bluuebunny/crossref_metadata_2025 huggingface dataset:
Correlation Between Soluble Vascular Endothelial Growth Factor-a and Vascular Endothelial Growth Factor-Receptor 1 with Response to Chemotherapy in Childhood Acute Leukemia
Authors: Mehrazma Mitra , Rafsanjani Khadije Arjomandi , Torkamanipoor Behzad , Keyhani Manoutchehr
Published: April 2020

Objective: Vascular endothelial growth factor (VEGF) and its receptors (VEGF-R1 and R2) are major regulators of angiogenesis, which plays a key role in the growth and dissemination of solid tumors and hematological neoplasms.

Materials and methods: To assess serum levels of VEGF and VEGF-R1 and their prognostic significance in newly diagnosed childhood acute leukemia, we used an Enzyme linked Immuno-Sorbant Assay (ELISA) to quantify their levels in stored samples obtained before treatment. Demographic data were recorded. Bone marrow blast percentage was counted at day of diagnosis and 2 weeks after induction therapy. The data entered and analyzed with SPSS11 software. P value under 0.05 assumed significant.

Results: Fifty-three children (22boys and 31girls) with newly diagnosed ALL with median age of 6.4±3.72 years were enrolled into the study. Most cases (56.6%) were pre B cell ALL. Mean value of VEFG-A in good responders was 55.13±24.96 pg/ml and in poor responders was 94.46±15.75 (P<0.0001). Mean valve of VEGF-R1 in good and poor responders were 0.132±0.0653 and 0.1665±0.0857 pg/ml, respectively (P>0.05). By using ROC curve, we found cut off point 76 pg/ml with sensitivity 99 % and specificity 81 % to discriminate poor response to chemotherapy.

Conclusion: Soluble VEGF-A is an independent factor of response to therapy in childhood leukemia and leukemic patients with sVEGF-A level over 76pg/ml, will have poor response to treatment.
Monosodium Urate Crystals Bind with Idiotype Protein and Promote Dendritic Cells to Induce Cytotoxic T Cells in Vitro
Authors: Sakamaki Ippei , Inai Kunihiro , Ueda Takanori , Tsutani Hiroshi
Published: April 2020

Monosodium urate (MSU) crystals have been studied to act as a key substance in local immunoreactions. MSU released from damaged cells works as an endogenous danger signal to antigen-presenting cells. MSU crystals evoke specific cell immunity and work as an adjuvant in a mouse model. The crystals also have another unique characteristic to bind with positively charged proteins, which could help to deliver some antigens into human dendritic cells (DCs). We focused on the application of MSU crystals as a not only an adjuvant but also as a carrier of positively charged antigenic protein to induce human cytotoxic T cells (CTLs) efficiently in vitro. We confirmed that MSU crystals facilitated human DCs to express the maturation marker, CD83, deliver (Fab′)2 attaching to the crystals. In order to determine whether MSU crystals facilitate the T-cell proliferation activity of DCs, the proliferative effects of DCs on allogeneic CD4+ cells were investigated. DCs pulsed with MSU crystals significantly facilitated the proliferation of allogeneic CD4+ cells when compared to DCs alone. The stimulation index (SI) was 2.5 ± 0.1 and 1.7 ± 0.1, respectively. When using DCs pulsed with the Fab attached to MSU crystals, the proliferation of CD4+ cells was significantly greater than when using DCs pulsed with Fab alone. The SI was 2.6 ± 0.2 and 1.9 ± 0.1, respectively. No significant differences were seen in the proliferation of allogeneic CD4+ cells between DCs pulsed with the Fab attached to MSU and DCs pulsed with MSU alone. We selected the multiple myeloma IM-9 cell line and its product idiotype (Id) protein as an ideal pair of target cells and positively charged tumor-specific antigen, respectively. After sensitizing DCs derived from HLA-A matched volunteers pulsed with tumor-specific monoclonal IgG-Fab fragments (IM-9 Fab) attached to MSU crystals, the CD8+ T cells stimulated by the DCs killed significantly more target cells (38.5 ± 3.5%, n=4) than those stimulated by DCs pulsed with IM-9 Fab alone (3.5 ± 7.5%). These cytotoxic effects of CD8+ cells stimulated by the DCs pulsed with IM-9 Fab attached to MSU crystals were reduced (3.6 ± 1.7%) when MSU crystals were pre-coated with fetal bovine serum to block to bind with IM-9 Fab. For efficient induction of CTLs, it is necessary for Id proteins to attach to MSU crystals. MSU crystals have some advantages of a protein carrier binding with positively charged proteins and delivering antigenic protein into DCs, as well as an adjuvant promoting DC maturation and inducing CTLs.
Innate Immunity in Idiopathic Thrombocytopenic Purpura (ITP)
Authors: Saito Akio , Yokohama Akihiko , Nakahashi Hirotaka , Toyama Kohtaro , Mitsui Takeki , Hashimoto Yoko , Koiso Hiromi , Saitoh Takayuki , Uchiumi Hideki , Handa Hiroshi , Sawamura Morio , Karasawa Masamitsu , Murakami Hirokazu , Nojima Yoshihisa , Tsukamoto Norifumi
Published: April 2020

Background: Dendritic cells (DCs), natural killer (NK) cells, and invariant NKT (iNKT) cells play important roles in innate immune systems. These cells have recently been shown to be involved in immunoregulation, and some studies have suggested associations with various kinds of autoimmune disease. Conversely, regulatory T cells (Tregs) that are important for peripheral tolerance and Th17 cells that play a central role in maintenance of chronic inflammation are also associated with the pathogenesis of several autoimmune diseases. Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease mediated by anti-platelet autoantibodies, but relationships to innate immunity are unclear. In addition, the pathogenesis of ITP associated with Helicobacter pylori remains obscure. In particular, the regulation of immune responses by these cells in patients infected with H. pylori has not been investigated. This study analyzed DCs, NK cells, iNKT cells, Tregs and Th17 cells in patients with ITP.

