bluuebunny/crossref_metadata_2025 huggingface dataset:
HLA Influence on NK Cell Expansion
Authors: Schellekens Jennifer , Stserbakova Anna , Tõns Madis , Everaus Hele , Tilanus Marcel GJ , Aints Alar
Published: April 2020

Natural Killer (NK) cells are effector cells in the innate immune system. The anti-leukaemic capacities of NK cells in haematopoietic stem cell transplantation make these cells a potential treatment modality to improve clinical outcome. Immunotherapy with NK cells requires transfusion of large quantities, which obviates the need for an in vitro culture system for NK cells. The killer cell immunoglobulin-like receptors (KIR) on NK cells recognise defined groups of HLA class I alleles. To elucidate the influence of these interactions on proliferation, the peripheral blood mononuclear cells (PBMCs) of 29 patients and donors were cultured in CellGro SCGM with IL-2 and OKT3 antibody to expand the NK cell fraction. The killer cell immunoglobulin-like receptor (KIR) and HLA repertoire were determined by sequence specific priming and sequence based typing respectively. The percentage of NK cell expansion from the total PBMC fraction varied between 5.4% and 71.6%. A significantly better NK cell expansion was observed for individuals homozygous for HLA-C epitope group 2 (p<0.05). For evaluation of cytolytic competence of the cultured NK cells, specific killing of an HLA class I expression deficient LCL 721.221 cell line and three 721.221 cell lines transfected with different HLA-C alleles was determined. A significantly better NK cell-induced specific cytotoxicity was observed towards the untransfected 721.221 cells compared to the HLA-C transfected 721.221 cells. No significant differences were observed between killing of the three HLA-C transfected 721.221 cell lines. We have shown that cytolytic capacities of the cultured NK cells are maintained and in vitro expansion of NK cells is dependant on the presence of HLA-C alleles.
KIR Gene and HLA-Cw Mismatches Affect on NK Cell Killer Activity
Authors: Cao Xiangshan , Li Jianyong
Published: April 2020

The KIRs were knew as natural killer (NK) cell inhibitory receptors with specificity for HLA molecules on their cellular targets. We investigated NK cell activation on the number of matches between cell killer immunoglobulin-like receptor (KIR) gene and HLA-Cw, and the level of inhibitory KIRs expressed on NK cell surface and the cytotoxicity of NK cell against AML leukemic cells in vitro. NK cell were isolated and purified from 27 healthy donors by isolation kit, Target cells were blasts derived from bone marrow of 30 patients with AML.Inhibitory KIRs expression knew as CD158a, CD158b was analyzed by flow cytometry to estimate the percentage of NK cells that could be inhibited by the HLA-Cw ligands..KIR and HLA gene typing were performed by PCR –SSP. from donors and patients respectively. NK cytotoxicity against AML leukemic cells demonstrated by MTT which showed the correlation between NK cytotoxicity and the number of KIR/HLA matches. the NK-susceptible K562 cell line which lacks HLA class I expression, was used as a positive control target in all cytotoxicity assays, autologous non-NK cell was used as negative control target cell. the cytotoxicity assays was performed in E:T 50:1 20:1 10:1 5:1 2.5:1. Results demonstrated the less number of KIR/HLA-Cw matches, the more NK cells are activated..0 match of NK cell/target cell KIR/HLA-Cw, cytotoxicity was (50.66±8.40)%,1 match and 2 matches were (38.28±6.71)%, (19.74±4.15)%, F=20.226, P<0.001. NK cells expressed KIRs also had relationship with cytotoxicity, inhibitory KIRs expressed >50%, the cytotoxicity is 10%, inhibitory KIRs expressed 20%–50%, the cytotoxicity is 20%, inhibitory KIRs <25%, the cytotoxicity is 55%, F=16.276,p<0.001. Therefore these data indicate NK cell kill AML leukemic cells mechanism follow KIR/HLA-Cw mismatch theory, the level of inhibitory KIRs expressed on NK cell surface showed the percentage of NK cells that could be inhibited by the HLA-Cw ligands. Key words: KIR NK cell CD158 HLA-Cw
A Comparative Oncogenomic Analysis Identifies Genetic Events Associated with Myeloma Progression and Drug Resistance
Authors: Chesi Marta , Braggio Esteban , Keats Jonathan J , Bergsagel P. Leif
Published: April 2020

We have successfully generated the first immuno-competent transgenic mouse model of multiple myeloma (MM), Vk*MYC, in which sporadic MYC activation in the germinal center is AID dependent and mediated by the somatic hypermutation process. All of Vk*MYC mice develop monoclonal PC expansion by 50 weeks of age, that is manifested by high levels of serum IgGs and major M-spikes by SPEP. The PCs in Vk*MYC mice are fully differentiated, somatically mutated, have a very low proliferation index and are found exclusively in the BM. Associated with the long lifespan (medium survival = 661d), Vk*MYC mice develop anemia and diffuse bone disease (osteoporosis with low bone mineral density), with sporadic occurrence of lytic bone lesions and hind limb paralysis. The phenotypic fidelity of the Vk*MYC mice translates in a therapeutic fidelity, as Vk*MYC mice show response to known clinically active anti-myeloma drugs (bortezomib, melphalane, dexamethasone), but not to drugs that have shown no clinical activity as single agents (vincristine, hydroxyurea, fludarabine). Furthermore, acquired bortezomib resistance could efficiently be induced in Vk*MYC mice after only four cycles of suboptimal treatment, and studies are in progress to identify genetic events associated with bortezomib resistance. Preliminary aCGH experiments on a high resolution Agilent 244K array, using genomic DNA from six Vk*MYC CD138+ selected PCs, showed that the genomic complexity of Vk*MYC tumors is quite low, with only few unbalanced rearrangements, such as trisomy of chromosome 15 detected in two samples or trisomy 9 present in another tumor. The most common abnormalities found are small focal monoallelic deletions, six of which are present in more than one tumor. A focal homozygous deletion of several exons of a gene was identified in two tumors samples. Remarkably, a comparative analysis of the syntenic chromosomal region in human MM cell lines and primary patient samples identified similar recurrent homozygous deletion in this putative novel tumor suppressor gene. Overall, these data suggest that the genomic complexity in this inbred strain of transgenic mice is significantly less then in patients with MM, simplifying the search for critical loci involved in MM tumorigenesis and drug resistance and further validate the Vk*MYC mice as a particularly faithful model of human MM.
Primary Central Nervous System Lymphoma of T-Cell Phenotype: A Rare Variant of Non-Hodgkin’s Lymphoma Treated with CNS Directed Therapy
Authors: Doolittle Nancy D , Neuwelt Edward A
Published: April 2020

