bluuebunny/crossref_metadata_2025 huggingface dataset:
Primary Extranodal Lymphoma: Revised Incidence, Location, Histological Classification and Survival in Zaragoza (Spain), 1992–2002
Authors: Giraldo Pilar , Franco-Garcia Esther , Alvarez Ramiro , Garcia-Carpintero Gloria , Pascual Mercedes , Martos Carmen
Published: April 2020

Background. Leukaemia and non-Hodgkin’s lymphomas (NHL) are the commonest haematological malignancies (HMs), accounting for about 10% of incident cases and 6% of all cancer deaths in the European Union (EU). There are few studies in order to estimate the differences in incidence and survival of primary extranodal Lymphoma (PEL). In Zaragoza (Spain) there is a population-based Cancer Registry (ZPCR), that includes all non-haematological and haematological (HMs) incident cases, conducted since 1960. The main aims of this study are:

To review all cases with PEL diagnosis. To estimate the incidence of PEL in the ZPCR registered cases during the period 1992–2002. 2. To calculate the survival of PEL

Methods. All PEL occurred in patients residing in Zaragoza during the period 1992–2002 were selected from ZPCR. All cases were reviewed in order to confirm the primary location and the morphology classification according to REAL. The population at risk was 9.266.609 person-years. The crude (CIR) and age standardized incidence rate (ASR) were calculated, using the European population as standard. Kaplan-Maier method was applied to calculate median survival time and their 95% confidence intervals, as well as 5-year survival. The end of follow-up was 31 December 2007 and Log-Rank was used to compare survival curves.

Results. Among all 4,340 HMs, a total of 1,757 (40.0%) were NHL (CIR: 19×105 person-year), 252 (14.0%) of them were classified as PEL, yielding an ASR of 2.1×105 person-year (males: 140 cases (53.0%), mean age: 59.2 years, ASR: 2.7×105 person-year and females 112 cases (47.0%), mean age: 66.8 years ASR: 1.6×105 person-year. According to topography the most frequent sites were digestive tract (50.4%), skin (19.8%), gland tissue (10.0%), oral cavity-pharynx (7.9%), lung (2.9%), CNS (2.4%), orbit (2.1%) and others (4.5%). The median survival for PEL was 6.61 years (95%CI: 3.7–9.5) and for nodal Lymphomas 5.01 years (95%CI: 4.1–5.9). The 5-year relative survival was 53.5% and 50.0% respectively. There are not significant differences between both groups. Moreover, non significant differences in survival were observed between males and females. Theses results are similar to those found in the EU.

Conclusions. The occurrence of PEL according to gender and mean age is similar to nodal NHL. No significant differences in survival were observed between nodal and extranodal NHL, probably the survival is more conditioned by the histology than topography.
A Systematic Review of the Cost-Effectiveness of Recombinant Factor VIIa (rFVIIa) and Activated Prothrombin Complex Concentrate (aPCC) in the Treatment of Mild to Moderate Bleeding Episodes for Haemophilia Patients with Inhibitors
Authors: Knight Chris , Danø Anne Møller , Kennedy-Martin Tessa
Published: April 2020

Objectives: Although haemophilia patients with inhibitors are rare, the clinical, humanistic and economic consequences associated with this disorder are considerable. The primary treatment for such patients is either rFVIIa or aPCC. The aim of this study was to identify, review and evaluate the quality of the published literature on the relative cost-effectiveness of rFVIIa and aPCC in treating haemophilia patients with inhibitors.

Methods: The review concentrates on the model type, the model design, model assumptions, and results.

Results: The results of this study suggest that rFVIIa may be the cost-effective alternative to treatment with aPCC due to the superior efficacy of rFVIIa and hence the avoidance of subsequent lines of treatment. In 7 of the 9 studies, rFVIIa had the lower average treatment cost. The adapted modelling framework is similar in all the economic models reviewed, suggesting clinical acceptability of the approach used. The estimates of efficacy varied between the models, especially for aPCC. The efficacy for aPCC derived from retrospective studies was lower than reported in the literature. Sensitivity analysis had been undertaken in the majority of the economic analyses and the results were found to be robust to realistic parameter variations. Only one of the studies was a cost-utility study, showing the lack of measuring health status within this area. The results showed the large impact appropriate treatment can have on the quality of life for haemophilia patients with inhibitors.

Conclusions: Ideally, there should be a systematic approach to identifying the relevant data and the lack of data from relevant randomized head-to-head trials is a contributing factor to the variation in efficacy rates and average dosages assumed. However, this systematic review has shown that despite differences in the estimates of efficacy, average dosage required, and unit costs the overall results are robust and appear to favour rFVIIa as the cost-effectiveness treatment for haemophilia patients with inhibitors.
Ambulatory Patient Preference for VTE Prophylaxis
Authors: Sousou Tarek , Khorana Alok
Published: April 2020

Introduction: Cancer-associated thrombosis leads to morbidity and mortality in cancer patients. Thromboembolism can be prevented with the use of existing and emerging anticoagulants. We sought to ascertain awareness as well as receptiveness to anticoagulation amongst ambulatory cancer patients receiving active therapy.

Methods: Two-hundred-fifty 12 question surveys were distributed to ambulatory cancer patients at the James P. Wilmot Cancer Center of the University of Rochester. Patients included in this study were those with active cancer, age ≥ 18 years and ambulatory. Hospitalized patients were excluded. Data was gathered regarding: age, gender, stage as well as type of malignancy, awareness of increased risk of venous thromboembolism and willingness to use various forms of anticoagulation.

