bluuebunny/crossref_metadata_2025 huggingface dataset:
Integrin Linked Kinase Is Important in Platelet Signalling and Function.
Authors: Tucker Katherine L. , Stevens Joanne M. , Jordan Peter A. , Sage Tanya , Jones Sarah , Barrett Natasha E. , St-Arnaud Rene , Frampton Jonathan , Dedhar Shoukat , Gibbins Jonathan M.
Published: October 2019

Integrin linked kinase (ILK) is a 59 kDa protein that has been previously implicated in both collagen- and thrombin-induced platelet activation. Platelet stimulation results in the transient activation of ILK kinase activity and movement to the plasma membrane where signaling complexes are formed with beta 1 and beta 3 integrins. The change in ILK activity and association with beta 1 and beta 3 integrins that occurs upon stimulation suggests that this protein may be important for the co-ordination of platelet responses. We have successfully developed a conditional ILK knockout mouse model using the Cre-Lox system to enable the study of platelets deficient in this protein. ILK deficient mice appear healthy and have normal platelet levels by day 8 following induction of gene deletion. ILK deficient platelets display reduced aggregation and fibrinogen binding in response to agonists such as collagen and thrombin. This is accompanied by a secretion defect, although early collagen stimulated signaling such as PLCγ2 phosphorylation and calcium mobilization are unaffected. Analysis of blood from ILK deficient mice using an in vitro flow system showed reduced thrombus formation under arterial conditions. Furthermore, extended bleeding times were observed in these mice. The data presented here demonstrates that ILK has a role in platelet regulation and is important for functional haemostasis.
The Novel Glycolysis Inhibitor, 3-BrOP, Has Broad Activity Against Acute Lymphoblastic Leukemias and Potentiates Methotrexate and Rapamycin.
Authors: Akers Lauren J. , Franklin Anna R. , Huang Peng , Zweidler-McKay Patrick A.
Published: October 2019

Background: Rapidly growing cancer cells are often dependent on glycolysis for ATP production, known as the Warburg effect. The novel glycolysis inhibitor, 3-bromo-2-oxopropionate-1-propyl ester (3-BrOP), has shown efficacy in models of glioblastoma, colon carcinoma and lymphoma. Evidence suggests that acute lymphoblastic leukemias (ALL) are also dependent on glycolysis. We proposed that potent inhibition of glycolysis would induce growth arrest and cell death in ALL blasts. Furthermore, we hypothesized that anti-metabolic therapies (methotrexate and rapamycin) may synergize with the ATP depletion caused by inhibition of glycolysis.

Objective: To determine the effect of 3-BrOP on human precursor-B and T cell leukemias, and to evaluate combination therapy with methotrexate (MTX) and/or rapamycin.

Design/Methods: A panel of nine human pre-B ALL and T-ALL cell lines were treated with 3-BrOP to determine IC50 via cell counts, cell cycle and MTT analysis under conditions of hypoxia and normoxia. Subsequently combinations with methotrexate and rapamycin were evaluated.

Results: All pre-B ALL and T-ALL cell lines demonstrate growth arrest, decreased MTT metabolism and significant apoptosis when treated with 3-BrOP alone (IC50 10–30mcM) in both normoxia and hypoxia (0.5% 02). Indeed 40mcM 3-BrOP induced >90% death in all cell lines tested. Both pre-B ALL and T-ALL cell lines undergo enhanced apoptosis when 3-BrOP is combined with modest doses of methotrexate (7.5nM) or rapamycin (50nM), as compared to these agents alone. Specifically, at 72 hours exposure T-ALL MTT proliferation decreased from 20% with 3-BrOP alone to 1% with 3-BrOP + rapamycin (p=0.037). Furthermore, sub-G0 content increased from 44% with 3-BrOP alone to 75% with 3-BrOP + rapamycin (p=0.032). Similarly, at 48 hours of exposure, pre-B ALL cells had an increased sub-G0 content from 36% with 3-BrOP alone to 47% with 3-BrOP + rapamycin (p=0.025).

Conclusions: Inhibition of glycolysis via 3-BrOP induces growth arrest and apoptosis across a panel of human pre-B and T cell ALL cell lines indicating a generalized dependence on glycolysis. 3-BrOP is equally effective in both hypoxia and normoxia. When combined with methotrexate and/or rapamycin, 3-BrOP potentiates their anti-leukemia effects. These combinations offer promising therapeutic approaches in ALL and support further pre-clinical evaluation.

Figure Figure
Potential Role of Dual NF-κB and Oxidative Stress Pathway Inhibitor, OT-304 as a Novel Drug Resistance-Reversing Agent.
Authors: Mousa Shaker A. , Patil Ghanshyam , Rebbaa Abdelhadi
Published: October 2019

The development of resistance to chemotherapy represents an adaptive biological response by tumor cells that leads to treatment failure and patient relapse. During the course of their evolution (intrinsic resistance) or in response to chemotherapy (acquired resistance), tumor cells may undergo genetic alterations to possess a drug resistant phenotype. Dysregulation of membrane transport proteins and cellular enzymes, as well as altered susceptibility to commit to apoptosis are among the mechanisms that contribute to the genesis of acquired drug resistance. Recently, the development of approaches to prevent and/or to reverse this phenomenon has attracted special interest and a number of drug candidates have been identified. Despite strong effects observed for these candidates in vitro, however, most of them fail in vivo. In the present study, we have identified a novel small molecule inhibitor of dual NF-κB and oxidative stress pathways, OT-304, as a potential candidate to reverse drug resistance. Initial investigations indicate that this compound effectively inhibits proliferation of doxorubicin-sensitive and doxorubicin-resistant cells to the same extent, suggesting that it is capable of bypassing the development of drug resistance. Additional experiments reveal that OT-304 enhances cancer cell sensitivity to doxorubicin and to etoposide, particularly in cells characterized by the over-expression of the drug transporter P-glycoprotein. These findings suggest that either the expression/and or the function of P-glycoprotein could be affected by OT-304. In vivo studies using tumor xenografts in nude mice showed that OT-304 is also capable of preventing the growth of drug resistant cancer cells. This later finding further confirms the role of OT-304 as a drug resistance-reversing agent and warrants further pre-clinical and clinical investigation to determine its efficacy in treating aggressive tumors.
Simple, Gel-Based Determination of Nucleophosmin Mutations in Acute Myeloid Leukemias.
Authors: Ahrens Kim P.
Published: October 2019