Primary central nervous system lymphoma (PCNSL) is a non-Hodgkin’s lymphoma confined to the craniospinal axis and/or eyes, without evidence of systemic spread. Almost all PCNSL’s are classified as large B-cell lymphoma. A rare variant of PCNSL includes lymphomas of the T-cell phenotype. Until the publication of a large retrospective series of patients (pts) (n = 45) with T-cell PCNSL (Shenkier TN, et al. J Clin Oncol2005;23:2233–2239),only case reports of T-PCNSL and literature reviews were available. The large retrospective series reported that ECOG Performance Status (PS) (0 or 1 versus 2, 3 or 4) at diagnosis was significantly associated with better outcome (Log rank P <.0001), as was primary treatment with high-dose methotrexate (MTX) (yes versus no) (Log rank P = .002). Median survival in the large series was 25 months (95% CI: 11 to 38 months). We reviewed our brain tumor database for pts seen between January 1980 and July 2008. Of 159 pts diagnosed with PCNSL, 3 (2M/1F) had histopathologically confirmed large T-cell phenotype. At diagnosis, the pts’ ages were 10, 23, and 64 years; in each case ECOG PS at diagnosis was 0. Brain parenchyma disease location was periventricular (1 pt), parietal (1 pt), and corpus callosum (1 pt); one pt had both brain and ocular lymphoma. First-line treatment was IA MTX-based chemotherapy with osmotic blood-brain barrier disruption (BBBD), without brain irradiation. The dose of MTX was 2.5 grams/day for 2 consecutive days, every 4 weeks for up to one year. A comprehensive neuropsychological test battery was administered at baseline prior to MTX-based BBBD treatment. Followup neuropsychological testing was conducted within 6 months after the final BBBD treatment. All patients attained CR and are alive 15.8, 8.9 and 1.2 years after diagnosis, with excellent PS and no evidence of disease progression to date. The 3 pts showed stable or improved neuropsychological function in all cognitive domains at follow-up testing. Side effects during BBBD treatment included focal seizures (1 pt) and atrial fibrillation (1 pt). In these 3 cases of T-cell PCNSL, encouraging tumor responses and survival were seen, with stable or improved neurocognitive functioning to date, warranting further investigation of CNS directed therapy in this rare variant of non-Hodgkin’s lymphoma.
A Prospective Study of Cardiotoxicity In Non-Hodgkin Lymphoma Patients with RAPID-Infusion Rituximab
Authors: Provencio Mariano , Sanchez Antonio , Mendez Miriam , Maximiano Constanza , Cantos Blanca , Vargas Juan A , Tutor Pablo , Mellor Susana , Canales Miguel A. , España Pilar
Published: April 2020

Background. Prospective study based on measuring the left ventricular ejection function (LVEF) of lymphoma patients treated with rapid infusion Rituximab (one hour).

Patients and Methods. 42 patients with non-Hodgkin lymphoma were treated with rapid infusion Rituximab-based chemotherapy (mostly CHOP-R). Basal LVEF was measured before chemotherapy and every six months after treatment.

Results. An average of 6.5 infusions per patient were administered (total of 273 infusions). Twenty-four (57%) patients had at least one cardiovascular risk factor. In 13 patients (31%) we observed a decreased in the post-treatment LVEF of over 10%. Patients (3) with drop > 10% recovered normally, but when LVEF decreased by over 15% (6 patients), no one recovered until normal level. None of them developed clinical congestive failure.

Conclusion. Rapid infusion Rituximab added to chemotherapy does not cause clinical cardiac toxicity. Patients recover LVEF when it decreased less than 15%.
Dual PI3K and mTOR Inhibition in Waldenstrom Macroglobulinemia
Authors: Husu Emanuel N. , Roccaro Aldo M. , Sacco Antonio , Melhem Molly R. , Azab Abdel kareem , Jia Xiaoying , Ngo Hai T. , Azab Feda , Runnels Judith , Quang Phong , Leleu Xavier , Anderson Kenneth C. , Ghobrial Irene M.
Published: April 2020

Background. We have previously showed that PI3K/Akt is constitutively active in Waldenström Macroglobulinemia (WM) malignancies, mediating growth, survival, cell cycle regulation, and migration in primary tumor cells. Once activated, Akt phosphorylates downstream targets, including mammalian target of rapamycin (mTOR). Both PI3K/Akt and mTOR represent valid targets for antitumor therapeutic strategies. We therefore evaluated the antitumor activity of NVP-BEZ235 (Novartis, MA) in WM.

Methods: WM cell lines (BCWM.1) and IgM secreting cell lines (MEK1, Namalwa) were used. Bone marrow primary CD19+ malignant cells and bone marrow stromal cells (BMSC) were obtained from WM patients. Cytotoxicity, DNA synthesis, and cell cycle were measured using MTT assay, [3H]-thymidine uptake, PI staining/flow cytometry, respectively. Effects of NVP-BEZ235 on cell signaling cascades were determined using immunoblotting and immunofluorescence. Adhesion on fibronectin has been evaluated in WM cells in the presence of NVP-BEZ235.