Results: One hundred ninety surveys (76%) were completed over a three week period. Study population consisted of 71 males (37%) and 119 females (63%) with mean age of 58. Malignancies surveyed include: lymphoma 26%, breast 20%, gastrointestinal 15%, leukemia 9% and lung cancer 7% among others. Nineteen percent of patients had stage IV malignancy. Of the patients surveyed, 53% reported being unaware of the increased risk of VTE with malignancy. Eighty-six percent of patients surveyed would be willing to use an oral anticoagulant 46% would be willing to perform daily anticoagulant injections.

Conclusions: Prophylaxis for VTE in ambulatory cancer patients is an area of active investigation and could lead to improvements in morbidity and mortality. This study reveals a lack of knowledge of the increased risk of VTE amongst cancer patients. However, informed patients expressed willingness to use prophylaxis if shown effective to reduce VTE in the ambulatory setting.
Bone Marrow Specific Scatter Pattern on Multiple Myeloma and the M:E Ratio and Cellularity Analysed by the CELL-DYN 4000® Methodology
Authors: Simoes Ana Teresa , Barreira Rogerio FC , Pereira Alexandra Alves , Cardeira Sara , Sanches Raquel , Costa Carla Silva , Gonsalves Ana Paula , Marques Adilia , Santos Susana , Salvado Ramon , Duarte Marta , Ribeiro Maria Leticia
Published: April 2020

INTRODUCTION: Optical microscopy is the reference method for the morphological evaluation of bone marrow aspirates; however, in spite of permitting a good qualitative evaluation, the relative quantification of the different type of cells is time consuming and not precise. Regardless the efforts to develop automatic methodologies, no precise and reliable methods are available. CELL-DYN 4000® haematological analyzer uses the MAPSS® technology, based on optical light dispersion, fluorescence and impedance, with great accuracy in differential cellular blood counts.

STUDY PURPOSE:

to establish the correlation between the optical microscopy and the automatically CELL-DYN 4000® bone marrow examination concerning the cellularity and the myeloid/erythroid ratio. to study the bone marrow scatter pattern on haematological diseases.

MATERIAL AND METHODS: 84 bone marrow samples (in K3EDTA) collected for diagnostic purposes among patients attending the Haematology clinic of Centro Hospitalar de Coimbra. Bone marrow films were stained by May-Grünwald-Giemsa’s, analysed by two different Haematologists and the diagnosis were confirmed by flow cytometry studies. Bone marrow automatic analysis were performed by CELL-DYN 4000® using CBC(N), Extended Count(W) and Resistant(R) methodologies.

Statistical analysis: Pearson’s correlation for agreement between automatic methodologies for erythroid lineage and lymphoid series counts; Pearson’s correlation for M:E ratio; Cohen’s Kappa Test for the cellularity; Fisher’s Exact Test to determine whether a specific graphical pattern was associated with a particular haematological disease.

RESULTS: Comparing bone marrow studies by manual and automatic methods we verified:

a good correlation (r=0,769, p<0,0001) with R methodology for erythroid lineage and lymphoid series, despite the interference of erythroblasts in the lymphoid series; an excellent agreement (k>0,808) in the cellularity assessment and an excellent correlation (r=0,955; p<0,0001) with the R methodology in M:E ratio.

We also found a very good association (c2=24; p<0,0001) of a specific scatter pattern and Multiple Myeloma (n=10) which is different from Plasmocytic Leukaemia (n=2) and Monoclonal Gammopathy of Undetermined Significance (MGUS) (n=2).

CONCLUSIONS: The present study shows that the bone marrow M:E ratio, as well as the cellularity evaluation by CELL-DYN® 4000 R methodology are reliable. It also shows the association between a specific graphic pattern and Multiple Myeloma. Morphological analysis is the gold standard for bone marrow examination and it will be, probably, never discharged, however, these data confirm the possibilities of having a good global evaluation of bone marrow samples by automatic methods.
TG-Interacting Factor Regulates Proliferation and Differentiation of Human Myeloid Leukemia Cells
Authors: Hamid Rizwan , Brandt Stephen J.
Published: April 2020

Transforming growth-interacting factor (TGIF) is a homeobox transcriptional repressor that has been implicated in holoprosencephaly and various types of cancer. TGIF is expressed in hematopoietic stem cells and modulates TGF-β and retinoic acid (RA) signaling, both of which play an important role in hematopoiesis. We recently reported that TGIF’s levels correlate inversely with survival in patients with acute myelogenous leukemia. Here we present the first direct evidence of a role for TGIF in myelopoiesis. We used short hairpin RNA interference to define the effects of TGIF knockdown on proliferation and differentiation of myeloid leukemia-derived cell lines. We show that TGIF levels increase with myeloid differentiation and its knockdown resulted in reduced proliferation and differentiation, and lower expression of CEBPβ, CEBPε, PU.1 and RUNX1, key myeloid transcription factors. The reduction in proliferation was not attributable to a block at a particular stage of cell cycle or a significant increase in apoptosis. Furthermore, we show that TGIF knockdown results in increased TGF-β and decreased RA signaling. Consistent with our progenitor cell line results, bone marrow cells derived from Tgif-null mice produced reduced number of colonies in methylcellulose colony forming assays. Insights into the molecular basis of TGIF’s effects were provided by a genome-wide chromatin immunoprecipitation-based elucidation of TGIF target genes. Together, these data suggest that TGIF has a role in regulating the balance between proliferation and differentiation. Reduced TGIF expression could tip the balance toward quiescent leukemic cells (including leukemic stem cells), providing them protection from anti-cycle agents.
Acute Splenic Sequestration in Sickle Cell Disease (SCD): Still a Life Threatening Complication
Authors: Brousse Valentine , Lesprit Emmanuelle , Bernaudin Francoise , Odievre Marie-Helene , Guitton Corinne , Quinet Beatrice , Dangiolo Silvana , De Montalembert Mariane
Published: April 2020

Background: Acute splenic sequestration (ASS) is a life threatening event in SCD during early childhood. Subsequent related mortality has been greatly reduced by both parental education and appropriate management. Moreover SCD screening in neonatal period has allowed early detection contributing to the decrease in fatal attacks.