Methods: Subjects comprised 31 patients with ITP and 22 healthy donors. Study protocols were approved by the Institutional Review Board of Gunma University Hospital, and written informed consent was obtained from all subjects. Flow cytometry was used to investigate amounts of circulating plasmacytoid DCs (pDCs) (CD123+ HLA−DR+) and myeloid DCs (mDCs) (CD11c+ HLA−DR+) from whole white blood cells, and NK cells (CD3− CD56+), iNKT cells (Vα24+ Vβ11+), Tregs (CD4+ CD25+ Foxp3+) and Th17 cells (CD4+ IL−17A+) from mononuclear cells. The intracellular interleukin (IL)-17A production in CD4+ T-cells activated by phorbol 12-myristate 13-acetate (PMA) and ionomycin was assessed to detect Th17 cells.

Results: Both the percentage and numbers of pDCs were significantly reduced in patients compared to healthy controls (p<0.001), while those of mDCs tended to be lower in patients than in controls, but with no significant differences. NK cell counts tended to be higher in patients than in controls and counts of iNKT cells tended to be lower in patients than in controls, but again no significant differences were demonstrated. Notably, Treg levels were comparable between patients and controls, while Th17 cells were significantly increased in patients compared with controls (p<0.002). In all cases, no significant differences were demonstrated between patients with H. pylori-positive and -negative results.

Conclusion: These results suggest that alterations in innate immunity as a reduction of pDCs could be associated with the pathogenesis of ITP. Furthermore, as in some autoimmune diseases that have been considered as Th1 diseases, Th17 cells may play an important role in ITP.
Inhibition of T-Cell Activation by a CEACAM1 (CD66a) Peptide
Authors: Skubitz Keith M. , Skubitz Amy P.N. , Kammerer Robert
Published: April 2020

Carcinoembryonic antigen family (CEACAM) members are widely expressed, and depending on the tissue, capable of regulating diverse functions, including tumor promotion, tumor suppression, angiogenesis, neutrophil activation, and T-cell activation. CEACAM1, a member of the carcinoembryonic antigen family, is expressed on human T-cells and is recognized by CD66a mAb. CD66a mAb binding to the T-cell surface inhibits T-cell activation. To identify active sites on CEACAM1, molecular modeling was performed using IgG and CD4 as models, and 28 peptides of 14 amino acids in length were synthesized that were predicted to be present at loops and turns between beta-sheets. The peptides were tested for their ability to alter T-cell activation by anti-CD3 and IL-2. One peptide from the internal domain inhibited T-cell activation. Scrambled versions of this peptide had no effect on T-cell activation. The data suggest that a peptide motif from the internal domain of CEACAM1 is involved in the interaction of CEACAM1 with other ligands and can alter signal transduction in T-cells. This peptide may serve as a template for the development of structurally related compounds that can regulate T-cell activation.
In Vitro Study of the Function of Dendritic Cells on Stimulating T Lymphocytes Proliferation in Severe Aplastic Anemia
Authors: Shao Zonghong , Wang Jun , Fu Rong , Li Lijuan , Liu Hui , Qu Wen , Rua Erbao , Liang Yong , Liu Hong , Wu Yuhong , Wang Guojin , Song Jia , Wang Huaquan , Xing Limin , Guan Jing , Wang Xiaoming
Published: April 2020

Objective To investigate the function of dendritic cells (DCs) in severe aplastic anemia (SAA).

Methods Fifteen untreated patients, 10 recovered patients with SAA and 12 normal controls were enrolled in this study. Their mDCs were induced from their bone marrow mononuclear cells with rhIL-4, rhGM-CSF and rhTNF in vitro. Then mDCs were co-cultured with allogenic lymphocytes (mixture lymphocyte reaction, MLR). The growth rate of lymphocyte was measured by MTT colorimetry and the concentrations of IL-12 and IFNγ in MLR supernatant were measured by ELISA. The correlation between the growth rate and the concentration of IL-12 or IFNγ was analyzed.

Results When mDCs and lymphocytes were co-cultured at the ratio of 1:50, the growth rate of lymphocytes stimulated with the mDCs from untreated cases was (322.13±171.07)%, which was higher than those stimulated with the mDCs from recovered and controls [(180.90±79.12)%, (192.25±91.93)%] (P<0.05). There was no statistical significance between the growth rates of lymphocytes stimulated with the mDCs from the recovered and controls (P>0.05). The mDCs of 8 recovered cases and 4 controls were co-cultured with allogenic lymphocytes at the ratio of 1:100, 1:50, 1:20 and 1:10, but no statistical significance was found between two groups at each ratio (P>0.05). Cross MLR showed only untreated cases versus normal had statistical significance (P<0.05). The concentrations of IL-12 and IFNγ in MLR supernatant of untreated cases were higher than those of the recovered or controls (P<0.05), but there was no statistical significance between the recovered and controls (P>0.05). The concentration of IL-12 in MLR supernatant correlated positively with the growth rate of lymphocyte (r = 0.529,P=0.001), which was also positively correlated with the concentration of IFNγ (r = 0.381,P=0.024).

Conclusion The function of mDCs to stimulate T lymphocytes proliferation by secretingt IL-12 in SAA was enhanced, which might play an important role in the immunopathogenesis of SAA.
Multicenter Trial of Photopheresis in Early Stage Mycosis Fungoides
Authors: Duvic Madeleine , Talpur Rakhshandra , Baron Elma D , Geskin Larisa J. , Demierre Marie-France , Zic John , Bohjanan Kimberly
Published: April 2020