Results: NVP-BEZ235 induced cytotoxicity and inhibited DNA synthesis with an IC50 of 20–25nM in BCWM.1 at 48 hours. Similar effects were demonstrated in all IgM secreting cell lines and in primary CD19+ WM cells, with an IC50 between 20nM and 50nM. No cytotoxicity was observed on peripheral blood mononuclear cells, indicating selective toxicity of the compound on the malignant lymphoplasmacytic clone. We observed that NVP-BEZ235 inhibited Akt (but not ERK phosphorylation) in a dose-dependent manner in BCWM.1 cells at 6 hours. Phosphorylation of GSK3α/β and ribosomal protein-S6, downstream target proteins of Akt, were also markedly inhibited. NVP-BEZ235-inhibited Akt phosphorylation was further confirmed by immunofluorescence. NVP-BEZ235 induced caspase-9, PARP cleavage and increased the release of Smac/DIABLO from the mitochondria to the cytosol, suggesting an induction of apoptosis in a caspase-dependent and –independent manner. We showed that NVP-BEZ235 inhibited adhesion of BCWM.1 cells to fibronectin in a dose-dependent fashion. Lastly, adherence to BMSCs did not confer protection to WM cells against NVP-BEZ235- induced cytotoxicity.

Conclusions. These data indicate that NVP-BEZ235 has significant antitumor activity in WM, thus providing the framework for clinical trials in this disease.
Triggered Release of Nanoparticulate Arsenic Trioxide for Treatment of Malignant Lymphomas: Preclinical Studies
Authors: Winter Jane N. , Waterhouse Dawn , Ahn Richard , Chen Haimei , Gordon Leo I. , Jovanovic Borko , Bally Marcel , O’Halloran Thomas
Published: April 2020

Lipid encapsulation of chemotherapeutic agents facilitates local delivery of highly concentrated antineoplastics to tumors while ensuring an adequate duration of drug exposure and reducing systemic toxicity. Using novel technology, we have encapsulated nanoparticulate (100 nM) arsenic trioxide (ATO) at high density in “nanobins,” biocompatible lipids and polymers that undergo a change in permeability when exposed to intracellular conditions, releasing drug cargo into the acidic tumor microenvironment (J. Am. Chem. Soc.2006, 128: 13348-9). At low concentrations, ATO is an effective agent in the treatment of acute promyelocytic leukemia, causing differentiation and turnover of PML. At higher but potentially toxic concentrations, ATO induces programmed cell death through the generation of reactive oxygen species and its effects on other proapoptotic signaling pathways. A preliminary dose-range finding study was completed comparing the toxicity profiles of free ATO and nanobin formulations of arsenic based on loading procedures using either cobalt (Co) or nickel (Ni). Female Balb/c mice were injected intraperitoneally with either a single dose of free or nanobin formulations of ATO at 20, 40, 60, 100 or 200 μmol/kg of elemental As. Mice were assessed daily for the first 5 days after injection and then every second day thereafter for a total study duration of 14 days and monitored for body weight and clinical signs. The maximum tolerated doses for both the Co- and Ni-ATO formulations were approximately 100 – 200 μmol/kg as a single intraperitoneal injection. Efficacy experiments for the Ni-ATO-nanobins were conducted in a murine (RAG2-M) xenograft model in which the mantle cell lymphoma cell line Z138C was injected subcutaneously. On day 21, when the average tumor volume was 50–100 mg, weekly injections of saline, empty nanobins, or the Ni-ATO nanobins at 60 μmol/kg were begun. Cohorts of mice received either two or three weekly injections. Mice bearing tumors of 1000 mm3 or greater were sacrificed. Results were normalized to the tumor size at the time that treatment was initiated (day 21) and are represented as fold-increase in tumor measured with calipers three times weekly. The results of the three weekly injections are illustrated below. Following two injections, the curves began to separate showing a significant inhibition of tumor growth that persists for two weeks beyond the last injection (p < 0.001). Two weekly injections also impacted tumor growth significantly (p < 0.01; data not shown). These results provide rationale for further preclinical investigation of this novel therapeutic.

Figure Figure
Prolonged Bone Marrow Failure Following Rituximab Therapy of Follicular Lymphoma
Authors: Debatin Laura , Veelken Hendrik , Schmitt-Graeff Annette
Published: April 2020

Prolonged Bone Marrow Failure following Rituximab Therapy of Follicular Lymphoma Laura Debatin, Annette Schmitt-Graeff*, Hendrik Veelken Depts. of Hematology/Oncology and *Pathology, University Medical Center Freiburg Rituximab (R) is a mainstay in B-NHL therapy. Apart from infusional reactions and prolonged B cell depletion, severe R side effects are relatively rare. A 65 year-old, previously healthy woman presented with anemia (Hb 9.3 mg/dl), thrombocytopenia, and cervical lymphadenopathy. A submandibular lymph node biopsy showed CD20+CD10+CD79a+ follicular infiltrates. Diagnosis of follicular lymphoma was supported by detection of an IgH-BCL2 gene rearrangement by FISH. CT scans revealed generalized lymphadenopathy and hepatosplenomegaly. The BM was infiltrated with 68% clonal CD20+CD10+CD79a+ B cells. Due to advanced (stage IVA) and high-risk disease (FLIPI 4/5), 200 mg/m2 cyclophosphamide/100 mg prednisone/day (d) were given for 5 days, followed by a single dose of 375 mg/m2 R. Grade 4 neutropenia developed 4 days later and precluded the planned CHOP chemotherapy. A BM biopsy on d+21 post R showed marked BM hypoplasia consistent with drug-induced changes, but no residual lymphoma. Serologic testing ruled out Parvovirus B19, HBV, HCV and HIV infections. Leukocytes recovered to >1/nl 3,5 weeks after R administration. No further cytoreduction was given due to prolonged cytopenia and documented PR. Despite this history, Bendamustin/R (200 mg) was given for eventual BM relapse at a different hospital 11 months later. Grade 4, filgrastim-refractory cytopenia with the same BM histology developed promptly and lasted for 4 weeks. The patient was transferred to our institution because of multiple opportunistic infections including pulmonary aspergillosis, E. faecium sepsis, and destructive mucor mycosis of the right maxillary sinus. Posaconazole and liposomal amphotericin were given for 5 months. 13 months after the 2nd R infusion, the patient had to undergo reconstructive surgery with a flap plasty to cover an extensive, disfiguring facial and maxillary necrosis. Since different alkylators were given at moderate doses, a causal relationship to similar episodes of rapid-onset, extended BM failure appears unlikely. R, administered immediately prior to both episodes, appears to be the causative agent in this case. Since hematopoietic precursors and the myeloid lineage do not express CD20, the mechanism for this near-fatal BM failure is unknown and suggestive of an idiosyncratic component. The BM findings are incompatible with a metamizol-type allergic agranulocytosis. Severe BM failure appears to be a rare but grave potential side-effect of rituximab.
Evaluation of the Pharmacokinetics, Immunogenicity, and Safety of XmAb®2513 in the Ongoing Study XmAb2513-01: A Phase 1 Study of Every Other Week XmAb2513 to Evaluate the Safety, Tolerability, and Pharmacokinetics in Patients with Hodgkin Lymphoma or Anaplastic Large Cell Lymphoma
Authors: Younes Anas , Zalevsky Jonathan , Blum Kristie A. , Smith Mitchell R , Fung Henry C. , Ramies David A , Lawrence Chris E , Combs Daniel , Bloss Jeffrey
Published: April 2020

