic resonance imaging (MRI).

METHODS: 35 patients with multiple myeloma (median age 68 yrs, range 46–81) underwent conventional RSS and whole body MRI. 19 of the patients had newly diagnosed multiple myeloma and 16 had relapsed multiple myeloma. The extent of myeloma bone involvement was evaluated in both RSS and MRI as follows: the body was divided up into ten areas: skull, cervical spine, ribs, thoracic spine, lumbar spine, pelvis, right arm, left arm, right leg, left leg. In each area the extent of myeloma bone involvement was scored in both RSS and MRI as follows: 0 = normal; 1 = one focus of abnormality; 2 = more than one focus of abnormality; 3 = diffuse disease. The scores for each of the ten areas were combined to give an overall score out of thirty for both RSS and MRI.

RESULTS: 30 of the 35 patients (85.7%) had evidence of bone involvement on MRI. This compares with 22 out of the 35 (62.9%) on RSS. The mean score for the extent of myeloma bone involvement on MRI was significantly higher than that for RSS (MRI mean score: 15.5 out of 30 (median 17, range 0–30); RSS mean score: 5.5 out of 30 (median 3, range 0 to 24); p<0.001). MRI was superior to RSS in all ten areas evaluated both in terms of lesion detection and extent of disease. The greatest difference between MRI and RSS was seen in the cervical, thoracic and lumbar spine, while the smallest difference was seen in the ribs and skull. Eight of the patients had no bone involvement detectable on RSS but did have bone involvement on MRI and this resulted in upstaging on Durie-Salmon staging in four patients.

SUMMARY/CONCLUSIONS: RSS has limited sensitivity and a significant ionising patient dose. It is a cumbersome procedure taking up to 30 minutes. Whole body MRI gives improved sensitivity and appreciation of anatomic location of disease. It is non-ionising and can be rapidly acquired at low cost. We conclude that whole body MRI is superior to conventional RSS in both the identification and evaluation of extent of bone involvement in multiple myeloma.
Establishment and Characterization of a New Human Myeloma Cell Line WuS1.
Authors: Sun Wanling , Wu Yongji , Li Hui , Wang Xuan , Zou Nong , Zhuang Junling
Published: October 2019

A new human myeloma cell line WuS1 was established from the bone marrow of a 45-year-old Chinese male patient with IgGλ type multiple myeloma (stage IIIB). The growth of WuS1 cells is constitutively independent of exogenous growth factors of feeder cells. The WuS1 cell line proliferated consistently as free-floating single cells in suspension, sometimes in small clusters or slightly adherent on the bottom of the plastic culture flask, without forming clumps. The cell line has been maintained without any external growth factors for over a year, and cells frozen in liquid nitrogen can be revived successfully. The doubling time of the cells was about 11 hours and the colony-forming rate was 55.56±6.33%. WuS1 displayed immature plasma cell features with an obvious heterogeneity in size and a high nuclear-cytoplasmic ratio in Wright-Giemsa staining.

Figure Figure

They were positive for ALP, CE, ACP(not inhibited by tartrate) and PAS stainings and negative for POX and NBE. By transmission electron microscopy, the cytoplasm of WuS1 contained abundant mitochondria, and parallel endoplasmic reticulum or Golgi apparatus in some cells. The monoclonal immunoglobulin G and λ light chain were positive in cell lysate and not in cell culture supernatants by immnuoelectrophoresis. The cell surface antigens were positive for CD3, CD59, CD106 and CD138, and negative for CD4, CD5, CD8, CD10, CD13, CD14, CD19, CD20, CD22, CD29, CD31, CD33, CD34, CD38, CD44, CD49d, CD45, CD54, CD56 and HLA-DR by flow cytometry. Chromosomal analysis revealed a hypodiploidy and complex karyotype. WuS1 cells were negative for Epstein-Barr virus by PCR using EBV nuclear antigen-1 specific primers. Twelve SCID mice were injected with WuS1 cells intravenously or subcutaneously, and obvious tumor infiltration in bone marrow, liver, spleen, lung, kidney and injection site (subcutaneously group) were observed by pathologic examination.