Objectives: Our aim was to update splenic sequestration epidemiology in a French cohort followed after neonatal screening.

Methods: we reviewed the medical files of SCD children born between 2000–2007 followed since birth in 5 pediatric centres. We included all SCD children diagnosed with acute splenic sequestration, defined as an acutely enlarging spleen with a fall of hemoglobin (Hb) concentration of at least 2g/dL.

Results: A total of 266 episodes of ASS occurred in120 children. There were 69 boys and 51 girls. Genotypes were as follows: 9 S beta° thalassemia, 2 SD-Punjab, 4 SC, 105 SS. In the SS subgroup, median age at first episode was 16 months [1–83] and 34,8% of these first episodes occurred before 12 months of age. Associated symptoms were found in 37 cases (isolated fever n=20, vaso occlusive crisis n=5, identified viral or bacterial infection n= 12) and did not influence the rate of recurrence. Mean Hb level during first crisis was 5.2g/dL [3.3–7]. Mean age at first episode for those who experienced only one episode (n=43) versus more than one episode (n=62) was statistically different (18 months (+/−10) versus 28 (+/−22); p=0.002, Student test). Median interval between first and second episode was 3.5 months [1–31]. 31 patients had 3 or more episodes with a mean interval between second and third interval of 2 months [1–17]. There was no significant difference in the mean age at first episode in children who experienced 2, 3 or more episodes. There was only one fatal case due to acute anemia which occurred in a girl at first recurrence (Hb:2,2g/dL). After the second attack 14(22.5%) patients were splenectomized, 26 patients (41.9%) were started on a transfusion program (followed in 13 cases by splenectomy) and 21 were followed up. Splenectomy was performed at a median age of 4.5years.

Conclusion: These results show that ASS remains a major concern in the management of SCD children. We show a high rate of recurrence and young age at first episode favours this recurrence threat. This study further confirms the effectiveness of early diagnosis and parental education in the decrease of mortality. Prospective studies on spleen dysfunction and additional research on predictive factors are warranted.
Different Subclones of Adult Acute Lymphoblastic Leukemia Detected by Clonal Igh and TCR Markers Behave Differently during Remission and at Relapse
Authors: Davidyan Yulya , Parovichnikova Elena , Surin Vadim , Savchenko Valeryi
Published: April 2020

INTRODUCTION. Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements are excellent patient–specific targets for the detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL). However they might be unstable during the disease course. False-negative results due to clonal evolution are the major disadvantage of using Ig/TCR gene rearrangements as PCR targets for MRD detection. In order to minimize false-negative results, it is necessary to use more PCR targets for MRD monitoring.

PATIENTS AND METHODS. Bone marrow samples of 34 ALL patients (27 B-ALL, 7 T-ALL) were analyzed for IgH and TCR gene rearrangement by means of PCR method at diagnosis, after induction and consolidation of remission, before and after stem cell transplantation and during maintenance therapy.

RESULTS. Clonal IgH and TCR gene targets for MRD detection were identified in 97% of the patients (33/34). In 70% of the patients (23/33) two or more specific markers for MRD monitoring were detected. Three clonal IgH gene rearrangements were discovered in 2 patients. We wished to investigate whether it was important to monitor two or more targets, because of the dynamics of various markers (subclones) may be different). Prolonged MRD monitoring with 2–3 markers was carried out in 9 patients on different stages of treatment. In 4 of 9 patients (3 precursor B-ALL, 1 pre-T-ALL) the results of MRD detection by 2–3 clonal markers at different time points were similar. And in 5 of 9 patients (3 precursor-B-ALL, 1 precursor T-ALL, 1 B-mature ALL) marker detectability during treatment was dissimilar and not parallel. Of note, 4 of those 5 patients with varying dynamics of different subclones relapsed. Relapse was not observed in the B-mature ALL patient. No relapses were detected in 4 patients with similar dynamics of MRD markers. Bone marrow samples of 8 adult ALL patients were subjected to a detailed analysis of IgH and TCR gene rearrangements by PCR method at diagnosis and relapse. In most patients (7/8) rearrangements identified at diagnosis were present also at relapse. In one patient with precursor-B-ALL one clonal incomplete IgH gene rearrangement (DH-JH) detected at diagnosis was lost at relapse and another one DH-JH rearrangement was preserved. Of note, acute myeloid leukemia was diagnosed at relapse in this patient.

CONCLUSIONS. Comparative PCR analyses of IgH and TCR gene rearrangements did not detect differences between newly diagnosed and relapsed ALL although in a small group of adult patients (unlike childhood ALL). Our data indicate the necessity to monitor MRD in ALL patients with more than one PCR target in order to minimize false-negative results and to predict the course of the disease. The detectability of various MRD markers (subclones) during treatment may differ and this fact could predict a higher probability of relapse.
Copper Levels in Patients with Hematological Malignancies
Authors: Saouli Zoi , Diamantidis Michael D. , Papadopoulos Athanasios , Kaiafa Georgia , Girtovitis Fotios I , Kontoninas Zisis , Ntaios George , Andreadis Charalambos , Savopoulos Christos , Hatzitolios Apostolos
Published: April 2020

INTRODUCTION: Copper is an essential trace element that plays an important role in human biochemistry. Its metabolic pathways involve cellular respiration, peptide biogenesis, connective tissue biosynthesis and antioxidant defense. It is known as an acute phase reactant increasing in response to infection, injure and in chronic inflammatory conditions.