Mutations in the Nucleophosmin, member 1 (NPM1) gene, have been shown to be significant for prognosis and treatment of cytogenetically normal (CN) patients with acute myeloid leukemia (AML). To increase sensitivity for minimal residual disease (MRD) detection, especially in AMLs without a distinct phenotype or genetic marker, some assays have specifically targeted the individual mutation, which has led to an increased complexity of these tests. Other assays use expensive equipment such as capillary electrophoresis to differentiate between the usual 4bp size difference in the mutated gene and the wildtype(wt) gene. We developed a simple PCR-based procedure that amplifies the exon 12 region of the NPM1 gene and produces a small product (156bp for the wt and 160 for the mutant genes) using RNA that can be separated on a 4% metaphor agarose gel. This approach is clinically relevant since it can easily distinguish greater than 95% of the mutations. Eight of 24 AMLs of various types were positive for the mutation using our technique and those that were cloned and sequenced yielded the most common insertion TCTG, confirming its accuracy.
A Novel Approach for Analyzing Gene Expression Profiles Defines Two Distinct Subgroups of t(4;11) Positive Infant Acute Lymphoblastic Leukemia Patients.
Authors: Stam Ronald W. , Bohlander Stefan , Kirschner-Schwabe Renate , Arentsen-Peters Susan , Pieters Rob , Dingermann Theo , Marschalek Rolf
Published: October 2019

Gene expression profiles (GEPs) are becoming increasingly more important for diagnostic procedures, allowing clinical predictions including treatment response and outcome for various types of cancer. However, established gene signatures not always appear helpful in understanding underlying disease mechanisms. Therefore, we explored an alternative approach for analyzing GEPs of a group of t(4;11) positive infant acute lymphoblastic leukemia patients (n=15), a highly aggressive type of MLL rearranged leukemia. We developed a method that uses a relational database program in combination with a normalization approach and specific discriminators. For normalization, every GEP of a given t(4;11) positive ALL sample was compared with 3 GEPs of normal bone marrow aspirates derived from healthy donors. Using the GeneChip Analysis Suite 5.0 program for single comparison analysis, the resulting gene lists were then compared with each other and only consistently identified genes (present on all three gene lists) were used for further analysis. This noise reduction decreased the amount of potentially deregulated target genes to about 30–40%. The remaining gene lists represented highly significant target genes that were then incorporated into a relational database program using specific discriminators. These discriminators were:

upregulation of HOXA9 and HOXA10, presence/absence of the AF4-MLL fusion transcripts in addition to the MLL-AF4 fusion, and the localization of the genomic breakpoint within the MLL gene.

This pilot study led to promising results, surprisingly classifying individual t(4;11) positive ALL patients into two distinct subgroups. Both subgroups share about 80 target genes, but also display particular sets of subgroup specific target genes. Importantly, these identified target genes can directly be linked to biological properties of t(4;11) positive leukemia cells, and therefore, allow important novel insights into this aggressive type of leukemia in infants.
Expression of the Human PIM-2 (hPIM-2) Gene in Patients with Acute Myeloblastic Leukaemia (AML) and Acute Lymphoblastic Leukaemia (ALL).
Authors: Kapelko-Slowik Katarzyna L. , Urbaniak-Kujda Donata M. , Jazwiec Bozena , Dybko Jaroslaw , Wolowiec Dariusz , Slowik Miroslaw , Frydecka Irena , Kuliczkowski Kazimierz
Published: October 2019

Background. A human PIM-2 is proto-oncogene that encodes serine/threonine kinase which interacts with various signalling molecules. hPIM-2 is highly expressed in haematopoietic tissues and in leukaemic and lymphoma cell lines which is consistent with a role during haematopoiesis and during oncogenic transformation. The aim. The aim of the study was to investigate whether the human PIM-2 is altered in acute myeloblastic leukaemia (AML) and acute lymphoblastic leukaemia (ALL). Patients. Thirty-seven patients were included: 22 with AML and 15 with ALL (10 with T and 5 with B subtype), aged 21–70 (x=41). Twenty-two patients reached complete remission (CR). Bone marrow samples were collected at the time of diagnosis. Control samples were obtained from 8 healthy donors.

Methods. We analyzed hPIM-2 expression by quantitative RT-PCR analysis. RNA was isolated from 3mlns mononuclear cells by using standard methods (TriPure Reagent, Roche Diagnostics). cDNA was synthesized by AMV reverse transcriptase (Finnzyme). Sequence for primers was forward, 5′-CTTTCCTTCCAATACCCCA-3′ and reverse, 5′-CCATCTTCCATTCCTTCCC-3′. As a n internal control c-ABL was used with primers c-ABL: 5′-CCCAACCTTTTCGTTGCACTGT-3′ and 5′-TGA CTGGCGTGATGTAGTTGCTT-3′. The intensity of each band was compared densitometrically.