Figure Figure

The novel WuS1 cell line will be useful in the study of the biology, etiology and treatment of multiple myeloma.
Efficacy of Prophylactic Anticoagulation To Prevent Thalidomide-Related Deep Venous Thrombosis.
Authors: Ikhlaque Nadeem
Published: October 2019

Deep venous thrombosis (DVT) is a known complication of treatment with thalidomide, occurring in up to 25% of patients. Although prophylactic anticoagulation has been recommended by some authors, there are few data available to assess whether this is beneficial. We reviewed the records of 131 patients receiving thalidomide for a variety of indications. Ninety-three patients had multiple myeloma, 12 had MDS or myelofibrosis and 26 had solid tumors. The mean and median dose of thalidomide was 200 mg/day. Fifty-five patients were prescribed warfarin with the intent of preventing DVT. Thirty-seven patients received warfarin at a dose of 1–2 mg per day (low dose) and 18 received a dose intended to raise the INR to 2–3 (high dose). Of 25 patients on single-agent thalidomide; only 4 received warfarin. Twenty-one of the 131 patients developed venous thrombosis during thalidomide treatment. All patients who developed DVT were receiving thalidomide in combination with dexamethasone or some form of chemotherapy. Only one patient who developed DVT was receiving thalidomide at more than 200 mg/day. Eighteen of the 76 patients (23.7%) who were not prescribed prophylactic anticoagulation developed DVT compared to three of the 55 patients (5.5%) who were prescribed any dose of prophylactic warfarin (p=.010). Only one of the 37 patients who received low dose warfarin developed DVT (p=.011). Bleeding complications occurred in 4 patients who were all receiving high dose warfarin. Two previous unrandomized studies suggested that low dose warfarin was not protective of DVT but both used high doses of thalidomide (250–400 mg) in combination with other agents which is no longer recommended. Our study suggests that low dose warfarin prophylaxis may be sufficient and safe for patients receiving thalidomide at a dose of 200 mg/day in combination with other agents.
Clinical Responses to Oral Vorinostat (Suberoylanilide Hydroxamic Acid, SAHA) Are Associated with Specific Gene Expression Signatures in Patients with Advanced Leukemias: Results of a Phase I Trial.
Authors: Loboda Andrey , Fantin Valeria R. , Bueso-Ramos Carlos E. , Randolph Sophia , Hardwick James S. , Ricker Justin L. , Richon Victoria M. , Issa Jean-Pierre , Kantarjian Hagop M. , Frankel Stanley R. , Garcia-Manero Guillermo
Published: October 2019

Vorinostat is a histone deacetylase inhibitor with potent in vitro and in vivo anti-leukemia activity. Several gene expression signatures were developed from preclinical models as biomarkers for clinical response to vorinostat. The signatures were evaluated in peripheral blood mononuclear cell (PBMC) samples from a Phase I study of oral vorinostat in patients with advanced leukemias or myelodysplastic syndromes (MDS). Of 41 registered patients, 31 had acute myeloid leukemia (AML), 4 had chronic lymphocytic leukemia, 3 had MDS, 2 had acute lymphoblastic leukemia, and 1 had chronic myeloid leukemia. Seven of 41 patients showed improvement with treatment, 2 complete responses, 2 complete responses that were not maintained for 28 days, and 3 with >50% decreases in blast counts without count recovery. All responses occurred in patients with AML at or below the MTD. The following three clinical gene expression signatures were shown to be predictive of clinical response in PBMC samples of AML patients. First, a positive prediction trend was observed when a preclinical 40-gene resistance prediction signature was evaluated in baseline (pre-dose and first day of dosing) AML PBMC samples (8 responder and 21 non-responder samples) or all PBMC samples (14 responder and 56 non-responder samples, including non-AML patients). If this trend is maintained in pretreatment samples of a larger patient population, it may allow for exclusion of up to 50% of non-responders from future clinical trials. Second, a similar prediction of resistance was obtained based on expression levels of known reactive oxygen species scavengers that were upregulated in non-responders relative to responders, indicating that cellular ability to withstand oxidative stress may be protective with respect to vorinostat exposure. This supports the hypotheses that oxidative stress pathways play an important role in the response to vorinostat. Finally, overexpression of p21 and several p53-responsive genes was also associated with resistance, consistent with preclinical observations. These results are being validated at the protein level in a tissue array platform. Post-dose downregulation of genes associated with cellular proliferation was also observed in responders but not in non-responder PBMC samples. This suggests that proliferation may be used as a post-dose efficacy biomarker. Further evaluation of vorinostat in patients with leukemias and MDS, including analysis of a potentially predictive gene expression signature for response to vorinostat, is warranted.
IVIg-Primed Splenocytes Ameliorate Murine ITP in an MHC Class II-Unrestricted Manner.
Authors: Crow Andrew R. , Siragam Vinayakumar , Freedman John , Lazarus Alan H.
Published: October 2019