XmAb2513 is a novel second-generation humanized monoclonal antibody (mAb) directed against the human cell surface antigen CD30, with an Fc region engineered to enhance cell killing activity via recruitment of effector cells through increased binding affinity to Fcγ receptors (FcγRs). The CD30 antigen (Ki-1) is a member of the tumor necrosis factor superfamily and was originally identified as a cell surface antigen on primary and cultured Reed-Sternberg cells of Hodgkin Lymphoma (HL). Expression of CD30 is a hallmark for the identification of HL and a subset of T-cell lymphomas, including Anaplastic Large Cell Lymphoma (ALCL). Preclinical in vitro data demonstrated XmAb2513 to be more potent with regards to antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) than the first generation anti- CD30 antibodies MDX-060 and SGN-30. Studies in cynomolgus monkeys demonstrated that XmAb2513 exposure was proportional to dose with half-lives (t½) ranging from 5 to 9 days in single dose studies and 12 to 17 days in repeat dose studies. The exposure and t½ data from these cynomolgus monkey studies supported an every other week dosing interval in clinical trials. Based on these favorable features, a Phase 1 study has been initiated to examine the safety and efficacy of XmAb2513 in patients with relapsed and refractory classical HL and ALCL. This ongoing Phase 1 Study is currently evaluating every other week dosing of XmAb2513 using a dose-escalation study design to establish the maximum tolerated dose (MTD). The dose levels under evaluation are 0.3, 1.0, 3.0, 6.0, 9.0, and 12.0 mg/kg, given intravenously over two hours. To date, seven patients are enrolled on the study in the first three dose levels. Pre-study simulation of human exposure based on the pharmacokinetic (PK) parameters obtained from cynomolgus monkeys predicted that 0.3 mg/kg XmAb2513 in humans would result in serum trough levels of 1–1.5 μg/mL after four infusions given every other week. Interim PK and immunogenicity results from the treated patients have been analysed and the results observed thus far are on track with these simulations. Additionally, immunogenicity assessment for human anti-XmAb2513 (HAHA) showed that the treated patients are so far negative for HAHA. Importantly XmAb2513 was well tolerated. As dose escalation continues, results for human PK, immunogenicity, and safety for additional dose cohorts of HL patients, and preliminary safety and response assessment data will be presented.
Experience in Using Hypercvad Combined with Alemtuzumab in Treating Peripheral T-Cell and T/NK-Cell Neoplasms
Authors: Kuan Jew Win , Lau Ngee Siang , Chang Kian Meng , Tan Sen Mui , Ong Tee Chuan , Purushothamaný Visalachy
Published: April 2020

Peripheral T-cell and T/NK-cell neoplasms (T/NK-cell neoplasms) are rare, representing less than 15% of non-Hodgkin lymphoma in the western hemisphere. The optimal therapy for peripheral T-cell and T/NK-cell neoplasms is an area of controversy due to rarity of the diseases, their variable clinical course, and the lack of randomized trials. Fractionated cyclophosphamide, vincristine, doxorubicin and dexamethasone (hyperCVAD) alternating with high doses of methotrexate and cytarabine has proven to be an effective cytoreductive regimen in patients with aggressive histopathologic variants of mantle cell lymphoma. Alemtuzumab (Campath®) is a humanized monoclonal antibody that targets CD52, a cell surface protein present at high density on most normal and malignant B and T lymphocytes. We report our experience in using six cycles of hyperCVAD in combination with alemtuzumab D1 +/− D4 for the treatment of newly diagnosed peripheral T-cell and T/NK-cell neoplasms in our institution. Seven females and five males diagnosed with peripheral T-cell and T/NK-cell neoplasms (NT and T/NK-cell lymphoma = 5, NT-cell leukemia = 5, NNK-cell leukemia = 2) from 2006 to 2008 were treated with alemtuzumab-hyperCVAD regime. Three patients completed alemtuzumab-hyperCVAD regime and achieved response within four cycles. Among the three patients who completed hyperCVAD regime with only three to five doses of alemtuzumab, two achieved complete response and one had progressive disease. There were six patients who did not complete the alemtuzumabhyperCVAD regime. Three of them stopped at fifth cycle due to cytomegalovisrus (CMV) complication. Two patients died after forth cycle due to neutropenic sepsis although they achieved complete response after two cycles. The other patient did not respond to the treatment, hence discontinued. Both the NK-cell leukemia patients did not respond to the treatment. It was noted that patients with at least one dose of alemtuzumab per hyperCVAD cycle were more likely to survive (62.5%) than the other (25.0%) (95%CI of difference = −0.176 – 0.689). Among the complete respondents, the median disease free duration (from the day achieved response to date or relapse) is 216 days. CMV reactivation occurred in four out of seven patients who received at least five doses of alemtuzumab. In conclusion, alemtuzumab-hyperCVAD seems to be useful in treating peripheral T-cell lymphoma, T/NK-cell lymphoma and T-cell leukemia but not NK-cell leukemia. At least one dose of alemtuzumab per hyperCVAD cycle should be administered to achieve favourable survival. Although CMV complication is common in alemtuzumab-hyperCVAD regime, it does not seem to affect the outcome of the treatment. A proper randomized controlled trial should be conducted to evaluate the effectiveness of alemtuzumab-hyperCVAD in the treatment of peripheral T-cell and T/NK-cell neoplasms.
A Novel Microarray Gene Expression Analysis of Murine AML Cell Lines Provides Clues to the Development of Drug Resistance to Ara-C
Authors: Rathe Susan K , Largaespada David
Published: April 2020