Results. Expression of the hPIM-2 in all leukemic patients (n=37) and both subgroups: AML (n=22) and ALL (n=15) was significantly higher than in normals (p=0,0001; p=0,0009; p=0.0002, respectively). Patients who reached CR expressed hPIM-2 significantly lower than patients with primary resistance to chemotherapy (with no CR) (p= 0,01). Moreover we have found the correlation between hPIM-2 expression and patients age (r=0,45, p=0,005) and WBC in peripheral blood (r=0,34, p=0,04). Summary. PIM-2 expression is increased in patients with AML and ALL Our data indicate that expression of hPIM-2 could be considered as an additional prognostic marker in patients with AML and ALL.
Expression Level of Granulocyte-Colony Stimulating Factor Receptor and Effect of Two Forms of G-CSF to in AML1/ETO+ Acute Myelogenous Leukemia.
Authors: Moon Heewon , Kim Tae Young , Min Hyun Chung , Oh Bo Ra , Yoon Sung Soo , Kim Byoung Kook , Cho Han Ik , Lee D.S. ,   AML/MDS Working Party
Published: October 2019

In our previous study, we have revealed that cases showing remission after treatment with G-CSF mostly had leukemia with AML1/ETO rearrangement and suggested that this finding might be explained by higher expression of G-CSF receptor (G-CSFR) in AML1/ETO+ cells than in AML1/ETO− cells. To confirm the relationship between AML1/ETO fusion gene and G-CSFR expression, we applied small interfering RNAs (siRNAs) for efficient suppression of AML1/ETO gene expression and observed the change of G-CSFR expression. Moreover, we investigated the effect of most commonly used two forms of G-CSF (glycosylated lenograstim and nonglycosylated filgrastim) on AML1/ETO positive leuekemic cells and other cell lines. Kasumi-1 cells (AML with AML1/ETO gene rearrangement) were transfected with AML1/ETO specific siRNAs (siAM, siAGF1) and isolated for investigation of G-CSFR gene expression using real time PCR. The proliferation effect of two different forms of G-CSF on was evaluated using Cell-Titer 96® Non-Radioactive Cell Proliferation Assay (Promega Co., Madison, WI, USA). G-CSFR expression was decreased in AML1/ETO gene suppressed cells by both siRNAs. The levels of G-CSFR mRNA were 27% of untreated control with siAGF, 23% with siAM at 16 hours after trasnfection. Both forms of G-CSF significantly stimulated the proliferation of Kasumi-1 cells compared to control cells without G-CSF treatment (40–80% of control increased). The degree of proliferation was similar with different forms of G-CSF (filgrastim and lenograstim) at different concentrations (10, 50 and 100 ng/mL). In K562 cells(chronic myelogenous leukemia), proliferation was increased only at 50ng/ml filgrastim after 72 hours. The CTV-1 cells (AML without AML1/ETO gene rearrangement) showed slightly increased proliferation activities only after 72 hours of lenograstim treatment. In U266 cells (Multiple myeloma) showed no significant response on proliferation with G-CSF. In conclusion, our study revealed that AML1/ETO rearrangement increased the G-CSFR expression and G-CSF had proliferation effect on AML1/ETO-positive cells. Thus, the effect of G-CSF should be considered when we decide the remission or treatment plan, especially for AML1/ETO+ AML with high G-CSFR expression.
Influence of High Expression of p73 and p53 Proteins on Clinical Outcome in Acute Myeloid Leukemia Patients.
Authors: Pluta Agnieszka , Smolewski Piotr , Wierzbowska Agnieszka , Cebula Barbara , Jamroziak Krzysztof , Robak Tadeusz
Published: October 2019

Background: Prognostic significance of apoptosis-regulating proteins, especially p73, is not clearly determined in acute myeloid leukemia (AML). The p73 protein is a member of p53 family implicated in the regulation of cell cycle, apoptosis and development. Overexpression of p73 protein, with prevalence of short TAp73 isoforms, has recently been described in patients with AML.

Aims: The main objective of this study was to verify whether expression of p73 and p53 proteins has a prognostic impact on response to induction chemotherapy and overall survival (OS) of adult patients with AML. Additionally, we aimed to correlate the expression of p73 and p53 proteins with spontaneous apoptosis of leukemic cells.

Material and Methods: Intracellular expression of p73 protein in leukemic blasts isolated from bone marrow or peripheral blood was examined in 73 AML patients (59 de novo, 14 refractory/relapsed) of median age 54 years (range 28–78 years). In parallel, analysis of cell cycle in leukemic blasts was performed to assess the subG1 fraction as a marker of spontaneous apoptosis. The control constituted leukemic cells incubated with isotype antibodies. All measurements were performed using multi-colour flow cytometry. Protein expression was expressed by both percentage of positive cells and mean fluorescence intensity.

Results: Fifty six patients received intensive induction chemotherapy. Twenty four (43%) patients achieved complete response (CR), 26 (46%) patients did not respond and 6 (11%) patients died in the early post-induction period. The median time of follow up reached 5.6 months (range 0.1–99 months). High expression of p73 showed a trend towards probability of CR achievement after induction regimen (p=0.08). Higher expression of p53 did not correlate with response to the treatment (p=0.28). In univariate analysis we found significantly better OS in patients less than 60 years old, with good or intermediate risk of kariotype and treated with intensive chemotherapy (p=0.00001; p=0.04; p=0.00001, respectively). Probability of better OS in AML patients with high p73 and p53 protein expression was p=0.1 and p=0.95, respectively. Simultaneous high expression of both p73 and p53 proteins on AML cell showed a trend toward longer OS (p=0.09). Moreover, a strong trend to better OS was observed in patients with high subG1 fraction. Importantly, high subG1 fraction correlated with high expression of p53 but not with p73 protein expression (p=0.03; p=0.15, respectively).