Extracorporeal photopheresis (ECP) has been approved for 20 years for patients with erythrodermic MF or Sézary syndrome (SS) with circulating atypical T-cells. The response rate of 54% was based on 25% improvement in erythema. ECP has not widely been used to treat patients with early stage MF. The objective of this multicenter single-arm open-label study was to determine the efficacy of UVAR® XTSÔ Photopheresis System in early stage MF with measurable skin lesions. Patients were eligible at stage IA if there was with minor blood involvement (B1), or at IB- IIA MF with or without minor blood involvement (B1). Patients had to be refractory to at least one treatment for early MF such as PUVA, electron beam, oral or topical high potency steroids, topical mustragen, oral methotrexate, alpha interferon, or bexarotene. Two photopheresis treatments were given on successive days every 4 weeks for six months. There were 13 evaluable patients treated with ECP (4 males/9 females; 12 Caucasians, 1 African-American) whose median age was 61 yrs (range 46–85). Their stages were IA (n=3), IB (n=8) and IIA (n=2). Patients received a median of 12 ECP sessions (range 4–42+) over a median of 6 months (range 5–22). The overall response rate of 54% (7 of 13) required a ≥50% improvement in the baseline body surface area involved with patches or plaques. The median time to response was 6 months (range 3–8 months) and median duration of response was 6 mos (range 2–30 mos). The protocol allowed additional therapy if patients failed to improve by month 3. ECP alone was effective in four of ten patients (40%) and the other three patients had partial response with combined therapy: two with low dose bexarotene (150 mg) added at 4 or 6 mos and a third with bexarotene, interferon, and PUVA at 6 mos. Only 2 of 13 patients progressed and four had stable disease. There were no photopheresis related adverse events other than mild post-ECP erythema in one patient. One patient developed central right arterial occlusion with left eye visual defect unrelated to the ECP. In conclusion, photopheresis had a 54% response rate for all 13 patients and a 40% response rate to ECP alone in 10 early stage MF patients without adverse side effects suggesting it could be successfully used to treat early MF patients.
Phase II Study of Rituximab Combined with Cladribine and Mitoxantrone in Relapsed or Refractory Indolent Non-Hodgkin’s Lymphoma
Authors: Ota Shuichi , Tanaka Junji , Nakata Masanobu , Imai Kiyotoshi , Ogasawara Masahiro , Kiyama Yoshio , Irie Tatsuro , Morioka Masanobu , Umehara Shintaro , Iwasaki Hiroshi , Chiba Koji , Kakinoki Yasutaka , Fukuhara Takashi , Kasai Masaharu , Imamura Masahiro
Published: April 2020

BACKGROUND. Synergistic or additive activities for rituximab and cladribine have been shown in preclinical studies. Indolent Non-Hodgkin’s lymphoma (I-NHL) tends to recur with shortening intervals of remission after standard chemotherapy. New modalities of treatment are necessary. Therefore, we evaluated the feasibility, efficacy, and toxicity of combined regimen that consisted of rituximab plus cladribine plus mitoxantrone (the RCM regimen) in the treatment of patients with relapsed or refractory I-NHL.

METHODS. The RCM protocol consisted of rituximab at a dose of 375 mg/m2 on Day 1, cladribine at a dose of 0.09 mg/kg per day on Days 2 through 6, and intravenous mitoxantrone at a dose of 6 mg or 10 mg/m2 per day on Day 2. The RCM courses were repeated at 4-week intervals, for up to 4 cycles.

RESULTS. Fourteen patients with I-NHL and one patient with mantle cell lymphoma entered in the study. The median age was 60 (range 47–77) and 8 were females. Histology was small lymphocytic lymphoma (n=1), follicular lymphoma (n=13), mantle cell lymphoma (n=1). Median time from diagnosis to RCM treatment was 3.6 (range 0.2–8.1) years and median number of prior treatment regimen was 2 (range 1–4). Twelve patients (80%) had recurrent disease after prior therapy including high dose therapy with autologous stem cell transplant, and 3 patients (20%) had refractory disease. Thirteen patients were treated on the RCM regimen, and 8 patients (61.5%) achieved a complete response, 3 patients (23.1%) achieved a partial response. Therefore, the overall response rate was 92.3%. Median time to response was 2.8 months (range 1.0–6.7). Median progression free survival of responders was 16.5 (range 1.3–25.5) months. The treatment revealed tolerability, with episodes of severe neutropenia (grade 3 and 4) observed in 12 patients (85.7%), episodes of grade 3 and 4 thrombocytopenia observed in 2 patients (15.4%). However, severe infections were not observed in any patients.

CONCLUSIONS. The RCM regimen is highly effective and well tolerated modalities of treatment in heavily pretreated and relapsed or refractory patients with I-NHL.
Cladribine in the Treatment of Relapsed or Refractory Non-Hodgkin Lymphoma
Authors: Noguchi Emi , Okamoto Rumiko , Shimoyama Tatsu , Sasaki Eisaku , Omuro Yasushi , Maeda Yoshiharu , Sakamaki Hisashi , Sasaki Tsuneo
Published: April 2020

BACKGROUND: The treatment of relapsed or refractory Non-Hodgkin lymphoma (NHL) depends on the number of drugs of choice, and additional treatments can achieve remission for many patients. Cladribine (2-chlorodeoxyadenosine, 2-CdA) is a purine nucleoside analogue, and generally approved for indolent NHL relapsed after or refractory to previous chemotherapy. We conducted this analysis to determine efficacy and toxicity of 2-CdA in the treatment of relapsed or refractory NHL.

PATIENTS AND METHODS: 29 relapsed or refractory NHL patients treated with cladribine in a single institution from 2002 to 2007 were entered in a retrospective analysis.

RESULTS: The median age was 64 years. Seventeen patients (59%) were male, twenty (69%) with indolent lymphoma consisting mainly of follicular lymphoma and mantle cell lymphoma, and twenty-eight (97%) previously treated with 1 to 10 regimens (mean 2.7). Cladribine was administrated 24% as a monotherapy and 22% with other chemotherapeutic agents. The dose and duration of administration were variable among the patients, however, most common regimen was 0.9mg/kg/day for seven consecutive days, for 1 to 6 cycles, depending on response. Response rate was 72% (CR and PR were 31%, 41%, respectively). Median overall survival was of 2.9 years. Patients showed improved survival rate in performance status 0 or 1 (Willcoxon: P<0.0001, Log-rank: P<0.0001), more than 3 cycles of cladribine (Willcoxon: P<0.028, Log-rank: P=0.126), or combination chemotherapy (Willcoxon: P=0.042, Log-rank: P=0.040). Main reason for death was disease progression. Myelosuppression was the most common adverse effect. Eight patients (28%) experienced CTCAE grade 4 neutropenia, fourteen (48%) lymphocytopenia, and three (10%) thrombocytopenia, respectively. Two episodes (6.9%) of grade 3–4 infections were observed. There was no treatment related mortality.