Acute myeloid leukemia (AML) has the ability to evade cell death in the presence of chemotherapeutic cocktails containing cytosine arabinoside (Ara-C). This lab previously developed two highly resistant murine AML cell lines, B117H and B140H, by introducing increasing concentrations of Ara-C to their parental cell lines, B117P and B140P, respectively. B117H and B140H can tolerate Ara-C concentrations ~1000X that of their drug sensitive parental cell lines. mRNA from all four cell lines were used in gene expression microarrays for the purpose of comparing Ara-C drug resistant murine AML cell lines with their Ara-C drug sensitive parental lines. A novel algorithm was developed to evaluate the changes in gene expression between the drug resistant and drug sensitive cells. The algorithm differed from more conventional algorithms in two key ways. First, the detection data was normalized by using ribosomal subunit 9 (Rsp9) as the normalization gene, and secondly it calculated fold change by comparing the minimum value of one population to the maximum value of the other population. The output of this algorithm was a list of genes with significant gene expression changes. These genes were next submitted to the Ingenuity Pathway Analysis (IPA) process. IPA implicated nuclear factor-κB (NFκB) in the Ara-C resistance process. Cell growth assays confirmed that the Ara-C drug resistant B117H cell line was significantly more sensitive to NFκB inhibition than its Ara-C sensitive parental cell line. This leads us to believe that the selection of Ara-C resistance may also concomitantly make some AML cells highly sensitive to killing by NFκB inhibition. This theory is being tested further through the use of drug combination assays, to determine if a synergistic or antagonistic relationship exists between Ara-C and various drugs that affect the NFκB pathway.
Pml and TAp73 Interacting at Nuclear Body and Activated p38MAPK Mediate Imatinib-Induced p53-Independent Apoptosis of Chronic Myeloid Leukemia Cells
Authors: Liu Jin-Hwang , Liu Chin-Cheng , Yen Chueh-Chuan , Gau Jyh-Pyng , Tzeng Cheng-Hwai
Published: April 2020

Imatinib induces apoptosis in chronic myeloid leukemic (CML) cells even with mutant p53. Ras provokes promyelocytic leukemia protein (pml), which promotes apoptosis and senescence in untransformed cells. In CML cells, ras is usually activated; however, the function of pml seems inadequate to induce apoptosis without imatinib. In both BCR-ABL+ p53mutant K562 and Meg-01 CML cells but not in BCR-ABL− HL60 cells, we found imatinib upregulated phosphorylation of p38 mitogen-activated protein kinase (MAPK), checkpoint kinase 2 (chk2) and transactivation-competent (TA) p73, formation of PML-nuclear body (NB), co-localization of TAp73/PML-NB, and expression of bax. Co-immpunoprecipitation of TAp73 and pml was also consistent with the TAp73/PML-NB co-localization. The induced co-localization also occurred to primary CML cells from three out of six patients, including the two with p53mutant. In K562 cells, both inhibiting p38MAPK with SB203580 and silencing pml or TAp73 with short interfering RNAs (siRNAs) abolished the imatinib-induced apoptosis whereas interferon alpha-2a induced additively with imatinib the apoptosis. Notably, interferon alpha-2a increased additively while SB203580 hindered the imatinib-induced phosphorylation of TAp73 and the TAp73/PML-NB co-localization. Together, imatinib induced in some CML cells, especially with mutant p53, a p53-independent proapoptotic mechanism via a p38MAPK/chk2-pml/TAp73 signaling.
The Antileukemic Potential of New Derivatives of 5-Fluoro-1H-Indole-2,3-Dione-3-Thiosemicarbazone on in Vitro Cell Lines
Authors: Kuruca Serap Erdem , Karali Nilgun , Cetin Beyza , Karadenizli Sabriye , Karakas Zeynep
Published: April 2020

1H-Indole-2,3-dione (isatin) is an endogenous compound identified in many organisms, possesses a wide range of biological activities. Biological properties of isatin include a range of actions in brain and offer protection against certain types of infections. This molecule has a versatile moiety that displays diverse biological activities, including anticancer activity. The discovery of numerous biologically active 3-substituted 2-indolinones led in the past decade to extensive synthesis of related compounds and as a result, anticancer agents were developed. In particular, among the 5-substituted analogs tested in the growth inhibitions against several human cancer cell lines, 5-halide, methoxy and trifluoromethoxy groups containing 3-substituted 2-indolinones show high antiproliferative effects. For this purpose, we first synthesized twelve 5-fluoro-1H-indole-2,3-dione-3-thiosemicarbazones that antituberculosis activities were shown previously by our teamwork and we researched anticancer drug potential in this study. The cytotoxic effects of twelve thiosemicarbazone derivatives were investigated by MTT assay in chronic myeloid leukemia cell lines (K562, HL60), B-lymphoma cell lines (P3HR1) and in vincristine resistant forms. The IC50 values (IC50 is a concentration that kills 50% of cells) were calculated from dose-response curve according to cytotoxicity index. The effectiveness of thiosemicarbazone derivatives were evaluated by comparing IC50 values in leukemic cell lines. All of the compounds were found cytotoxic in B-lymphoma cell lines (P3HR1, P3HR1Vin) in range 0.95–2.41 μM. However, the allyl derivative of thiosemicarbazones has cytotoxic activity in all the cancer cell lines (K562, K562Vin, HL-60, P3HR1, P3HR1Vin) that were tested. As a result, 5-fluoro-1H-indole-2,3-dione-3-thiosemicarbazones derivates might have chemotherapeutic drug potential in B-lymphoma patients. The allyl derivative of thiosemicarbazones has benefit both B-lymphoma and chronic myeloid leukemia patients in a large spectrum.
MYC Responsible for down-Regulation of CD33 Expression in Human Myeloma Cells
Authors: Shamsasenjan Karim , Otsuyama Ken-ichiro , Iqbal Mohd S. , Mahmoud Maged S. , Kawano Michio M.
Published: April 2020