AIM: The aim of this study is to observe the alterations of serum copper levels in patients with various hematological malignancies and also to examine the relation of its levels with the extent of the disease and assess the role of copper determination in staging these patients.

MATERIALS AND METHODS: 77 patients (32 male, 45 female) with various hematological diseases were studied. 50 normal, healthy subjects were studied along. Age ranged from 18–75 years (mean age: 63 years). There were 15 patients suffering from chronic lymphocytic leukaemia (CLL), 17 from non-Hodgkin lymphoma (NHL), 7 from Hodgkin’s disease, 13 from acute myeloid leukaemia (AML), 8 from multiple myeloma and 17 from remainder hematologic malignancies. Copper was measured by flame atomic absorption spectrometry (normal range 0,8–1,3 mg/l). Statistical analysis was conducted by SPSS 12.0. Student’s t-test was used.

RESULTS: Copper levels of the healthy control group were within normal range. When all patients were studied, 51 patients with relapsing disease had elevated copper levels (mean value 1,8 mg/l), whereas 26 patients with remission of their disease had normal copper levels (mean value 1,03 mg/l). Student’s t-test (Sig. two-tailed = p value was 0,000<0,001). When separate disease groups were studied, the results were in accordance with the aforementioned assumption made on all patients: 17 NHL patients (11 relapse − mean copper value = 1,87, 6 remission − mean copper value = 1,12, p<0,001), 15 CLL patients (8 relapse − copper = 1,57, 7 remission − copper = 0,86, p<0,001), 13 AML patients (9 relapse − copper = 1,9, 4 remission − copper = 1,09, p<0,001).

CONCLUSIONS: Our study indicates that elevated copper levels are associated with relapsing hematological malignancies, whereas normal copper levels are associated with remission of the underlying disease. Copper determination might prove to be beneficial in staging such patients, which will lead to proper therapeutic decisions.
Evaluation of Central Nervous System Recurrence of Intravascular Large B-Cell Lymphoma Treated with Rituximab-Containing Chemotherapy
Authors: Shimada Kazuyuki , Murase Takuhei , Matsue Kosei , Okamoto Masataka , Ichikawa Naoaki , Tsukamoto Norifumi , Kikuchi Ako , Saburi Yoshio , Masaki Yasufumi , Niitsu Nozomi , Miwa Hiroshi , Asaoku Hideki , Kosugi Hiroshi , Matsumoto Morio , Kashimura Makoto , Yoshida Takashi , Tamura Kazuo , Yamamoto Kazuhito , Yamaguchi Motoko , Nakamura Shigeo , Naoe Tomoki , Kinoshita Tomohiro
Published: April 2020

Central nervous system (CNS) recurrence is a serious and usually fatal complication in the clinical course of aggressive lymphoma. Although our recent report suggested that the improvement of clinical outcomes in intravascular large B-cell lymphoma (IVLBCL) by the application of rituximab, the clinical impact of CNS recurrence in IVLBCL is unknown. To evaluate risks and risk factors associated with development of CNS recurrence in IVLBCL, we retrospectively analyzed 106 patients with IVLBCL receiving chemotherapies with or without rituximab, using our recent cohort (Shimada et al. J Clin Oncol. 2008). Clinical outcomes for our cohort were updated as of December 2007. For this analysis, we excluded 21 of 106 patients (20%) with CNS involvement at diagnosis, which was diagnosed by imaging modalities including computed tomography (CT) and/or magnetic resonance imaging (MRI), and/or cerebrospinal fluid (CSF) analysis. In all, 46 patients (54%) and 39 patients (46%) received chemotherapies without rituximab (chemotherapy) and with rituximab (R-chemotherapy), respectively. With a median follow-up for surviving patients of 39 months (range, 2–158 months), 11 of 46 patients (24%) in the chemotherapy group and 7 of 39 patients (18%) in the R-chemotherapy group developed CNS recurrence after beginning chemotherapy. Median duration between diagnosis and CNS recurrence in the both groups was 9 months (range, 1–44 months). Three patients in the each group displayed CNS progression on the initial series of chemotherapies. Risk of CNS recurrence at 3 years reached 29% in the chemotherapy group and 21% in the R-chemotherapy group, respectively (log-rank test, P = 0.41) (shown as figure). In univariate analysis, use of rituximab combined with initial chemotherapies was not identified as favorable predictive factors. In 12 patients who received autologous stem cell transplantation before CNS recurrence, 1 patient developed CNS relapse by the end of the study. Median survival after CNS recurrence was 6 months (range, 1–58 months), and survival rate at 2 years after CNS recurrence was 14%. This study indicates that CNS recurrence remains a critical problem in the rituximab era. Further studies are required to establish the optimal treatment strategy for CNS prophylaxis in IVLBCL.

Figure Figure
Evolving First-Line Treatments -from Chemotherapy to Immunotherapy Alone- in 143 Consecutive Follicular Lymphoma Patients Treated in the Last 14 Years at the European Institute of Oncology, Milano
Authors: Bassi Simona , Gigli Federica , Rabascio Cristina , Sammassimo Simona , Mancuso Patrizia , Cocorocchio Emilia , Bertazzoni Paola , Laszlo Daniele , Pruneri Giancarlo , Calabrese Liliana , Lionetti Maria Teresa , Vanazzi Anna , Alietti Alessandra , Nassi Luca , Agazzi Alberto , Pastano Rocco , Peccatori Fedro Alessandro , Martinelli Giovanni
Published: April 2020