CONCLUSIONS: Cladribine is effective even in heavily pretreated patients with NHL. It also has favorable safety, and can be considered as a good alternative treatment for relapsed or refractory NHL.
Proteomic Analysis of Pkc-Beta Inhibitor Enzastaurin-Treated Mantle Cell Lymphoma
Authors: Weinkauf Marc , Rieken Malte , Pastore Alessandro , Hiddemann Wolfgang , Dreyling Martin H.
Published: April 2020

Background: Activation of the PI3K/AKT pathway is involved in pathogenesis of MCL and accordingly, protein kinase C beta (PKCbeta), a pivotal enzyme in B-cell signalling and survival, is over- expressed in most cases of mantle cell lymphoma (MCL). Enzastaurin, an oral serine/threonine kinase inhibitor, suppresses signalling through PKCbeta/PI3K/AKT pathways, induces apoptosis, reduces proliferation, and suppresses tumor-induced angiogenesis. Recently, a phase II study showed that enzastaurin is able to prolong the progression-free survival in a subset of relapsed MCL (Morschhauser et al, Ann Oncol 2008).

Methods: Four MCL cell lines (GRANTA 519, HBL-2, Jeko-1, Rec-1) were treated with Enzastaurin at a proliferation inhibiting dose of 10μM, defined in previous pilot experiments. After 2–8h of treatment, cells were harvested and a two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE)-based protein screening was performed.

Results: Already 1h after treatment, distinct alterations of the protein pattern were recognized in the 2D-PAGE gels of all cell lines. 115 (12%) of 977 concurrent protein spots exhibited significantly (>3fold) altered protein levels after 4h of enzastaurin exposure, i.e. increased (57 spots; 5,8%) or decreased (58 spots; 5,9%) protein expression, and after 8h the percentage of affected protein spots further increased up to 22% of all concurrent protein spots, due to decreased protein levels. Based on previously defined selection criteria (Weinkauf et al, Electrophoresis 2007) 62 differentially expressed protein spots at 4h (increased/decreased expression: 39/23 spots) were chosen for mass spectrometric identification. Mass spectrometry identified 108 different candidate proteins with significant identification confidence (p<0.05). 29 candidate proteins were detected in multiple protein spots, while 79 candidate proteins were identified in only single protein spots. The identified candidate proteins could be classified in distinct cell programs, including DNA repair and cell division, cytoskeleton (centrosome and spindle), apoptosis, signal transduction and transcription factors. Interestingly, some of the identified candidates had been also previously identified after proteasome inhibition by bortezomib, e.g. ENOA, TOP1, CE290, SMC1A, HSP60 (CH60), ATPB, KAD5 and TPIS.

Conclusion: The identified candidate proteins are currently confirmed in MCL patient samples. Characterization of essential signal pathways after Enzastaurin exposure will not only expand the understanding of the molecular pathogenesis of MCL, but also hint towards promising targets for new molecular therapies.
TLR-9 Activated Lymphoma B Cells Induce Anti-Tumor Immunity in a Murine Model
Authors: Goldstein Matthew J. , Brody Joshua , Levy Ronald
Published: April 2020

Background: Tumor vaccines typically combine unique, tumor antigens with immune stimulants in an effort to elicit a tumor-specific immune response. In prior work we have described a vaccination maneuver that combines cytotoxic chemotherapy to release tumor antigens with intra-tumor injection of a Toll-like Receptor 9 (TLR9) ligand, CpG oligonucleotide (Li and Levy, J. Immunology, 2007). In this therapy, CpG can activate both host antigen presenting cells as well as the tumor itself. Stimulation of tumor B cells through TLR9 induces up-regulation of immune co-stimulatory molecules including CD80, CD86, and CD40 as well as increasing expression of MHC Class I and II. We have now developed an alternative vaccination approach in which B cell lymphoma tumor cells are stimulated with CpG ex vivo and administered as a whole-cell vaccine.

Methods: A20 B cell lymphoma tumor cells were incubated with CpG for 72 hours, irradiated, and administered to naïve Balb/C mice. The complete vaccination regimen included six doses of 1×106 cells administered daily at a sub-cutaneous (s.c.) site. Vaccine-induced immune responses were assessed by measuring IFN-γ expression of peripheral blood lymphocytes (PBLs) in response to co-culture with A20 tumor cells. Tumor protection studies were conducted by challenging vaccinated mice with a lethal dose of 10×106 A20 tumor cells. Anti-tumor immunity generated by vaccination was also tested in adoptive cell transfer studies.

Results: Sub-cutaneous vaccination with CpG-stimulated, whole-cell vaccine induces robust anti-tumor T cell immunity comparable to that induced by intra-tumor vaccination with CpG. This immunizing effect was dependent on tumor cell activation, since native tumor cells were less efficient at inducing anti-tumor immune responses. Both CD4+ and CD8+ T cells participated in this response. Mice vaccinated with this regimen were protected against tumor challenge. Splenocytes from vaccine-primed donors were adoptively transferred into irradiated, syngeneic recipients. These ‘immunotransplanted’ recipients had even greater immune protection against tumor challenge than the directly vaccinated donor mice.