Conclusions: These data indicate that p73 protein may be implicated in apoptosis of AML in response to chemotherapy, especially when p53 pathway is abrogated. Further studies are needed to establish exact role of p73 and p53 proteins in this process and its influence on OS of AML patients.
A Rapid Assay of Cytosine Arabinoside Uptake and Metabolism by Acute Myeloid Leukaemic Cells Using a Bioluminescent Bacterial Biosensor.
Authors: Smith Graham , Alloush Habib , Smith Margaret A. , Hill Philip J. , Salisbury Vyvian
Published: October 2019

We have already reported the construction and evaluation of a self-bioluminescent reporter strain of E. coli with high sensititivity to the nucleoside analogue cytosine arabinoside (Ara-C) the mainstay of treatment in patients with Acute Myeloid Leukaemia (Blood, 106 (11), Abstr. 2473; 695a). This rapid assay, which uses a bioluminescent biosensor to measure intracellular levels of Ara-C and its active metabolite Ara-CTP, has been utilised in leukaemic cell lines KG1a (FAB type M0) which is sensitive to Ara-C and THP-1 (FAB type M5) which is partially insensitive to Ara-C due to over expression of cytidine deaminase (cdd). It has also been used in primary cultures of AML cells from six patients with M0 (n=1), M2 (n=2), M3 (n=1) and M4 (n=2). Intracellular concentrations of Ara-C as low as 0.05 μM can be detected by an increase in light output from the bacterial biosensor. The technology is suitable for the quantitation of response to clinical Ara-C doses (between 25–100μM) in less than 8 hours. Results from the KG1a cell line with known sensitivity to Ara-C showed correlation between the rapid assay and a 3 day cytotoxicity test of Ara-C sensitivity. Preliminary tests with the six clinical marrow samples showed 100% concordance with clinical outcome within eight hours of commencing the assay (vide infra: Table 1). In newly diagnosed AML patients, only 70% are sensitive to Ara-C therapy. Interestingly, in our hands, the M3 patient was Ara-C insensitive despite the fact that, most commonly, M3 patients do not express p-glycoprotein and therefore do not require Ara-C. Our small cohort of patients were disproportionately insensitive to Ara-C and therefore had a poor outcome overall. This assay system is potentially suitable for prescreening AML samples prior to commencement of Ara-C containing chemotherapy regimens. It may also give information on relative Ara-C sensitivity thus permitting dose adjustment and prevention of unnecessary toxicity to other organs. The assay could be repeated at relapse to document clonal evolution and decide whether Ara-C can be used to achieve a second remission. Work is currently underway with an industrial partner to look at commercialisation of this assay.

TABLE 1: Correlation between light output and clinical response in six patients with primary AML Patient Diagnosis Clinical Outcome Differential Light Output: cytotoxicity test Bioluminescent Assay: Ara-C sensitivity 1 M0 No response 5% no 2 M2 No Response 84% no 3 M2 No Response 79% no 4 M3 Remission Death 91% no 5 M4 Complete Response 99% yes 6 M4 No Response 62% no
Preliminary Study on Effects of Marsdenia Tenacissima Injection on NB4 Cells.
Authors: Li Dong , OuYang Jian , Li Cuiping , Chen Junhao
Published: October 2019

Purpose To study the effect of Marsdenia tenacissima injection on NB4 leukemia cells and its mechanisms.

Methods After treated with Marsdenia tenacissima, viability of NB4 leukemia cells were determined by trypan blue assay. Cell cycle was assessed by FACS. Cell differentiation were determined by cell morphology, cell surface differentiation antigen and NBT reduction test. Cell cycle and apoptosis associated proteins were measured by western blot.

Results Marsdenia tenacissima inhibits the growth of leukemia cells in a dose and time dependent manner. Marsdenia tenacissima induces G0/G1 phase arrest of NB4 cells, accompanied by P21 up-regulation. Marsdenia tenacissima did not change the protein level of PARP and p27. Marsdenia tenacissima could increase the expression of CD11b and percent of NBT positive cells, induces differentiation of NB4 cells.

Conclusion Marsdenia tenacissima could inhibit the proliferation of NB4 cells through inducing cell cycle arrest and cell differentiation.
Prognostic Value of Immunophenotyping in Elderly Patients with Acute Myeloid Leukemia: A Single Institution Experience.
Authors: Plesa Claudiu , Chelghoum Youcef , Plesa Adriana , Elhamri Mohamed , Tigaud Isabelle , Michallet Mauricette , Dumontet Charles , Thomas Xavier
Published: October 2019

The poor prognosis of elderly patients with acute myeloid leukemia (AML) raises the question about the benefit of intensive chemotherapy in these patients. The impact of initial characteristics on prognosis has previously been addressed in elderly paitents, but very few data are available regarding the prognostic value of immunophenotypic characteristics in this setting. We investigated the expression of the membrane antigens CD13, CD15, CD33, and CD34 by flow cytometry in elderly patients with newly diagnosed AML, and analyzed whether these parameters possessed clinical or prognostic relevance, in order to help physicians in their choice of therapy. Immunophenotyping was performed in 273 patients aged 60 years or more (median 69 years). CD13 was expressed in 73%, CD15 in 43%, CD33 in 64%, and CD34 in 66% of cases. Complete remission was obtained in 157 cases (58%). The median overall survival was 8.1 months with a 3-year survival rate of 14%. Three risk groups were defined based on CD34 and CD33 antigen expression: Poor risk in patients with CD34+ CD33+ or CD34− CD33− disease, intermediate risk in patients with CD34+ CD33− disease, and favorable risk in patients CD34− CD33+ disease. Immunophenotype was, after cytogenetics, the most significant prognostic factor in terms of survival in a multivariate analysis (p = 0.03 and p < 0.0001 respectively). When combining immunophenotypic and cytogenetic parameters, patients were classified into four prognostic groups: Group A (3-year survival: 33%) including favorable and normal karyotypes with a favorable immunophenotype; Group B (3-year survival: 28%) including normal karyotypes with an intermediate immunophenotype; Group C (3-year survival: 8%) including intermediate or normal karyotypes with an unfavorable immunophenotype; and Group D (3-year survival: 2%) including all unfavorable cytogenetics. Immunophenotypic characteristics appeared to be a major prognostic factor in this patient population. Using two simple parameters assessed at time of diagnosis, we devised a prognostic system of immediate clinical utility for prognostic stratification and risk-adapted therapeutic choices.
Efficacy of Mitoxantrone and Etoposide in Patients with Acute Myeloid Leukemia Refractory to Initial Standard Induction Therapy.
Authors: Sehgal Rajesh , McHayleh Wassim , Natale James , Raptis Anastasios , Agha Mounzer , Redner Robert L. , Richard Gene , Welsh Ann , Foon Kenneth A. , Boyiadzis Michael
Published: October 2019