Here we present the clinical results and the outcomes of 143 consecutive newly-diagnosed follicular lymphoma (FL) patients (pts) treated with 3 different modalities of treatment in our institution from 1994 until 2007. During this period, from 1994 to 2002, 55 pts received Chlorambucil and Prednisone (Chl+PDN), subsequently 31 pts Rituximab with Chlorambucil (R-Chl) and 57 pts a 4-weekly standard-dose of Rituximab alone (R). Chlorambucil was given in an induction phase at 10mg/day for 6 consecutive weeks followed by a longer maintenance phase in the first group: 2-week pulses of 10 mg daily with 2-week intervals for a total of 12-months of treatment versus four 2-week pulses with monthly-Rituximab. In the single agent R group 36 pts received maintenance with at least 4-bimonthly administrations. The 3 groups were comparable for demographic and prognostic factors: median age at diagnosis was 55 yrs, over 60% of pts were in advanced stages and mainly asyptomatic (about 90%) and over 50% of pts in each group were low-risk FLIPI. A third of pts in both Rituximab-based treatment groups had bone marrow involvement and bulky disease. In terms of ORR 78% of pts treated with Chl+PDN, 97% with R-Chl and 72% with R single agent, obtained a clinical response, with a percentage of complete response of 58%, 90% and 47% respectively. No significant incidence of adverse events were reported and none discontinued the therapy because of toxicity. One case of myelodysplatic syndrome was described in a relapsed patient of the Chl-PDN group. With a median follow up of 72, 56 and 27 months in the 3 groups evaluated, the CR rate was maintained in 29%, 74% and 37% of pts respectively with a median duration of response of 36, 40 and 24 months. Rituximab as first-line in FL pts seems safe, feasible and able to induce a high rate of clinical response similar to those achieved with standard chemotherapy alone. On the other hand, the immunochemotherapy seems to confirm its superiority in terms of response rate and duration of response.
Selective Abrogation of STAT5 Expression Impairs the Proliferation of Acute Myeloid Leukemia Cells with Various Cytogenetic Alterations
Authors: Kim Youngsoo , Zhou Tianyuan , Guo Shuling , Siwkowski Andy , Witchell Donna , Pandey Sanjay , Monia Brett P.
Published: April 2020

STAT5 is a key common downstream mediator of multiple signaling pathways which are often dysregulated in various hematologic malignancies, including acute myeloid leukemia (AML). Due to the heterogeneity and high relapse rate of the disease, the treatment options for AML are currently limited. Although the approach of treating the disease by inhibiting upstream kinases such as FLT3 within these signaling pathways appeared promising, the clinical efficacy of these drugs as mono-therapy have been disappointing. We hypothesized that this lack of efficacy might be due to the residual STAT5 activity that is present even in the presence of these inhibitors in vivo. Therefore, abrogating the expression of the final regulator of these pathways, STAT5, might be a much more efficient way of blocking signaling, thus inhibiting the proliferation and survival of AML cells. In this study, we first investigated the role of STAT5 in the proliferation of AML cells by selectively suppressing the expression of the gene using 2nd-Generation antisense oligonucleotides (ASOs). Suppression of STAT5 following ASO treatment (>80% over control ASO) led to a significant inhibition of cell proliferation (50~70% over control ASO), a decrease in colony formation, and a modest induction of apoptosis in a range of AML lines including KG-1α, MV-4-11, and MOLM-13. STAT5 ASO treatment was highly specific for the STAT5 target and produced predictable effects on gene expression, as demonstrated by the downregulation of Pim-1 and cyclin D1, well-known STAT5 regulated genes. No changes in the expression levels of Bcl-XL, STAT1, and STAT3 were observed. Furthermore, relative anti-proliferative activity within the various AML lines correlated well with the relative levels of STAT5 activity. Interestingly, there was a strong correlation between the extent of STAT5/Pim-1 downregulation and the degree of anti-proliferation, suggesting a possible role of Pim-1 as a downstream effector of STAT5 ASO anti-proliferative activity. Studies comparing the relative effects of the STAT5 ASO inhibitor with the potent multi kinase inhibitor CEP701 in various AML cell lines demonstrated potent anti-proliferative activity for the STAT5 inhibitor in the cell lines including KG-1α that display resistance to the multi kinase inhibitor. Taken together, these results suggest that a STAT5 ASO therapeutic approach may have utility for the treatment of AML and related hematologic disorders.
Human Myeloma Cells Undergo Differentiation and Apoptosis upon Exposure of Rosiglitazone and All-Trans Retinoic Acid Via Apoptosis Signaling Pathway Modulation
Authors: Huang Haiwen , Depei Wu , Chen Guanghua , Chang Weirong , Leung Anska Y.H. , Liang Raymond , Chow Howard C.H.
Published: April 2020

Background Myeloma cells often develop drug resistance leading to treatment failure in the patients. New agents that can overcome drug resistance through induction of apoptosis and differentiation are needed. PPARg serves as a transcription factor and functions as a heterodimer with Retinoid X receptor a (RXRa). Activation of PPARg by its ligands has shown potential anti-neoplastic effects in solid tumors. In this study, we investigate the effects of RGZ as well as combined with all trans-retinoic acid (ATRA) on human myeloma cell lines and try to address its potential mechanism.

Method U266 and RPMI-8226 cells were treated with different concentration of RGZ in the presence or absence of ATRA and various biological responses were studied by the methods of [3H] thymidine incorporation, MTT, cell cycle analysis, Annexin V-PI stanning, Wright-Gemsa staining, RT-PCR and caspase-3 activity assay.

Results We report that exposure to RGZ induced proliferation inhibition and viability loss in a dose-dependent manner in both U266 and RPMI-8226 cells. A similar exposure to RGZ also induced cell cycle arrest, cell differentiation and apoptosis of myeloma cells. A combination of RGZ with ATRA enhanced the effects of RGZ and induced cell differentiation in myeloma cells. The mRNA expressions of FLIP, XIAP and survivin were detected in both cell lines and the levels decreased significantly after cultured with RGZ, similar synergism effect of ATRA and RGZ on mRNA level of these apoptosis related genes was also observed. Caspase-3 activity increased substantially along with the increase of RGZ concentration and the addition of ATRA in culture medium shows similar synergism effect on caspase-3 activation.