Human myeloma cells from about 10% of cases with multiple myeloma expressed CD33 and have monocytoid morphology with convoluted nuclei, and all these patients had no increase in serum CRP values. In CD33(+) myeloma cells as well as myeloma cell lines, CD33 expression levels were correlated with the increased expression levels of CEBPA (C/EBPα) gene. This correlation was confirmed by the finding that transfection with the CEBPA gene induced CD33 expression in a CD33(−) myeloma cell line. As suggested by the lack of an increase in serum CRP values in CD33(+) myelomas, IL-6 down-regulated the expression of CD33 in CD33(+) myeloma cell lines along with the down-regulation of CEBPA gene expression. Cucurbitacin I (STAT3 inhibitor) but not U0126 (MAPK inhibitor) could abolish the effect of IL-6. Furthermore, IL-6 up-regulated the expression of MYC via STAT3 phosphorylation and MYC bound to the promoter region of CEBPA gene followed by the down-regulation of the CEBPA expression. It was confirmed that introduction of shRNA for MYC into a CD33(+) myeloma cell line blocked the IL6-induced down-regulation of CD33 and CEBPA expression. Therefore, these results indicate that IL-6 can reverse the expression level of CD33 by up-regulating MYC followed by the down-regulation of CEBPA expression.
Nonsense Suppression Approaches in Treating Hemophilia
Authors: Liu Yi-Lin , Margaritis Paris , Khazi Fayaz , Downey Harre , Kadauke Stephan , Hasbrouck Nicole , Sheedy Josephine , Welch Ellen , Weetall Marla , High Katherine A.
Published: April 2020

Genetic diseases can result from nonsense mutations that cause premature translation termination. Nonsense mutations account for ~10–15% of all cases of hemophilia A and B and this population may benefit from small molecule-induced readthrough of nonsense codons. Recent studies document the ability of an orally bioavailable small molecule, PTC124, to facilitate dose-dependent readthrough of nonsense codons in a variety of in vitro and in vivo systems, including reporter gene constructs, mdx mice (a murine model of Duchenne muscular dystrophy; Nature447:87–91, 2007), and in a mouse model of cystic fibrosis (PNAS105: 2064–2069, 2008). PTC124 was well-tolerated in healthy human volunteers (J. Clin. Pharmacol.47:430–444, 2007) and in patients with cystic fibrosis (The Lancet, online August 21, 2008). We previously constructed murine models of severe hemophilia B with nonsense mutations in a transgenic human factor IX (hF.IX) gene resulting in nonsense codons at amino acid positions 29 or 338 (R29X and R338X; Blood104:2767–2774, 2004). These mice have no circulating hF.IX detectable by ELISA. To evaluate the effect of small molecules on readthrough in the hemophilia B model, we administered PTC124 to mice carrying the R338X mutation by subcutaneous injection for three days. Measurable plasma levels of hF.IX were detected by ELISA (n=3) and high levels of hF.IX were detected in the liver by immunohistochemistry in the single mouse that was sacrificed. Following oral administration of PTC124 to R338X mice for three days, 20% of mice demonstrated detectable circulating hF.IX levels in the range of 3–5 ng/mL. In a separate experiment, we administered another small molecule, PTC-EMK, by IP injection for three days to R338X mice. With this regimen, 40% of mice showed detectable circulating hF.IX levels. To examine the effect of PTC124 or PTC-EMK on other nonsense mutations, we prepared ten of the most frequently reported nonsense mutations in the hF.IX gene (R29X, R116X, W194X, R248X, R252X, Y266X, W310X, R333X, R338X, and W407X) and transiently transfected these constructs into HEK293 cells. The cells were treated with PTC124 or PTCEMK at various concentrations for 72 hrs and the level of hF.IX protein in the medium was quantified using the ELISA. Of the nonsense containing constructs analyzed, 6/10 mutants showed positive responses with the largest response noted for cells containing the R29X mutation treated with PTC-EMK. In summary, our results demonstrate the ability of PTC124 and PTC-EMK to read through nonsense codons in the hF.IX mRNA and support the potential use of orally bioavailable small molecules as a therapeutic option for patients with hemophilia due to nonsense mutations.
Proteomic Analysis of Multiple Myeloma Cells Targeted with Bortezomib
Authors: Cumova Jana , Potacova Anna , Kasalova Irena , Sedo Ondrej , Konecna Hana , Zdrahal Zbynek , Hajek Roman
Published: April 2020

Introduction: Multiple myeloma (MM) is still an incurable disease characterized by clonal expansion of malignant plasma cells. New anticancer drugs further improve prognosis of myeloma patients. Despite promising clinical activity, some patients with MM failed to respond to bortezomib therapy. The aim of this study was to evaluate changes in protein expression of myeloma cell line ARH-77 after bortezomib treatment.

Materials and methods: Myeloma cell line ARH-77 was treated with bortezomib (5–20nM) for various periods of time. The proteins contained in total myeloma cell lysate were separated by 2-dimensional polyacrylamide gel electrophoresis (2-DE) and the differentially expressed proteins between the untreated (control) and treated cell lines were excised and identified by mass spectrometry.