Conclusions: We have developed a vaccination approach that takes advantage of the antigen presentation capability of malignant B cells. Vaccination with CpG-activated lymphoma cells induced anti-tumor immune responses that were further enhanced by adoptive transfer of immune cells into lymphodepleted recipients. These vaccine maneuvers are directly translatable into therapeutic, human clinical trials.
Forodesine (Immucillin H, BCX-1777) Shows Activity on Mantle Cell Lymphoma Primary Cells
Authors: Rinaldi Andrea , Ceresa Emilia , Rossi Davide , Gaidano Gianluca , Bantia Shanta , Manz Chantal Y. , Catapano Carlo , Zucca Emanuele , Bertoni Francesco
Published: April 2020

Mantle cell lymphoma (MCL) represents a subtype of B-cell lymphoma associated with a very unfavourable clinical outcome. Currently no therapy can be considered as standard, and new therapeutic approaches are needed. Forodesine is a potent inhibitor of purine nucleoside phosphorylase (PNP), whose major role is to catalyze the cleavage of inosine, deoxyinosine guanosine, and deoxyguanosine (dGuo) to their corresponding base and sugar 1-phosphate by phosphorolysis. In the presence of deoxycytidine kinase, PNP inhibition leads to an increase in the concentration of dGuo triphosphate (dGTP), followed by inhibition of DNA synthesis and cell death by apoptosis. When combined with dGuo, forodesine has been shown to have in vitro cytotoxic activity on T-cell (T-ALL, T-PLL) and on B-cell malignancies (CLL, B-ALL), and Phase I/II trials are on going in CLL and CTCL patients. Here, we report the first data on in vitro activity of forodesine in MCL. Primary MCL cells, derived from six patients, were exposed to forodesine (0, 2, 20 μM) in combination with dGuo (0, 10, 20 μM), for 48 hrs. Cells were cultured in X-VIVO 10 medium (Cambrex) with 10% FBS. Cell viability was assessed by flow cytometry with the Annexin V - propidium iodide assay. Four patient samples (67%) showed an increase in the number of Annexin V positive cells ranging from 1.9 to 5.3 times compared to untreated cells. The effect was larger for 20 μM forodesine compared with 2 μM. There was no effect of dGuo alone and only a minimal effect of increasing dGuo concentration from 10 μM to 20 μM. Cell lines did not appear to be ideal models to evaluate the efficacy of forodesine in vitro. Three established MCL cell lines (Granta-519, Rec, JeKo1) were treated with escalating doses of forodesine, but the results were not reproducible, while the same cells showed expected IC50 values between 25–30 μM when exposed to bendamustine for 72 hrs. In conclusion, the in vitro data reported here with 4/6 MCL patients primary samples sensitive to forodesine and the results from various groups on other T- and B-cell malignancies suggest that clinical trials of forodesine in MCL may be warranted.
A Therapeutic Approach for Acute Leukemias: Glycolysis Inhibition Revisited
Authors: Akers Lauren J. , Levy Alejandro G. , Franklin Anna R. , Fang Wendy , Huang Peng , Zweidler-McKay Patrick
Published: April 2020

Background: Recent evidence suggests that myeloid and lymphoid leukemia cells are dependent on aerobic glycolysis for ATP production, known as the Warburg effect. Early glycolysis inhibitors were effective but only at high mM concentrations. The novel third generation glycolysis inhibitor, 3-bromo-2-oxopropionate-1-propyl ester (3-BrOP), has shown efficacy in models of neuroblastoma, glioblastoma, colon carcinoma, and lymphoma in the mcM range and has improved oral bioavailability and biodistribution. We proposed that potent inhibition of glycolysis would lead to ATP depletion and induce growth arrest and cell death in acute lymphoblastic leukemia (ALL) and acute myelogenous leukemia (AML) cells. Objective: To determine the feasibility and effect of 3-BrOP on human AML, precursor-B, and T cell leukemia cell lines and patient samples.

Design/Methods: A panel of fifteen human pre-B ALL, T-ALL, and AML cell lines were treated with 3-BrOP to determine IC50s via cell counts, cell cycle, and Alamarblue. In addition, western blots and ATP assays were performed to determine caspase activation and ATP depletion.

Results: All cell lines demonstrate growth arrest, decreased alamar blue metabolism and significant apoptosis when treated with 3-BrOP. The five human precursor-B ALL cell lines had IC50s between 10–30mcM, while the T ALL cell lines had IC50s between 15–30mcM. The human AML cell line panel had IC50s between 20–40mcM. In addition, patient samples representing ALL (IC50 15 mcM) and AML (IC50 30mcM) were sensitive to 3-BrOP mediated cell death. Of note, caspase-3 cleavage was detected by 2 hours of exposure, and was nearly complete by 6 hours in both pre-B and T-ALL suggesting a potent an early pro-apoptotic signal. Overall, cell cycle analysis at 48 hours of treatment, showed the majority of cells had died (83–97%, p<0.001). The remaining cells were apoptotic with >80% of Nalm6 (pre-B ALL) and 72% of U937 (AML) cells having sub-G0 DNA content (p<0.001). As expected, exposure to 3-BrOP reduced intracellular ATP concentrations in a dose and time dependent manner. In Nalm6 cells (pre-B ALL), by 48 hours of exposure, the ATP levels were 50% of control at a dose of 15 mcM. At a dose of 22.5 mcM, the ATP level was 13% of control, and by 30 mcM there was no detectable ATP. Importantly, at higher doses (>30mcM) ATP levels were reduced significantly within 24 hours. These effects on ATP levels correlate with dose and time dependent viability.

Conclusions: These data demonstrate that a broad range of acute leukemia cell lines and patient samples appear to be dependent on glycolysis for ATP production and survival. The novel 3rd generation glycolysis inhibitor, 3-BrOP, induces cell death across all subtypes of human acute leukemia cell lines. Glycolysis inhibition via 3-BrOP represents a novel and clinically feasible therapeutic approach for acute leukemias and warrants further pre-clinical evaluation.
Proteome Analysis of the Proteins Associated with All-Trans Retinoic Acid Resistance in Acute Promyelocytic Leukemia Cells
Authors: He Pengcheng , Zhang Mei , Qi Jun , Wang Xiaoning , Xi Jieying , Wang Huaiyu , Li Jing , Wu Di , Wei Kaihua , Liu Feng , Wang Hongli
Published: April 2020