Conclusion These results suggested that RGZ may represent a promising candidate for the treatment of multiple myeloma and ATRA may be useful as a combination therapy for multiple myeloma.
Mechanism of CAG Regimen Eliminating Human T Cell Acute Lymphoblastic Leukemia Cell Line, A3
Authors: Sun Aining , Wu Yanling , Xue Shengli , Depei Wu , Chang Weirong
Published: April 2020

Objective To explore the mechanism of CAG regimen eliminating human T cell acute lymphoblastic leukemia cell line, A3 and evaluate the role played by G-CSF/G-CSFR system in this process.

Methods

The expression of G-CSFR on A3 cells was detected by flow cytometric analysis. Cell cycle parameters of A3 cells treated with different concentration of G-CSF(5ng/ml A10ng/ml A15ng/ml A20ng/ml G0ng/ml as control) were examined by propidium iodide staining. The inhibition and apoptosis rates of A3 cells caused by treatment with various combination of G-CSF, cytarabine (Ara-C), and aclarubicin (ACR) after incubation for 48h were analyzed by Cell Counting Kit (CCK-8) and AnnexinV staining, respectively. After incubation for 48 hours with G-CSF and PD98059(the specific inhibitor of MEK in Ras-MAPK signaling pathway), cell cycle and cell dynamic change were examined.

Results

The expression frenquency of G-CSFR on A3 cells was 94.2% which was comparable to that of KG-1 cells. The proportion of A3 cells in S-phase was elevated concomitantly with the increasing G-CSF concentrations within 0–20ng/ml, highest at 15ng/ml of G-CSF. After incubation with Ara-C and G-CSF for 48 hours, the proliferation of A3 cells was inhibited more significantly than incubation with incubation with Ara-C alone (P<0.05, Ara-C 10−5M and 10−6M) by CCK-8 assay. Incubated with Ara-C, ACR, and G-CSF for 48 hours, the apoptosis of A3 cells was increased than that treated with Ara-C and ACR. With the concentration of PD98059 increased gradually, the proportion of A3 cells in S-phase and OD values of A3 cells decreased, which was less than that of control group (p<0.05).

Conclusion

G-CSFR was expressed on A3 cells. G-CSF/G-CSFR system had a synergetic effect on eliminating A3 cells when administrated simultaneously with chemical agents by driving G0-phase cells into S-phase. Apoptosis was one of the mechanisms of CAG regimen eliminating A3 cells. The interaction between G-CSF and G-CSFR activates a series of signaling pathways which includes Ras-MAPK. The inhibition of MAPK phosphorylation by PD98059 contributed partially to the effect of G-CSF on A3 cells.
Role of Zoledronic Acid in the Apoptotic Cell Death of Oral Fibroblasts and Epithelial Cells: A Mechanism of Bisphosphonate-Associated Osteonecrosis
Authors: Scheper Mark A , Badros Ashraf Z. , Chaisuparat Risa , Cullen Kevin , Meiller Timothy F
Published: April 2020

Purpose: Osteonecrosis of the jaw is reported in patients receiving IV zoledronic acid (ZA). The purpose of this study was to investigate the effects of ZA on oral mucosal cells using an in vitro model as an alternative mode for the induction of bone necrosis.

Experimental Design: Human gingival fibroblast and keratinocyte cell lines were exposed to different concentrations (0.25–3μM) of ZA and siRNA versus caspase 3 or 9, using 1μM as the expected baseline concentration. Apoptotic effects were determined by TUNEL and Annexin V studies. Effects on cell proliferation were determined by Coulter Counter and MTS assay. Validation of the apoptotic effects of ZA were obtained via RT2 Profiler PCR Arrays of human apoptosis and Western blot.

Results: A dose response effect on apoptosis and cell proliferation was observed with increasing ZA concentrations; both reversed using siRNA against caspase 3 or 9. Initial apoptotic effects were observed microscopically at 8 hours, with increasing apoptotic events measured out to 24 hours. Gene expression analysis demonstrated the differential expression of multiple genes involved in apoptosis including: TNF, Bcl-2, caspase, IAP, TRAF, and death domain families. Western blot analysis confirmed the presence of activated forms of caspase 3 and 9 and underexpression of survivin protein expression.

Conclusions: This study demonstrates that low concentrations of ZA rapidly and directly affect the oral mucosal tissues though the induction of a gene-regulated apoptotic process. These findings provide the establishment of an oral cell based model supporting a soft tissue mechanism in the pathogenesis of osteonecrosis.
Bone Marrow Microenvironmental Overexpression of DKK1, HGF, Frizzled-Related Protein B, and Syndecan-1 Are Associated with the Degree of Osteolytic Bone Disease in Multiple Myeloma Patients
Authors: Haaber Jacob , Kerndrup Gitte Birk , Lyng Maria , Andersen Thomas L. , Pallisgaard Niels , Abildgaard Niels
Published: April 2020