The most effective reinduction regimen for acute myeloid leukemia (AML) patients who do not achieve complete remission (CR) after one cycle of cytarabine combined with an anthracycline is not well established. In an effort to search for new synergistic and non-cross resistant antileukemic regimens different chemotherapeutic combinations have been investigated in refractory AML patients. Multiple regimens including high dose cytarabine, anthracyclines, fludarabine and etoposide have been used with CR rates up to 40%. Mitoxantrone and etoposide have activity in AML as induction agents but their role in reinduction in patients not responding to first line therapy has not been fully established. In the current retrospective study we evaluated the efficacy and toxicity of mitoxantrone and etoposide in AML patients treated at our institution who did not respond to first induction therapy with cytarabine and an anthracycline. A total of fifty seven AML patients were treated with mitoxantrone and etoposide (mean age 55 years, range 18–75 years). Twenty four patients were treated with mitoxantrone 10 mg/m2/d and etoposide 100 mg/m2/d both administered intravenously, days 1 to 5 (regimen 5+5) and thirty three patients were treated with mitoxantrone 10 mg/m2/d administered intravenously days 1 to 3 and etoposide 100 mg/m2/d administered intravenously, days 1 to 5 (regimen 3+5). Twenty six of fifty seven patients (46%) achieved CR. CR was achieved in 38% of patients (9/24) treated with the 5+5 regimen and 52% of patients (17/33) treated with the 3+5 regimen. Mean blast percentage before treatment with mitoxantrone and etoposide was 25% in patients who achieved CR vs 40% in patients who did not achieve CR (p < 0.03). Grade 3/4 hepatic toxicities were seen in 5% (3/57) of patients and there were no grade 3 or 4 renal toxicities. The median duration of neutropenia in patients achieving CR was 29 days after reinduction. 10% (6/57) of the patients died from infectious complications. Cytogenetic analyses were performed prior to first-line therapy in all patients. Patients with favorable cytogenetics treated with etoposide and mitoxantorne had 100% CR (3/3), patients with standard cytogenetics had 58% CR (19/33) and patients with unfavorable cytogenetics had 19% CR (4/21). Overall CR was achieved in 61% (22/36) of patients with favorable and standard cytogenetics. Our data suggest that the combination of etoposide and mitoxantorne is an active and well tolerated regimen, especially in patients with favorable and standard cytogenetics, and warrants further studies in prospective trials.
Combination Methyltransferase and Histone Deacetylase Inhibition in Elderly Patients with Secondary Acute Myelogenous Leukemia.
Authors: Thatikonda Chandanda , Rossetti James M. , Shadduck Richard K. , Lister John
Published: October 2019

Background: Secondary acute myelogenous leukemia (AML) carries a poor prognosis when compared to de novo AML. Therapeutic options are typically limited as patients are often elderly with comorbidities that preclude intensive chemotherapy. Moreover, resistance to therapy is common. Studies have shown that methyltransferase inhibitors (MTI) have activity in myeloid malignancy. It is not known if addition of histone deacetylase (HDAC) inhibitors improves or prolongs the activity of MTI. We present our experience of 3 elderly patients with secondary AML treated with MTI (azacitidine or decitabine) in combination with an HDAC inhibitor (vorinostat).

Methods: Patients include 2 males and 1 female, ages 78 yrs, 82 yrs and 71 yrs. All were initially diagnosed with high-grade myelodysplastic syndrome (MDS), which transformed to CD34+ secondary AML at 33, 14 and 4 months from diagnosis of MDS, respectively. One patient had trisomy 8. Patients received 8, 3 and 4 cycles of MTI before leukemic transformation, respectively. At transformation, one patient failed standard induction therapy and 1 patient failed arsenic trioxide. Both of these patients were then placed back on MTI as maintenance therapy. Vorinostat was added at a dosage of 400 mg daily at MTI cycles 8, 3 and 1 post transformation, respectively. The number of cycles of combination therapy was 6, 9 and 4. Gastrointestinal intolerance was an issue in 2 of these patients: nausea (n=2), diarrhea (n=2), vomiting (n=1) and loss of appetite (n=1). Dose reduction to 100mg in one patient and 300 mg in another resulted in some symptomatic relief. The addition of 5-HT3 antagonists or low dose prednisone allowed dose escalation. One patient had 50% reduction in marrow blasts, 1 had near clearance of the peripheral blasts and 1 had a transient minor platelet response. All 3 patients are alive at 14, 10 and 9 months post transformation (6, 8 and 7 months after addition of vorinostat) with no disease progression, respectively.

Conclusion: The combination of MTI and HDAC could be an option in elderly patients with secondary AML without increasing morbidity and mortality. Treatment appears well tolerated when premedication with 5-HT3 antagonists is employed. As the treatment options are limited in high-risk elderly patients with secondary AML, larger studies are needed to investigate the utility of this combination.
Bortezomib (Velcade™) Observational Study in China - Efficacy and Safety of Bortezomib Based Regimen in 223 Relapsed or Refractory Multiple Myeloma Patients.
Authors: Shen Zhi-xiang
Published: October 2019

Introduction: Multiple myeloma (MM) is a plasma-cell malignancy with approximately three years’ median survival. Patients usually relapse or become refractory to existing treatments. Clinical studies have confirmed that bortezomib is an effective agent for relapsed MM patients and has potent synergy with other chemotherapeutic agents, which provides a new clinical regimen that may improve response. In this observational study, we intend to observe the safety/tolerability and efficacy of bortezomib-based regimens in Chinese patients with relapsed/refractory MM.