Although 90% patients with untreated acute promyelocytic leukemia(APL) obtain complete remission because of the usage of all-trans retinoic acid(ATRA), patients with ATRA-resistance are increased gradually. ATRA-resistance has become one of the main causes which affect the long-term therapeutic efficacy of APL. The mechanisms of ATRA-resistance are complex, which probably involve the metabolism of ATRA, abnormal expression of cellular retinoic acid binding protein(CRABP) and P-glycoprotein(P-gp), mutation of RARα and aberration translocation of APL. However, in these previous researches, it was one or a few proteins but not the entirety proteins that were emphasized on the mechanisms of ATRA-resistance. Comparative proteomics can analyze the entire protein expression in cells in whole and has the superiority in screening the drug-resistance proteins differentially expressed. In order to investigate the mechanisms of ATRA-resistance in APL in whole, we compared and analyzed the protein expression profiles between MR2 cells(APL cell line with ATRA-resistance) and NB4 cells(APL cell line with ATRA-sensitiveness) by comparative proteomics. After the total proteins of MR2 cells and NB4 cells were extracted respectively, they were separated by two-dimensional electrophoresis(2-DE). The differences in proteome profile between MR2 cells and NB4 cells analyzed by ImageMaster™ 2D Platinum software. The average protein spots in 2-DE maps of MR2 and NB4 cells were 1160±51 and 1068±33 respectively. 8 protein spots were selected to be identified by Matrix-assisted laser desorption/ionization time of flight-mass spectrometry (MALDI-TOF-MS), in which the quantity of the protein differentially expressed was more than two times(≥2 or ≤0.5) between MR2 and NB4 cells’ 2-DE map. They were all successfully identified and their definite information was obtained. Among them, 6 proteins were probably involved in the mechanisms of ATRA-resistance in APL and they were Cofilin-1, Elongation factor 1-beta (EF-1β), Tropomyosin isoform(TM), High mobility group protein B1(HMGB1), Ran-specific GTPase-activating protein (RanGAP1) and Galectin-1. Moreover, so far there was no related report on the roles of HMGB1, RanGAP1 and Galectin-1 in the mechanisms of ATRA-resistance in APL. These differential proteins identified provide the new clues for us to further elucidate the mechanisms of ATRA-resistance from multiple factor.
Differential Protein Expressions Between Leukemia HL-60 and Adriamycin-Resistant HL-60 Cell Lines by Comparative Preteomic Analysis
Authors: Hu Jian Da , Lin Min-Hui , Chen Xin-Ji , Liu Ting-Bo , Lv Lian-Huang
Published: April 2020

Resistance is a major problem of chemotherapy failure in acute leukemia. Although multi-drug resistance is an important factor, the exact mechanisms of resistance remain to be clarified. With the aim of better understanding the protein involvement in development of resistance mechanisms, a comparative proteomic analysis——two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) was used to compare differential protein expression profiles in adriamycin-resistant acute myeloid leukemia (AML) HL-60/ADR and sensitive HL-60 cell line. Total cellular proteins extracted from HL-60 and adriamycin- resistant HL-60 cells were separated by 2-D gel electrophoresis.Differential expressed proteins were analyzed by mass spectrometry (MALDI-TOF/TOF) and database searching. 16 significantly differentially expressed protein spots were identified, among which 13 protein spots were identified as up-regulated and 3 as down-regulated. The identified proteins were categorized into: (±)metabolic enzymes; (II)proteins related with signal transduction;(III)cell cycle regulators; and(‡W)cellular proliferation and apoptosis proteins. Some of these differential protein expression were confirmed by western blot. In addition, we investigated the expression of distinct proteins in the primary leukemia cells. The results revealed that nucleolar phosphoprotein (B23) over-expressed in the relapsed patients with acute monocytic leukemia (M5). It suggests that B23 might be a useful indicator of prognosis of M5, but the exact role in resistant mechanism is still to be investigated. 2-D DIGE is a useful approach to investigate differential protein expression related treatment resistance.
Magnetic Nanoparticle of Fe3O4 Loaded with Daunorubicin Reverse Leukemia Multidrug Resistance in Vivo
Authors: Chen Bao-An , Lai Bin-bin , Cheng Jian , Gao Feng , Xu Wen-lin , Shen Hui-lin , Ding Jia-hua , Gao Chong , Sun Yun-yu , Wang Jun , Zhao Gang , Song Hui-hui , Bao Wen , Sun Xin-chen , Cheng Hong-yan , Deng Yu-xia , Li Guo-hong , Liu Li-jie , Chen Wen-ji , Shao Ze-ye , Xia Guo-hua , Wang Jue-qiong , Yuan Peng , Pei Xiao-ping , Chen Ning-na , Li Qin-ning , Wang Xue-mei
Published: April 2020

Object: To the effect of Fe3O4-magnetic nanoparticle loaded with DNR on In order to study the multidrug-resistant reversal effect of Fe3O4-magnetic nanoparticle loaded with DNR in vivo.

Methods: K562-n,a leukemia cell line with high tumorigenicity in nude mice and its multidrug-resistant counterpart K562-n/VCR cells were inoculated subcutaneously into each groins of nude mice (5×106 cells/each) to establish a human leukemia xenograft model. The mice were randomly divided into group A receiving normal saline, group B receiving anti-tumor drugs daunorubicin (DNR), group C receiving Fe3O4-magnetic nanoparticle, group D receiving Fe3O4-magnetic nanoparticle loaded with DNR, and group E receiving Fe3O4-magnetic nanoparticle containing DNR with a magnetic field built on the surface of tumor tissues. After 20 days treatments, mice were killed and tumor tissues were isolated for pathological observation. The volume and weight of tumors were measured, then tumor suppression rate was calculated. The side effects of DNR loaded Fe3O4-magnetic nanoparticle were also evaluated.

Results: The results showed that DNR loaded Fe3O4-magnetic nanoparticle can significantly suppress the growth of K562-n/VCR tumor in vivo, DNR alone can greatly suppress the growth of K562-n, Fe3O4-magnetic nanoparticle loaded with DNR can not further inhibit the K562-n tumor.