Gene expression analysis of 8 different genes in homogenized bone marrow trephine biopsies from 47 newly diagnosed, untreated MM patients with different degrees of osteolytic bone disease (OBD) and 19 MGUS patients were analysed by quantitative PCR technique. The analysed genes represent both genes suspected to be involved in osteoblast inhibition (DKK1, HGF, Frizzled-related protein B, Syndecan-1) and osteoclast activation (RANKL, MIP-1α), and genes involved in the regulation of these genes (OPG, RANK). The expression levels of these 8 genes in the biopsies represent the net gene expression in bone marrow microenvironment including MM cells, hematopoietic cells, stromal cells, osteoblasts, and osteoclasts. OBD was graded into no, limited, or advanced by standard radiography. The expression level of DKK1 increased significantly with more advanced OBD (p=0.005), whereas the expression level between the MGUS group and the MM group with no/limited OBD was not significantly different (p= 0.097). Syndecan-1 was expressed in all BM-samples from MGUS and MM patients. Syndecan-1 expression was significantly increased with more advanced OBD and was significantly different between the three bone morbidity groups. The expression of Syndecan-1 was, furthermore, closely correlated to PC numbers in the BM samples (p< 0.0001). HGF and FRPB expressions in the MM group with no/low OBD and MM group with advanced OBD was not statistically different but HGF and FRPB expression was significantly increased in the MM groups compared with the MGUS group. OPG expression showed a trend towards being decreased with more advanced OBD. There were no correlation between the degree of OBD and the expression levels of MIP1a, RANKL, and RANK. Thus, in this BM microenvironment expression profile study we found that inhibitors of the Wnt signalling pathways, DKK1 and Frizzled-related protein B, and the inhibition of osteoblastogenesis by HGF, seem to play an important role in the pathogenesis of OBD in MM. In the contrary, we did not find a significant correlation between up-regulation of the osteoclast activating cytokines, RANKL and MIP1α, with more advanced OBD, and we did not find a correlation between the expression level of the RANKL receptor, RANK, and the degree of OBD.
Dysregulation of Pax5 Activity Contributes to the Extinction of the B-Cell Phenotype in Reed-Sternberg Cells
Authors: Collins Graham P , Paterson Jennifer C , May Gillian E , Gupta Rajeev , Marafioti Teresa , Mason David Y , Enver Tariq
Published: April 2020

Hodgkin/Reed-Sternberg cells (HRS cells) are thought to be derived from post-germinal centre B-cells and yet have down-regulated the B-cell phenotype. The B-cell transcription factor Pax5 is important in the maintenance of B-cell identity and we demonstrate that it is down-regulated in HRS cells lines and in HRS cells of the majority of primary classical Hodgkin Lymphoma (cHL) cases. Specifically, 3/30 cases were negative for Pax5, 16/30 were weakly positive, 10/30 cases were moderately positive and 1/30 showed Pax5 staining of equivalent intensity to infiltrating, polyclonal B-cells. In order to functionally test the relevance of a reduced Pax5 expression level, the cHL cell lines L428 and L1236 were stably transfected with Pax5 using a lentiviral transfection system. Transfection of L1236 resulted in up-regulation of CD79a protein expression. However, CD79a was not upregulated in L428 and expression of the Pax5 target genes Cd19 and Blnk was unaffected by Pax5 transfection in both cell lines. Chromatin immunoprecipitation demonstrated that Pax5 failed to bind the high affinity binding site within the Cd19 promoter in the cHL lines despite high levels of Pax5 expression, appropriately localised to the nucleus. Pax5 could, however, bind synthetic oligonucleotide corresponding to this site (as shown by electrophoretic mobility shift assays) raising the possibility that epigenetic modification in vivo may be responsible for the failure to bind DNA. Bisulphite genome sequencing confirmed that in cHL cell lines, the region surrounding the Pax5 binding site in the Cd19 promoter was extensively methylated. Moreover, histone modification analysis also demonstrated an absence of markers of accessible, active chromatin (di- and trimethylated H3K4) and an enrichment of a marker indicating closed, repressive chromatin (trimethylated H3K27). Within the Cd79a promoter, previous studies have implicated the methylation status of a single cytosine residue within the binding site for a Pax5-Ets1 complex to be an important determinant of activation of the Cd79a gene. Interestingly, this residue was shown to be largely methylated in L428 cells but largely unmethyated in L1236 cells, providing a likely mechanism for the differential activation of this gene by transfected Pax5 protein. To investigate whether the observed epigenetic changes were responsible for preventing Pax5 binding and activity at the Cd19 and Cd79a promoters, Pax5 transfected cHL cell lines were cultured in the presence of the demethylating agent 5-aza-2-deoxycytidine. Up-regulation of Cd19 and Cd79a expression was significantly greater in Pax5 transfected cells than in control transfected cells. To conclude: our data suggests that dysregulation of Pax5 activity (at the levels of protein expression and epigenetic modification of the Pax5 binding sites) is important in mediating the extinction of the B-cell programme in HRS cells.
Antitumor Effects of JBD57 in Human Multiple Myeloma Cells in Vitro
Authors: Pereira Juliana , Levy Debora , Ruiz Jorge Luis Maria , Maciel Felipe Vieira Rodrigues , de Alencar Fisher Chamone Dalton , Bydlowski Sergio Paulo
Published: April 2020