Methods: This was a multicenter, open-label, phase IV observational study for patients with relapsed or refractory MM. Bortezomib (0.7 to 1.6 mg/m2 i.v.) was given on days 1, 4, 8, and 11 of a 21-day cycle, for a maximum of 8 cycles, combined with other agents, mainly of dexamethasone addition. If pyretic neutropenia, grade 4 hematologic adverse events (AEs), or grade ≥3 non-hematologic AEs developed, bortezomib treatment was temporarily discontinued and re-administered at 75% of the initial dosage after remittance of the AEs. Responses were classified mainly by European Group for Blood and Marrow Transplantation criteria (EBMT).

Results: Between Mar 2006 and May 2007, 223 patients with MM were enrolled at 31 medical centers. Median age was 58 years (range 35–82), 96% of patients were in stage II/III, and the most common subtype was IgG (46%). Patients had received various prior therapies such as VAD (29% of patients), VBMCP (M2) (16%), and thalidomide combinations (15%). There were 198 patients evaluable for response, of whom 54 (27%) achieved a complete response (CR), 99 (50%) achieved a partial response (PR), 20 (10%) had minimal response (MR), 16 (8%) had stable disease (SD), and the other 9 (5%) had progressive disease (PD). Patients who received 4 or more cycles of bortezomib achieved a higher CR rate (56%) compared with patients who received fewer cycles (partly due to adverse event). Rapid responses were seen with the median time to response being 1 cycle (range 1–5). Common predictive factors including age, isotype, number of previous therapies, concentration of C-reactive protein, or beta2-microglobulin had no significant influence on treatment response. The incidence of adverse events (AEs) was 54%, including 18% of patients with hematologic AEs (33% of them grade 3–4), 27% with gastrointestinal AEs (50% of them grade 3–4), and 20% with peripheral neuropathy (40% of them grade 3–4). Serious AEs occurred in 25 (11%) patients. Most AEs were predictable and manageable.

Conclusions: These data demonstrate that a bortezomib-based regimen is a promising regimen with high response rate and is well tolerated in most relapsed and refractory MM patients. Additional clinical trials and long-term follow-up on Chinese patients are warranted.
Oral Acyclovir Is an Effective in Decreasing the Incidence of Bortezomib Associated Herpes Zoster in Patients with Multiple Myeloma.
Authors: DePaolo Dawn , Miller Kena C. , Whitworth Amy , Mohr Alice , Mashtare Terry L. , Wilding Gregory , Ailawadhi Sikander , Padmanabhan Swaminathan , Chanan-Khan Asher A.
Published: October 2019

Introduction: Bortezomib, a proteasome inhibitor is an effective antimyeloma therapy. We recently reported that multiple myeloma patients treated with bortezomib or bortezomib based regimen resulted in a higher incidence of herpes zoster (HZ). The exact etiology of this side effect remains unknown though our investigation demonstrated prolonged lymphopenia among patients treated with bortezomib. The high incidence of HZ among bortezomib treated patients prompted to prospectively investigate the role of antiviral prophylaxis in this patient population. Here we report for the first time the efficacy of acyclovir as prophylaxis for bortezomib associated HZ.

Patients and Methods: We prospectively evaluated the impact of oral acyclovir (400mg PO twice daily for the duration of bortezomib therapy) on the incidence of HZ. All patients with multiple myeloma who were treated with bortezomib or bortezomib based regimens, and received prophylactic acyclovir were evaluable for this analysis. To compare the overall proportion of HZ among patients who received acyclovir prophylaxis and the historical control, Fisher exact test was used.

Results: A total of 51 consecutive patients (27 M and 24 F) received acyclovir as prophylaxis. The median age was 61 (range 40–81 years), with advance stage MM noted in 86% (n=44) patients. Among these 69% had previously untreated MM while 31% had relapsed or refractory disease. Single agent bortezomib was given to 11 patients and 40 patients received bortezomib in combination with other antimyeloma agents (such as thalidomide, pegylated liposomal doxorubicin, dexamethasone, cyclophosphamide and/or lenalidomide). The overall incidence of HZ was 0% among patients receiving the acyclovir prophylaxis vs. 13% in the historical control. There was a significant difference in the overall proportion of HZ among patients who received acyclovir prophylaxis and the historical control (p = 0.0026).

Conclusion: This is the first report on the efficacy of acyclovir for the prevention of bortezomib associated HZ in MM patients. While bortezomib is an effective antimyeloma therapy, an important side effect with significant morbidity is reactivation of HZ. Here we demonstrate for the first time that this side effect can be effectively prevented by oral acyclovir. Our observation warrants further evaluation. Considering significant morbidity associated with HZ we recommend the routine use of prophylactic acyclovir (or other antiviral agents) for patients being treated with bortezomib or bortezomib-based therapies.
Regulatory T Cells Do Not Affect Human Hematopoietic Stem Cell Engraftment and Prevent T Cell Alloreactivity Against CD34+ Cells: A Preclinical Study.
Authors: Mahmud Dolores , Chunduri Sandeep , Mahmud Nadim , Van Den Dries Lennert , Maciejewski John J. , Rondelli Damiano
Published: October 2019