Conclusion: In conclusion, Fe3O4-magnetic nanoparticle loaded with DNR can reverse the leukemia multidrug resistance in vivo.
Mechanism Research of Reversal in Multidrug Resistance by Magnetic Nanoparticle Fe3O4 Syndesis with 5-Bromotetrandrine and Daunomycin
Authors: Chen Bao-An , Wu Wei-wei , Cheng Jian , Gao Feng , Xu Wen-lin , Shen Hui-lin , Ding Jia-hua , Gao Chong , Sun Yun-Yu , Wang Jun , Zhao Gang , Song Huihui , Bao Wen , Sun Xin-chen , Cheng Hong-yan , Deng Yu-xia , Li Guo-hong , Liu Li-jie , Chen Wen-ji , Wang Jue-qiong , Yuan Peng , Pei Xiao-ping , Chen Ning-na , Wang Xue-mei
Published: April 2020

Objective To evaluate the MDR reversal activity of magnetic nanoparticle Fe3O4 (Nano-Fe3O4) and 5-bromotetrandrine (BrTet) on multidrug resistance cell line K562/A02 solitarily or symphysially, and to investigate the reversal mechanism of this coopration.

Methods The proliferation of K562 and K562/A02 cells which were cultured with daunomycin (DNR) alone or in combination with Nano-Fe3O4, BrTet or both for 48h were evaluated by MTT assay. DNR accumulation and P-gp of K562 and K562/A02 were analyzed by fluorospectrophotometry.

Results The IC50 of DNR for K562 and K562/A02 cells were 2.74±0.19μM and 32.33±8.40μM, respectively; but the sensitivity of K562/A02 cells to DNR was partially restored after culturing with Nano-Fe3O4, BrTet solitarily and symphysially (the IC50 were 7.04±0.85μM, 4.25±2.16μM, 1.80±0.30μM; the FR were 4.32, 7.61, 17.96, respectively), but had no effects on K562 cell lines. The differences had statistical significance. Flow cytometry assay showed that Nano-Fe3O4 and BrTet increased the DNR accumulation in K562/A02 cells, especially in the group of synergia of these two agents. The mean fluorescence intensity of endocellular DNR increased from 2614 pretreated with DNR only to 4783 incubated with these two reversal reagents, while the values were 3364, 4077 for incubating with Nano-Fe3O4 and BrTet respectively. P-gp were down regulated through pretreating with Nano-Fe3O4, BrTet alone and symphysially in K562/A02 cells by fluorospectrophotometry assay.

Conclusion Nano-Fe3O4 and BrTet synergisticly showed significant MDR reversal activity in vitro. The reversal activity may be related to the inhibition of P-gp overexpression and the increasing of the anticancer drug accumulation intracelluarly.
Pediatric Therapy-Related Myelodysplastic Syndrome/Acute Myeloid Leukemia: The M. D. Anderson Cancer Center Experience
Authors: Aguilera Dolly G , Vaklavas Christos , Tsimberidou Apostolia-Maria , Wen Sijin , Medeiros L. Jeffrey , Corey Seth J.
Published: April 2020

Background: Therapy-related myelodysplastic syndrome/acute myeloid leukemia (t-MDS/AML) is a long-term complication of pediatric cancer and it carries a poor prognosis.

Patients and Methods: We retrospectively studied pediatric t-MDS/AML patients treated at M. D. Anderson from 1975 to 2007. We also compared these patients to pediatric patients with de novo MDS/AML during this time interval.

Results: Among 2589 children with cancer treated at M. D. Anderson, we identified 22 (0.85%) patients with t-MDS/AML and 141 (5.4%) patients with de novo MDS/AML. Patients with t-MDS/AML had a median age of 14 years (range, 3–20). There was a male and Hispanic predominance. The most common primary malignancies were osteosarcoma and Hodgkin lymphoma. The median latency period was 4.1 years. Fourteen patients received AML-type chemotherapy, 5 underwent allogeneic stem cell transplantation (SCT) as induction therapy, and 3 received supportive care. Fourteen patients underwent SCT as induction (n=5), post-remission (n=5), or salvage therapy (n=4). Their respective 2-year survival rates were 20%, 40%, and 25% (p= 0.85). Patients with de novo AML were younger (p=0.001), and had higher rates of CR (p=0.03), and survival (p<0.0001). Independent factors predicting shorter survival were poor/intermediate-risk cytogenetics (p=0.01), lower hemoglobin level (p=0.0001), and t-MDS/AML (vs. de novo) (p=0.003).

Conclusion: Childhood t-MDS/AML has a poor prognosis. Although patients benefited from AML-type induction chemotherapy followed by SCT as post-remission therapy, effective therapies are needed.
Incidence of HFE Gene Mutations in Chinese Patients with Myelodysplastic Syndrome and Aplastic Anemia
Authors: Nie Ling , Yang Lin , Li Qinghua , Wang Jianxiang , Xiao Zhijian
Published: April 2020

The C282Y and H63D mutations of HFE gene responsible for hereditary hemochromatosis lead to absorption of excess dietary iron, tissue iron deposition and occurrence of clinical complications such as congestive heart failure, arrhythmia, hepatocellular cirrhosis, insulin resistance and diabetes. Iron overload is one important clinical feature in patients with myelodysplastic syndrome (MDS) and aplastic anemia(AA). However, the conflict has been existing about influence of HFE gene mutations on iron overload in MDS patients. In the present study, we analyzed the incidence of the C282Y and H63D mutations of HFE gene in 271 MDS patients, 402 AA patients and 1615 healthy controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). No C282Y mutations and compound heterozygote were observed in the entire cohort. The genotype distribution of H63D heterozygous and homozygous did not differ significantly in AA cases from those in controls(9.7%vs. 10.2%, 0.25%vs. 0.24% respectively, Pearson Chi-square p both >0.05). While the frequency of the H63D heterozygous in MDS patients was significantly lower than controls (4.1%vs. 10.7%, Pearson Chi-square p=0.002). H63D homozygous was not found in MDS patients. The incidence of C282Y and H63D mutations of HFE gene in Chinese MDS cases is lower than those reported in the literature. Comparing the pretransfusion serum ferritin(SF), serum iron concentration(SI) and transferrin saturation values(TS) between HFE-mutation and HFE-wild MDS groups, we did not find a significant difference (all P>0.05); However, Only SI values were significantly higher in the HFE-mutation AA cases than those in HFE-wild ones[42.6(24.6–60.4)umol/Lvs. 32.0(8.4–63.3)umol/L, P=0.011]. We further estimated the function of important organs in MDS and AA patients. There is no significantly difference in Alanine amino transferase (ALT), Aspartate amino transferase (AST), fasting blood sugar, and electrocardiogram(ECG) between HFE-mutation and HFE-wild MDS and AA groups irrespective of the red blood cell transfusion history. The results suggest that the distribution of C282Y and H63D mutations has ethnic and genetic differences, and is very rare in Chinese population. The mutations of HFE gene are not main factors of iron overload in Chinese patients with myelodysplastic syndrome and aplastic anemia.
Impact of Baseline Renal Function on Transfusion-Independence Response, Survival, and Transformation to Acute Myeloid Leukemia in Patients with Deletion 5q: A Sub-Analysis of the MDS-003 Study
Authors: Battiwalla Minoo , Fu Tommy , Knight Robert D. , List Alan F.
Published: April 2020