Results: There were analyzed 94 proteins differentially expressed between treated and control cells; total of 34 protein spots were upregulated: proteins involved in regulation of apoptosis, chaperons/stress related proteins, proteolysis of ubiquitin/protein degradation and cytoskeleton proteins. Sixty protein spots were downregulated: proteins involved in synthesis, regulation of apoptosis, chaperons/stress related proteins, regulation of cell cycle proteins, proteins connected to glycolysis and proteolysis of ubiquitin/protein degradation and antioxidant/redox proteins.

Conclusion: Employing optimized proteomic approach we identified 94 proteins with altered expression after bortezomib treatment.
IgG and IgA Monoclonal Gammopathies of Undetermined Significance (MGUS) at Low Risk of Evolution: Proposal and Validation of a Prognostic Scoring System
Authors: Rossi Francesca Gaia , Marcheselli Luigi , Petrucci Maria Teresa , Rossi Davide , Callea Vincenzo , Guffanti Andrea , De Muro Marianna , Federico Vincenzo , Baraldi Anna , Musto Pellegrino , Bacigalupo Andrea , Gaidano Gianluca , Goldaniga Maria , Avvisati Giuseppe , Deliliers Giorgio Lambertenghi , Baldini Luca
Published: April 2020

PURPOSE: The presenting clinico-hematologic features of 1283 patients with IgG and IgA class monoclonal gammopathies of undetermined significance (MGUS) were correlated with the frequency of malignant transformation to evaluate the most important variables prognostically associated with its evolution into multiple myeloma (MM).

PATIENTS AND METHODS: Two IgG MGUS patient populations were evaluated: a study sample (553 pts) and a test sample (378 pts). The IgA MGUS population included 352 cases.

RESULTS: Considering IgG cases the median follow-up was 6.7 yrs in the study group vs 3.6 yrs in test group; in the first 47/553 pts developed MM vs 22/378 in the latter. At multivariate analysis serum monoclonal component (MC) £1.5 g/dl, absence of BJ proteinuria, normal serum polyclonal Ig levels and age less than 70 defined a prognostically favourable subset of patients. On the basis of these four variables, pts could be stratified into 3 different 10 yrs-evolution risk groups (HR 1.0, 4.28, 11.6; P<0.001). This scoring system was validated in the test sample. Considering IgA cases, thirty out of 352 patients developed MM after a median follow-up of 4.8 yrs. At multivariate analysis, Hb < 12.5 g/dl and serum polyclonal Ig reduction resulted related with neoplastic progression.

CONCLUSIONS: using simple variables, whose prognostic role we have previously described, we could validate a prognostic model useful to identify situations with different evolution risk in IgG MGUS. Considering IgA cases a possible prognostic role of Hb level emerged, while the negative one of monoclonal component class or serum levels was not confirmed.
Phenotypic Correction of Hemophilia a Using An Ips-Based Cellular Therapy
Authors: Xu Dan , Alipio Zaida , Yang Jianchang , Fink Louis M. , Xu Wilson , Ward David C , Ma Yupo
Published: April 2020

Hemophilia A, a sex-linked bleeding disorder, is caused by mutations within the genomic sequence of the Factor VIII (FVIII) gene. Mutations of FVIII lead to depleted or reduced protein production, and inefficient clotting. Because hemophilia A is caused by a single gene deficiency, several attempts at gene therapy have been made, including several phase I clinical trials. However, these have all failed for various reasons--including immune rejection. However, with the recent discovery of induced pluripotent stem (iPS) cells the immune rejection barrier can be circumvented. iPS cells can be generated from somatic cells by introducing the ectopic expression of three transcription factors Oct4, Sox2, Klf4 (Nakagawa et al., Nat Biotechnol.26: 101–106, 2008) To date, iPS cells have been indistinguishable from ES cells, and thus provide tremendous therapeutic potential. In this study, we differentiated iPS cells to endothelial and endothelial progenitor cells using the EB (embryonic bodies) differentiation method. iPS derived endothelial or endothelial progenitor cells express both endothelial or endothelial progenitor cell specific markers, such as CD31, and Flk1, as well as FVIII. These iPS-derived cells were directly injecting into the liver of irradiated hemophilia A model mice. Seven days after transplantation, hemophilia A and control mice were challenged by a tail-clip bleeding assay. Non-transplanted hemophilia A mice died within few hours, while transplanted mice survived for many weeks and more. Plasma FVIII expression in transplanted hemophilia A mice is significantly higher than in non-transplanted hemophilia mice and this elevated expression confers a correction on the hemophilia A phenotype in mice following a tail-clip. Our studies suggest that transplantation of iPS derived endothelial progenitor cells can rescue the phenotype of hemophilia in mouse models and thus potentially suitable for the future development of cell therapy of monogenetic disorders.
Serum free Light Chains Profile as Early Biomarker with Predictive Value after autologous Transplant in Multiple Myeloma
Authors: Recasens Valle , Rubio-Escuin Rebeca , Rubio-Martinez Araceli , Pascual Transito , Lopez Natividad , Sanjuan Mjose , Giraldo Pilar
Published: April 2020

Background: Serum free light chain Freelite® assays, is a quantitative method to measure serum free light chain (FLC) concentration, and could be a simple and accurately indicator to predict the early relapse in Multiple Myeloma(MM) patients with apparently complete response. FLC are small proteins that cross easily through the glomeruli pores, entering the proximal tubule, where they are reabsorbed and metabolised. In patients with a low tumour burden, the amount of FLC produced can remain below this reabsorption capacity and so have no detectable in urine. However, serum levels of FLC continue to rise and therefore become a good biomarker to detect an early progression.

Aims of study: To determine the predictive value of serum FLC concentration in MM patients in complete response after autologous stem cell transplant as a early biomarker of relapse.