We have previously demonstrated that allogeneic blood T cells stimulate cord blood (CB) CD34+ cell differentiation into professional antigen presenting cells (APC) in-vitro and in-vivo (Abbasian J, Blood2006:108:203–208). In this study we immunomagnetically selected human CD4+CD25+ regulatory T cells (Tregs) and showed that >80% of these cells were positive for FoxP3 intracellular expression. Then we tested whether Tregs may affect CB CD34+ cell clonogenic activity in-vitro an in-vivo, and if co-incubation of Tregs and CD34+ cells may modify the phenotype and function of Tregs. A colony-forming cells (CFU-C) assay performed with CD34+ cells mixed with allogeneic Tregs at 1:2 ratio resulted in comparable numbers of Granulocyte- Macrophage CFU (CFU-GM), burst-forming unit-erythroid (BFU-E) and CFU-Mix as compared to cultures with CD34+ cells alone (p=0.2, p=0.5 and p=0.5, respectively)(n=3 exps). Human CD34+ cells were co-transplanted with human CD4+CD25+ allogeneic Tregs into NOD/SCID mice at 1:1 and 1:2 ratio. After 6 weeks mice marrow was harvested and showed a 1.3±1.1% (n=3 mice) and 1.6±0.8% (n=4 mice) engraftment of huCD45+ cells, respectively, which was comparable to the engraftment observed in control animals transplanted with CD34+ cells alone (1.4±0.4). In addition, among the engrafted huCD45+ cells similar proportion of CD33+ myeloid cells, CD14+ monocytes and CD1c+ dendritic cells were observed in the three groups of animals. Mixed lymphocyte culture (MLC) experiments showed that irradiated CD34+ cells stimulated brisk proliferative responses of CD4+CD25- cells (S:R=1:2), but did not induce any proliferation of Tregs (n=3 exps). After incubation with CD34+ cells in the presence of IL2, on average >80% CD4+CD25+ cells maintained the intracellular expression of FoxP3 and surface expression of CD62L and CD152 (n=3 exps). Then, Tregs autologous to CD34+ cells were isolated from the CB CD34- cell fraction while allogeneic Tregs were isolated from healthy individuals’ peripheral blood. When 2.5 x 104 autologous or allogeneic Tregs were added to an MLC with 2.5 x 104 irradiated CD34+ stimulator cells and allogeneic responders at 1:2 ratio, they suppressed T cell alloreactivity to CD34+ cells on average by 68±14% and 41±16%, respectively (n=3 exps). Our findings suggest that co-transplantation of CD34+ cells and autologous or allogeneic Tregs may allow normal stem cell engraftment while limiting T cell alloreactivity. These results will prompt the design of new strategies in allogeneic hematopoietic stem cell transplantation, particularly in an HLA incompatible setting.
A New Mice Model of Graft Versus Host Disease Based on Chemotherapy Conditioning.
Authors: Sadeghi Behnam , Hassan Zuzana , Aghdami Nasser , Rozzel Bjorn , Abedi-Valugerdi Manuchehr , Hassan Moustapha
Published: October 2019

Background: Transplantation related complications such as graft versus host disease (GVHD) are limiting factors for allogeneic haematopoietic stem cell transplantation (HSCT). The majority of mice models of GVHD are based on the use of radiotherapy as a conditioning regimen. However, these models may not explain mechanisms of HSCT related complications following chemotherapy-based conditioning regimen.

Aims: The aim of this study was establishment of a mice model of GVHD using the chemotherapy as conditioning regimen.

Methods: Recipient female BALB/c (H-2Kd) were conditioned using busulfan (Bu) in total dose of 80 or 100 mg/kg followed by cyclophosphamide (Cy) in total dose of 200 or 300 mg/kg. Twenty millions of bone marrow (BM) cells and 30×106 spleen cells of male C57BL/6 (H-2Kb) were injected into the recipient mice in allogeneic setting. As a control, syngeneic HSCT using female BALB/c as recipients and donors and the same conditioning regimen and cell doses as in allogeneic setting was used. Acute GVHD manifestations and histo-pathological changes in skin, liver and intestine were evaluated at different time points after BMT. The chimerism and tissue mapping were studied by FACS, immunohistochemistry and FISH analysis. Affected skin was examined by FACS analysis to find donor T cells. Cytokines level were measured by using luminex assay.

Results: Infusion of 20×106 BM cells and 30×106 spleen cells from C57BL/6 mice to BALB/c mice conditioned with 80 or 100mg/kg Bu plus 200mg/kg Cy induced lethal acute GVHD between days 5–7 post transplantation. Histological and clinical signs of GVHD were found in 85% and 100% of the allogeneic transplanted mice based on the intensity of conditioning. About 35% of mice in allogeneic group died due to GVHD progression. The number of donor T cell in the recipient skin increased with GVHD progression and was correlated to the level of chimerism in the spleen but not in the BM. CD3+ CD8+ T cells infiltration and apoptosis were found in the skin, intestine and liver of mice suffering from GVHD. None of the syngeneic transplanted mice died or developed any sign of GVHD even in long term follow up, and CD3+CD8+ T cells were absent in their tissue samples. Increasing the dose of Cy in conditioning regimen in allogeneic transplanted mice increased mortality of animals.

Conclusion: We have established a new mouse model of acute GVHD using chemotherapy based conditioning. This model can be utilized to study the basic mechanisms underlying GVHD triggered by cytostatics used in the conditioning regimen and affecting different cell sub-populations and cytokine storm.