JBD57 is a nucleoside/nucleotide analogue that in human cells causes depletion of mitochondrial DNA by disrupting oxidative phosphorylation pathways leading to toxic accumulation of nonesterified fatty acids, dicarboxylic acids and free radicals. Human 26S proteasome is also a target for JBD57. Here we evaluated JBD57 citotoxicity in several human tumor cell lines in vitro. Human MM cell line RPMI 8226/S (CCL-155), human T-cell lymphoblastic-like (Jurkat) and human T-cell leukemia (1301) were grown in RPMI 1640 medium; uterine sarcoma (MES-S (CRL-1976) cells were grown in McCoy medium; HUV-EC-C (CRL-1730) cells were grown in 199/EBSS medium. Media were supplemented with 10 % FBS. Cells were incubated at 37°C in a water-jacketed incubator with 5 % CO2. To evaluate JBD57 citotoxicity in RPMI 8226/S, MES-S, Jurkat, 1301 and HUV-EC-C cells, 104cells/well were grown in flat-bottomed 96-well tissue culture plates for 24, 48 and 72 hr; JBD57 was added to the media in several concentrations (0μM, 32.25μM, 62.5μM, 125μM, 250μM and 500μM). At the end of the experimental periods, cell viability was determined by the MTT method. JBD57 inhibited the growth of MM cell line RPMI 8226/S in a dose- and time-dependent manner. Cell viability decreased progressively with increasing concentrations of JBD57 as well as with increasing time periods. The IC50 (inhibitory concentration at 50%) was 125 μM at 72 hr. The viability of the MM cells after 72 hr incubation with JBD57 500μM was 33%, whereas 100% viability was observed when no drug was added. On the other hand, JBD57 did not affect cell viability of any of the other studied cell lines (uterine sarcoma, Jurkat, 1301 and HUVEC-C). JBD57 promotes a significant human MM cell death in a dose and time dependent manner but do not affect neither normal cell HUV-EC-C nor the tumoral cells MES-S, Jurkat and 1301, at least in the studied conditions. These results suggest that the potent antitumoral activity of JBD57 observed against MM cells could be potentially useful in the treatment of multiple myeloma.
Bortezomib in Combination with Epirubicin, Dexamethasone and Thalidomide Is a Highly Effective Regimen for Multiple Myeloma
Authors: Lü Shuqing , Wang Jianmin , Xu Xiaoqian , Ni Xiong , Qiu Huiying , He Yi , Wang Bin , Huang Chongmei , Yang Jianmin
Published: April 2020

Promising efficacy of bortezomib, the first therapeutic proteasome inhibitor, has been demonstrated in the treatment of multiple myeloma (MM). In this study, we evaluate the therapeutic effects and adverse reactions of bortezomib combination with epirubicin, dexamethasone and thalidomide (BADT) for the treatment of multiple myeloma (MM). Using BADT regimen (bortezomib 1.0mg/m2 on days 1, 4, 8 and 11; epirubicin 12mg/m2 on days 1, 4, 8, 11; dexamethasone 20mg on days 1–2, 4–5, 8–9, 11–12; thalidomide 100mg/m2 on days 1–28) for a maximum of eight 4-week cycles. 12 MM patients were treated, including 5 previously untreated patients and 7 previously treated patients. Each patient completed at least 2 cycles with a mean of 4.5 cycles. Of the 12 patients, 9 patients (75%) achieved complete remission (CR)and 2 patient (16.7%) achieved partial remission (PR), with an overall response (OR) of 91.5%. The mean number of cycles to CR was 2.8 (1–6), and the mean number of cycles to PR was 1.3 (1–3). 7 patients achieved PR after one cycle treatment, and 4 patients achieved CR after one cycle treatment. The main adverse reactions included peripheral neuropathy (4/12), thrombocytopenia (3/12), abnormal change of ECG (4/12), neutropenia (5/12), and liver function impairment (3/12), mainly grade ±-II. Infection occurred in three patients with neutropenia, including sepsis in one patient and herpes zoster in another patient. One patient died of diseases progression, another patient died of secondly sarcoma after achievement CR. The median progression-free time was 14 months (2–26) months, including 2 patients who have been in CR for 15 months since discontinuation of the treatment. In Conclusion, the BADT regimen is highly effective with low toxicity in the treatment of MM. It is warranted to conduct further study on this regimen in more cases.
The Investigation of JAK2 and C-MPL Mutations of Myeloproliferative Disorders Patiens
Authors: Xia Jun , Shen Yunfeng , Li Jianyong
Published: April 2020

Most studies have shown that JAK2V617F was found in nearly all the patients with polycythemia vera (PV) and half of patients with essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). However, JAK2 exon 12 mutations in JAK2V61F-negetive PV patiens and MPLW515L/K mutations in IMF patiens implied other pathogenesis in these myeloproliferative disorders (MPD). Two parts were included in our research to investigate the JAK2 and c-MPL mutations of MPD patients. Firstly, to evaluate the JAK2 exon 12 mutations and its clinical significance in patients with PV, polymerase chain reaction was used to amply the region of JAK2 exon 12 and direct sequencing was performed to detect mutations of JAK2 exon 12. Clinical features of the PV patients with JAK2 exon 12 mutations and JAK2V617F mutation were retrospectively reviewed. The results showed that the JAK2 exon 12 mutations were detected in 3 of 12 JAK2V617F-negtive PV patients (25.0%). Three JAK2 exon 12 mutations were H538QK539L, K539L and N542-E543del respectively. The patients carrying the mutations of JAK2 exon 12 displayed higher hemoglobin(223.6±15.0 vs190.3±20.2g/l, P<0.05), lower white blood cell counts (7.8±2.3 vs16.2±6.7 *109/l, P<0.05)compared with JAK2V617F-positive PV patients (n=31). Secondly, allele specific polymerase chain reaction (AS-PCR) and gene sequencing were used to detected JAK2V617F, MPLW515L and four types of JAK2 exon 12 mutations (F537-K539delinsL, H538QK539L, K539L, N542-E543del) in 30 IMF patients. Results showed that 15 of 30 (50.0%) patients had JAK2V617F mutation and 2 of 30 (6.7%) patients had MPLW515L mutation. JAK2 exon 12 mutations were not detected in IMF patients. Literature reviews show that this is the first report of JAK2 and c-MPL mutations in a number of Chinese patients with BCR/ABL-negative MPD. It is concluded that the JAK2 exon 12 mutations exist in PV patient and the patients with JAK2 exon 12 mutations are characterized by isolated erythrocytosis. The frequency of MPLW515L mutations is high in IMF, which supports recent studies.