We have previously demonstrated that IVIg-primed leukocytes can, upon transfer to naïve mice, completley recapitulate the therapeutic effects of IVIg in treating immune thrombocytopenia in a murine model. Dendritic cells (DC) appear to be the primary cellular target of IVIg in this model. The exact pathway by which IVIg-primed DC inhibit platelet clearance remains, however, unknown. DC are professional antigen presenting cells of haematopoietic origin that are specialized for the capture, processing and presentation of antigens to T cells via their MHC Class II. This primary function of DC is absolutely dependent on MHC Class II expression. Herein, we have evaluated the efficacy of action of IVIg-primed splenocytes from MHC mismatched mice in the amelioration of murine ITP. Mice expressing the Class II haplotype I-Ab (C57BL/6) were injected intravenously with IVIg-primed splenocytes from syngeneic mice, or Balb/C mice (Class II haplotype I-Ad /I-Ed) or CD1 mice (outbred, mixed Class II haplotype I-A/E) prior to the induction of thrombocytopenia by an anti-platelet antibody. We found that IVIg-primed splenocytes from all strains of mice tested were able to successfully ameliorate thrombocytopenia in C57BL/6 mice, regardless of Class II haplotype. None of the splenocytes primed with a control protein, BSA, ameliorated thrombocytopenia. To confirm our findings that IVIg-primed splenocytes function independent of MHC haplotype, we next employed IVIg-primed splenocytes from MHC Class II deficient mice and found these splenocytes were also able to successfully ameliorate murine thrombocytopenia. These data demonstrate a non-MHC-restricted function for DC in IVIg function, and suggest that IVIg-primed DC function occurs independent of MHC Class II expression in the amelioration of murine ITP. In addition, since this potential new cell-based therapy for ITP is not MHC-restricted, we speculate that IVIg-primed DC from any donor (including autologous) could theoretically be used to treat individuals with ITP or other autoimmune diseases without the requirement for donor matching.
Importance of Early Intervention with Dasatinib at Cytogenetic Rather Than Hematologic Resistance to Imatinib.
Authors: Kantarjian Hagop M. , Quintas-Cardama Alfonso , O’Brien Susan , Ravandi Farhad , Borthakur Gautam , Liu David , Chen Tai-Tsang , Cortes Jorge
Published: October 2019

Recent data for patients with chronic-phase (CP) CML indicate that development of resistance or intolerance to imatinib and treatment intervention following progression to advanced-phase disease are both associated with poor clinical outcome (with 3-year survival rates of 72% for chronic-phase disease, 30% for accelerated-phase CML, and 7% for blast-phase CML) (Kantarjian et al. Cancer2007;109:1556–60). Results also demonstrate that intervention with a second-generation tyrosine kinase inhibitor is more effective for individuals with chronic-phase CML than for those with advanced-phase disease. Of note, development of hematologic resistance (loss of a complete hematologic response [CHR]) was identified as an independent prognostic factor with a 3-year survival rate of 57% (compared with 92% for patients who experienced cytogenetic resistance [loss of a major cytogenetic response (MCyR)]). The objective of the current data analysis was to assess whether the development of hematologic resistance to imatinib would be a predictor of poor response to dasatinib (SPRYCEL®) relative to the development of cytogenetic resistance. Data from three Phase-II/III dasatinib clinical studies (CA180–013 [70 mg BID], CA180–017 [70 mg BID], and CA180–034 [100mg QD, 50 mg BID, 140 mg QD, 70 mg BID]) in patients with CP CML with resistance to prior imatinib were evaluated. Consistent with our earlier findings, significantly higher complete cytogenetic response (CCyR) rates were reported for patients who had developed secondary or acquried cytogenetic resistance to prior imatinib than for those with hematologic resistance. The improved rates of CCyR in patients with cytogenetic resistance to imatinib were associated with prolongation of progression-free survival compared to patients with hematologic resistance, thereby confirming the importance of early intervention in the event of imatinib resistance and the significance of achieving CCyR with dasatinib.

Loss of MCyR Loss of CHR N n % N n % n = number of patients with [1] complete cytogenetic response or [2] progression Complete cytogenetic response [1] CA180–013 47 34 72 71 27 38 CA180–017 19 12 63 22 5 23 CA180–034     100 mg QD 28 21 75 14 2 14     50 mg BID 17 11 65 12 3 25     140 mg QD 18 9 50 16 8 50     70 mg BID 23 19 83 12 2 17 Progression-free survival at 1 year [2] CA180–013 47 3 95 71 15 84 CA180–017 19 1 94 22 3 85 CA180–034     100 mg QD 28 1 96 14 1 93     50 mg BID 17 0 100 12 6 58     140 mg QD 18 2 87 16 2 93     70 mg BID 23 2 95 12 4 69

These results confirm previous observations and

emphasize the importance of close monitoring of treatment response to imatinib, and argue for early intervention in case of imatinib resistance.

Late intervention, ie waiting for patients to lose a hematologic response, appears to significantly diminish the effectiveness of dasatinib.