Patients and Methods: Observational, analytical study performed in 24 MM patients included in an autologous stem cell transplant program during 2002–2006 in one center. All patients received induction therapy with VBCMP/VBAD (×4), follow by G-CSF movilization and collection of CD34+ cells. The infussion of CD34+(mean: 5.39×106/kg; range: 2.24–18.8) was performed after conditioning with high doses of Melphalan. Serum samples were collected on 0, +4, +7, +21, +30, +60 y +90 days and stored frozen at −80°C. Serum FLC were analysed in a nephelometer system following the manufacturer instructions. We have compare the increase of FLC according M-component subtype, number of CD34+ cells administered, time until immune reconstitution, administration of coadyuvant AM3-glycopeptical therapy, infections in early postrasplant period, maintenance therapy and time free relapsed survival. Descriptive statistical analysis, ANOVA comparative test and Kaplan-Meier survival study was performed.

Results: 24 patients, 47.0 % females, mean age 63.2 y (38–72), IgG (46.1%), IgA (15.4%), light chains (23.0%), non secretory (7.7%), time to immune reconstitution 15–17 days, kappa/lambda ratio at baseline 57.0 (CI95% 0.8,20.3), days: +4:37.3(CI95% 0.1,3.2), +7:1.85(CI95% 1,3), +21:2.37 (CI95% 0.4,2.0), +30:0.87 (CI95% 0.2,0.9), +60:0.4 (0.2, 2.1) and +90:1.26 (CI95% 0.3, 2.9). Mean relapsed free survival 27.6 m (CI95% 9,55), OS 59.0 m (CI95% 27,76), 7 patients have dead for progression. At day +21 opposite correlation kappa/lambda ratio and RFS was observed.

Comments: The early increase of free serum kappa/lambda chains ratio could be an useful biomarker to predict RFS and OS in MM patients in complete response after therapy.
CCL5 (RANTES) Is Suspected to Be the Key Chemokine to Understand the Bone Pathology of Multiple Myeloma
Authors: Hayashi Kunio , Nomura Shosaku , Hashimoto Seiji , Hideshima Teru
Published: April 2020

Background: It has been shown that CCL5 recruits Osteoclasts and Osteoblasts to active bone turnover regions to maintain bone remodeling. In healthy volunteers, CCL5 is higher in male than in female. Although CCL5 is highest in 20ys men, it becomes same level at 60ys in both male and female. These results suggest that the value of CCL5 correlates with the activity of the bone metabolism. In this study, we examined the significance of CCL5 level in MM, which may provide new insights in the bone pathology of this disease.

Patients and Methods: fourteen MM were enrolled in our study. Twelve were newly diagnosed and 2 were relapsed patients. We evaluated CCL5(serum) and bone alkaline phosphatase(BAP, serum), as well as type-I collagen N-telopeptide(NTX, urine) when patients were diagnosed and after the treatment. Regimens of the treatment are

Thalidomide 200mg/day and Dexamethasone(Dex) 40mg every 8th day within 21 days cycle, Velcade 1.3mg/m2 on days 1, 4, 8, 11 of a 3 weekly cycle with Dex 40mg, combination of Thalidomide, Velcade and Dex.

Result: We compare CCL5 of 60ys healthy volunteers, ITP patients, Stage 1 MM, Stage2 MM and Stage3 MM. We found highest CCL5 level in Stage 3 MM. Interestingly, BAP was almost same level between Stage1 and Stage3; however NTX level is higher in Stage3 than in Stage1, suggesting that increased CCL5 in Stage3 was predominantly from activated osteoclasts. Importantly increased CCL5 and BAP were recognized in patients who had CR or PR. Moreover, the increase of the number of megakaryocyts in the bone marrow was recognized in 7 patients. These megakaryocytes were strongly stained with anti-CCL5 Ab, suggesting that successful MM treatments could increase production of CCL5 by megakaryocytes.

Conclusion: CCL5 of Osteoclasts in active myeloma contributes bone resorption. On the other hand, in successfuly treated myeloma, CCL5 is produced by megakaryocytes which make Osteoblasts activate. CCL5 may be a novel marker of bone disease in myeloma.
Quality of Life Assessment at Diagnosis and after Different Treatment Phases in Multiple Myeloma Brazilian Patients
Authors: Silva Vanderleia Costa , Almeida Manuella Sampaio , Hungria Vania T. M. , Etto Leina Yukari , de Oliveira José Salvador Rodrigues , Barros José Carlos , Ciconelli Rozana , Colleoni Gisele W.B.
Published: April 2020

Multiple myeloma (MM) causes great impact in quality of life (Q/L), since patients become dependant of others even for routine activity execution and personal care.

Objectives: To characterize the impact of MM in the Q/L of patients treated in two public institutions of Sao Paulo State, Brazil, using a generic (SF-36) and a specific questionnaire for oncologic patient (QLQ-C30), applied in three different moments: at diagnosis, after the end of clinical treatment and at day +100 after autologous hematopoietic stem cell transplant (ASCT).

Patients and methods: From March 2006 to August 2007 we evaluated 49 patients with MM, using the two questionnaires. Analysis was made through ANOVA, Post hoc and T-paired test comparing the three groups.

Results: 88.6% of included patients have family budget lower than US$ 600.00/month (Economic Class C, D or E). The generic questionnaire SF-36 demonstrated that Physical Functioning, Role-Physical and Bodily Pain were statistically different in all three groups, favoring the day +100 post-ASCT group (ANOVA). SF-36 still demonstrated improvement in Role-Emotional when MM post treatment group was compared with the day + 100 post-ASCT group (T-paired Test). The QLQ-C30 questionnaire confirmed what had been demonstrated by the SF-36 in relation to the Physical Functioning and Bodily Pain plus improvement in the following aspects: Role Functioning, Fatigue, Lack of Appetite and Constipation, favoring the day + 100 post-ASCT group (ANOVA). QLQ-C30 also detected a significant improvement in Social Aspect in patients with MM after day +100 of ASCT.

Conclusion: The specific questionnaire for cancer patients QLQ-C30 seems to be more informative than the generic questionnaire SF-36 and reflects the real benefit of ASCT in Q/L of MM patients from two public Brazilian institutions, which provide assistance for economically challenged population.