Introduction: Lenalidomide reduced transfusion requirements and reversed cytologic and cytogenetic abnormalities in patients who have myelodysplastic syndrome (MDS) with the chromosome 5q31 deletion del(5q)] (List et al., NEJM 2006). Dose-adjusted lenalidomide is safe and effective in MDS patients with del(5q) and severe renal impairment (Knop et al., Leuk Lymph 2008), but the impact of baseline renal function on outcomes has not been specifically studied. This MDS-003 clinical trial sub-analysis investigates the impact of baseline renal function on the transfusion-independence (TI) response, overall survival, and transformation to acute myeloid leukemia (AML).

Methods: Transfusion-dependent, Low/Int-1-risk patients with primary MDS and del(5q) with/without additional cytogenetic abnormalities were treated with lenalidomide 10 mg daily for 21 days every 28 days. Patients were treated until disease progression, treatment failure, or treatment-limiting adverse events. This sub-analysis included patients with normal renal function (creatinine clearance [CrCl] >80 mL/min) and patients with mild (CrCl 50–80 mL/min), moderate (30–50 mL/min), or severe renal impairment (CrCl <30 mL/min). CrCl was calculated according to the Cockcroft-Gault formula. Red blood cell (RBC) transfusion-dependent anemia was defined as having received ≥2 units of RBCs within 8 weeks of the first day of study treatment. Neutropenia was defined as a baseline absolute neutrophil count of <1,000/μL. Thrombocytopenia was defined as a baseline platelet count of < 100,000/μL. TI response, overall survival, and transformation to AML were assessed.

Results: Of 148 patients enrolled in the study, 70 patients had normal renal function and 70 patients had renal impairment (47 mild, 19 moderate and 4 severe impairment; with renal data missing for 8 patients). The TI response was 44/70 (63%) for patients with normal renal function and 48/70 (69%) for patients with impaired renal function (P=0.47). In the subcategories, the TI responses were 79%, 53% and 25% for mild, moderate and severe impairment, respectively (P=0.07, P=0.42 and P=0.13 vs. normal function). Renal impairment resulted in shorter median overall survival compared with no renal impairment (hazard ratio of 2.88, 95% confidence interval, 2.24–3.45). The transformation to AML rate was 19% for normal patients and 20% for patients with renal impairment.

Conclusion: In this analysis, baseline renal function does not have a significant impact on achieving transfusion independence or transformation to AML. Patients with moderate to severe renal impairment had a shorter survival than patients with normal renal function.
Clinical Efficacy of Decitabine in Patients with Myelodysplastic Syndrome (MDS)
Authors: Golenkov Anatoli , Dudina Galina , Buravtsova Iolanta , Voronova Olga , Chukavina Marina
Published: April 2020

Decitabine (DACOGEN, Janssen Pharmaceutica N.V. and Eisai Inc.) is an efficient hypomethylating agent approved for the treatment of MDS in the United States and other countries. Previous studies from North America reported the efficacy and safety of decitabine 15 mg/m2 every 8 hours for 3 days every 6 weeks (3-d schedule; Kantarjian, Cancer2006;106:1794) and 20 mg/m2 once daily for 5 days every 4 weeks (5-d schedule; Kantarjian Blood2007;109:52 and Steensma, ASH2007;110:1450) in MDS patients. The aim of the study reported here was to assess the clinical efficacy and safety of the 3-d schedule of decitabine in MDS patients in Russia. A total of 15 patients with MDS were enrolled: 9 male and 6 female; average age 62 years. Diagnoses included refractory anemia with ringed sideroblasts (RARS) in 3 patients, refractory anemia with excess blasts (RAEB) in 9 patients, refractory anemia with excess blasts in transformation (RAEB-T) in 2 patients, and chronic myelomonocytic leukemia (CMML) in 1 patient. Cytogenetic abnormalities included: -Y in 2 patients, del(5q) in 2 patients, and del(5q)+t(11;17) in 1 patient. At the time of this analysis, decitabine treatment had been administered for 1–2 cycles in 7 patients and for 3 cycles in 8 patients. The most frequent grade ≥3 adverse events were neutropenia (n=8, including 5 with neutropenic fever) and thrombocytopenia (n=4). Two patients with RAEB-T had severe neutropenia with infection: a necrotic lesion of hemorrhoids (n=1) and lower limb cellulitis (n=1). No other grade ≥3 adverse events were observed. Among the 8 patients who had received 3 cycles of decitabine, transfusion dependence was 100% before treatment and 38% (3/8) after decitabine treatment, with prolongation of transfusion independence periods in all 8 patients. Average hemoglobin level was 6.8 g/dL before treatment and 8.4 g/dL after 3 cycles of decitabine treatment. Overall response rate was 50% after 3 cycles of decitabine, including complete response (CR) in 1 (13%) patient and partial response (PR) in 3 (38%) patients. The other 4 patients who had received 3 cycles of decitabine had stable disease. The median time to first treatment response was 2 cycles. In this study of MDS patients in Russia, toxicity was consistent with previous studies and patients responded well to decitabine treatment on a 3-day dosing schedule.