Figure Figure Figure Figure
Delivery of mRNA into Dendritic Cells Using Novel Transfer Peptides Leads to Prolonged Antigen Expression and Potent Immune Responses In Vivo.
Authors: Woan Karrune , Heiser Axel , Dahm Philipp , Vieweg Johannes , Su Zhen
Published: October 2019

We have previously shown that vaccination with RNA-transfected DC is a potent strategy to stimulate CTL and antitumor immunity in cancer patients. In this study, we investigated whether novel transfer peptides derived from the RNA-binding region of the HIV-1 nucleocapsid protein could be utilized for effective delivery of mRNA into human monocyte-derived dendritic cells (DC). Here we show that both peptide-mediated mRNA delivery and electroporation of DC with mRNA resulted in efficient gene transfer. However, the use of transfer peptides led to prolonged antigen expression and did not negatively affect the viability of DC, the migratory capacity of matured DC, and the production of cytokines by these cells in vitro. In murine studies, DC loaded with transfer peptide-mRNA complexes were clearly superior, compared to mRNA-electroporated DC, in stimulating antigen-specific CTL, CD4+ T cell, and antibody responses. Importantly, no transfer peptide-specific cellular or humoral immune responses were detected in vaccinated mice. Our data suggest that vaccination with transfer peptide-mRNA-loaded DC may represent a promising strategy to stimulate potent anti-tumor immune responses in a vaccination setting.
Lenalidomide Enhancement of NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity (ADCC) Is Mediated by Granzyme B and FasL and Is Associated with Modification of SHIP-1, PLC-g2 and pERK and Enhanced Chemokine Production.
Authors: Wu Lei , Adams Mary , Schafer Peter , Muller George , Stirling David , Bartlett Blake J.
Published: October 2019

Lenalidomide is an oral anti-angiogenic, anti-proliferative and immunomodulatory drug that is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk MDS associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone for the treatment of previously treated multiple myeloma patients. Encouraging early results suggest a potential for clinical efficacy in B cell non-Hodgkin’s lymphoma (NHL) and chronic lymphocytic leukemia (CLL). Lenalidomide has been shown to enhance Th1-type cytokines and T cell and NK cell activation markers in patients with advanced cancers. Lenalidomide has been shown to enhance rituximab-mediated protection in a SCID mouse lymphoma model in vivo and we have previously shown that in an in vitro ADCC system lenalidomide is able to directly enhance human NK cell and monocyte function in response to rituximab (chimeric anti-CD20 mAb) and other IgG1 antibodies. We have now addressed the effect of lenalidomide on NK cell signaling pathways and have shown that compared to the levels seen with FC gR stimulation (with IgG) plus IL-12, there is an inhibitory effect of lenalidomide on SHIP-1 with concomitant enhancement of PLC g2 and pERK. This is associated with increased secretion of a variety of chemokines as well as GM-CSF and decreased IL-6 production. In functional ADCC assays, NHL cell lines were pre-coated with rituximab and exposed to either NK cells or monocytes pre-treated with lenalidomide. After 4 (NK cells) or 15 (monocytes) hours in culture the viability of the tumor cells was assessed. The consistent enhancement of ADCC in the presence of lenalidomide is associated with greatly increased expression of granzyme B and FasL, although perforin is maximally elevated without lenalidomide. We found that ADCC is totally blocked by an inhibitor of granzyme B and is partially blocked by anti-FasL. In conclusion, we have shown that the ability of lenalidomide to enhance ADCC-mediated killing of rituximab treated CD20+ve NHL cell lines is mediated by granzyme B and FasL and is associated with clear effects on NK cell signaling and chemokine production.
Monosodium Urate Crystal as a Carrier for Idiotype Tumor Vaccine.
Authors: Sakamaki Ippei , Inai Kunihiro , Ueda Takanori , Tsutani Hiroshi
Published: October 2019

Monosodium urate (MSU) crystal works as a danger signal to evoke specific cell immunity. It stimulates dendritic cells (DCs) maturation and enhances the generation of cytotoxic T cells (CTL), so that it could help specific immune therapy for tumor. On the other hand, MSU crystals are found to attach IgG in gouty arthritis and has been reported that the site of attachment is the Fab moiety of IgG. We focused on the application of MSU as a carrier of IgG idiotype protein to induce cytotoxic T cells to cell line producing the idiotype protein. Multiple myeloma cell line, IM-9, which is characterized by having HLA-A2 and producing monoclonal IgG-k protein, was used as a target cell. Monoclonal Fab fragments were used as idiotype protein; those were made by digesting with papain of IgG collected from the medium harvesting IM-9 cells. MSU crystals were made by sitting at refrigerator of uric acid dissolved by sodium hydroxide solution with boiling. Allogenic dendritic cells were derived from monocytes in peripheral blood of HL-A matched volunteer by treatment with GM-CSF and IL-4. They were pulsed by MSU attached with Fab fragments (MSU-Fab), by Fab fragments (Fab), or by none. Following education by pulsed DCs to CD3+ T cells collected from peripheral blood of same volunteer, CD8+ T cells were collected and were used as effector cells. Cell cytotoxic ability of the effector cells was determined by lactate dehydrogenase release assay under mixed culture of the target cells and the effector cells (CD8+ cells: IM-9 cells = 5: 1). Compared to Fab, MSU-Fab were easily phagocytosed by DCs. DCs pulsed with MSU-Fab or MSU were induced to express CD83 molecule, a marker for mature human blood dendritic cells, whereas neither Fab-pulsed nor untreated-control DCs did not express CD83 molecule. CD8+ T cells educated by DCs pulsed with MSU-Fab significantly killed more IM-9 myeloma cells than CD8+ T cells educated by DCs pulsed with MSU, DCs pulsed with Fab or DCs alone. These cytotoxic effect of CD8+ T cells educated by DCs pulsed with MSU-Fab were reduced when MSU crystals were bathed 10% fetal bovine serum solution before mixing with Fab in order to block MSU to be attached with Fab. The present study revealed that MSU crystals work as carrier of idiotype tumor vaccine not only by facilitating the utilization of the vaccine protein in DCs but also by enhancing the ability to induce tumor specific CTL. The safety and efficacy of MSU-Fab idiotype vaccine will be determined by clinical setting in patients with multiple myeloma.