bluuebunny/crossref_metadata_2025_split huggingface dataset:
Importance of JAK2 V617F Allele Burden in the Diagnosis of Myeloproliferative Diseases and Its Association to Age.
Authors: Rapado Inmaculada , Albizua Enriqueta , Ayala Rosa , Hernandez Jose Angel , Garcia-Alonso Luis , Grande Silvia , Gallardo Miguel , Gilsanz Florinda , Martinez-Lopez Joaquin 
Published: October 2019

Abstract
The aim of this study is to compare the diagnostic value of several methods, with different sensitivities, to detect JAK2 V617F mutation. Three new methods based on Real Time PCR to detect JAK2 V617F mutation were employed. The first two, based on hybridization probes and Peptide Nucleic Acid Probe (PNA) specific for wild allele; and the third employing Allele Specific Oligonucleotide (ASO) primers for JAK2 V617F mutant allele and MGB TaqMan probe. Healthy subjects (n 149) and patients with Essential Thrombocythemia (ET) (n 63), Polycythemia Vera (PV) (n 31), Secondary Thrombocythosis (ST) (n 38), Secondary Erytrocythosis (SE) (n 35) and other haematological malignancies (n 72) were included in the study. Results. Validity test study for JAK2 617 using HP PCR showed: A-. For PV patients: Sensitivity (Se) was 87.1%, Specificity (Sp) 100%, Positive Predictive value (PPV) 100%, and Negative Predictive Value (NPV) 97.9%. B-. For ET patients: Se was 61.3%, Sp 100%, PPV 100%, and NPV 93.9%. With JAK2 617 PNA PCR: A.- For PV patients: Se was 93.6%, Sp 97.8%, PPV 87.9%, and NPV 98.9%. B. - For ET patients: Se was 71%, Sp 95.7%, PPV 73.3%, and NPV 95.2%. With JAK2 V671F ASO quantitative PCR (JAK2 617 ASO qPCR): A.- For PV patients: Se was 93.5%, Sp 98.5%, PPV 90.6%, and NPV 98.9%. B.-For ET patients: Se was 80.6%, Sp 95.9%, PPV 75.8%, and NPV 96.7%. Cutoff point of 1% was established by ROC curves as mutation burden to distinguish PV or ET versus secondary causes. Three healthy subjects were positive (2%, 3/149) by JAK2 617 PNA PCR method. With JAK2 617 ASO qPCR, 16 healthy subjects would be positive (10%) if cutoff burden established in 0.1%. Significant differences were obtained when comparing Myeloproliferative Disease (MPD) patients over 40 years (n 64) to patients under 40 (n 15). JAK2 V617F median allele burden was 18.41% and 7.26% respectivily (p=0.016).
Conclusions: JAK2 617 HP and ASO qPCR are the best tests in differential diagnosis of Polyglobulias and Thrombocytosis. When JAK2 V617F allele burden is low, the best test for MPD diagnosis is JAK2 617 ASO qPCR. The number of JAK2 V617F mutated cells is higher in patients over 40, which leads to think that age may influence in tumour burden.
Bleeding Risk in Patients with Essential Thrombocythemia and a Platelet Count of More Than 1.5 Million/μl: A Challenge of the Dogma of Mandatory Initiation of Cytoreductive Therapy.
Authors: Petrides Petro E. , Siegel Fabian 
Published: October 2019

Abstract
Background: The most important limiting factors of patient survival in essential thrombocythemia (ET) are thromboembolic and hemorrhagic complications [1]. It has been postulated that the paradox of hemorrhagic events in a disease with highly elevated platelet counts is caused by an acquired von Willebrand Factor (vWF) deficiency syndrome which is found in ET patients with very high platelet counts [2]. Therefore, cytoreductive therapy is usually recommended by several international expert groups when platelets exceed 1,5 Million/μl.
Aims: To critically examine the concept of cytoreductive therapy in high-platelet ET based upon the postulated increased bleeding risk.
Methods: In a prospective study we observed 3 patients with a continously elevated platelet counts of more than 1.5 million/μl and secondary loss of large von Willebrand factor multimers, as shown in SDS electrophoresis, that did not receive cytoreductive therapy, but have been treated only with aspirin.
Results: Patient 1 is a female which was 48 years at the time of diagnosis. She is negative for the JAK2V617F mutation and shows a secondary loss of large vWF multimers. We have now observed her for 7 years without any bleeding complications on aspirin therapy. During that period she has had more than 1.5 million platelets/μl for more than 46 months with a zenith of 2.27 million/μl. The second patient is a male who was 60 years old at the time of diagnosis. He is JAK2V617F negative and shows a relative decrease in large vWF multimers. He has been bleeding free since his diagnosis 6 years ago and had platelet levels of more than 1.5 million/μl for a cumulative period of more than 30 months. He has been treated only with aspirin. Patient 3 is a 59 year old male who has been diagnosed with ET 8 months ago. He is heterozygous positive for the JAK2V617F mutation. He also has a relative decrease in large multimers including a complete loss of the largest multimers. His platelet count has been elevated over 1.5 Million/μl (maximum: 2.18 Million) for a cumulative period of 6 months during which he has been treated only with aspirin. After that period he has been switched to anagrelide due to headaches. He has not shown any hemorrhagic complications except an increased bleeding tendency during shaving.
Conclusion: Our observations challenge the recommendations by several groups to automatically initiate platelet reducing therapy above a platelet count of 1.5 million/μl. Acquired von Willebrand factor deficiency caused by ET does apparently not lead to hemorrhagic complications in all patients and therapy with aspirin should not be automatically excluded based on this dogma.
T Cell Pathway Deficiencies in Chronic Lymphocityc Leukemia: Partial Restoration with OKT3/IL-2 Activation.
Authors: Di Ianni Mauro , Moretti Lorenzo , Del Papa Beatrice , De Ioanni Maria , Terenzi Adelmo , Bazzucchi Moira , Ciurnelli Raffaella , Falzetti Franca , Tabilio Antonio 
Published: October 2019

Abstract
As Chronic Lymphocytic Leukemia (CLL) is associated with several defects in the T cell compartment, the impact of tumour burden on the autologous immune system was studied. Gene expression profiles (using Applied Biosystem Human Genome Microarray) identified 237 genes with significantly increased expression and 221 genes with significantly decreased expression (p<0.05) in CD3+ cells from CLL patients compared with healthy donors. Panther software analysis identified 34/237 upregulated genes and 26/221 downregulated genes that were involved in specific pathways, mainly cell differentiation and proliferation, survival, apoptosis, cytoskeleton formation, vesicle trafficking and T cell activation. The 26 dowregulated genes included Zap70, a member of the syk family protein tyrosine kinase, which is involved in T-cell activation. Zap-70 results were validated by mRNA quantification by RT-PCR (−1.77 fold in comparison with healthy controls) and by flow-cytometric analysis (Mean Intensity Fluorescence=33±12 vs 80±23.62 in controls, p<0.05). To test the hypothesis that activation with OKT3 /IL-2 could bypass these T cell deficiencies, activated T cells from 20 patients with CLL were tested in vitro for cytotoxicity (using the 51chromium release assay) against mutated and unmutated (according to IgVH mutational status) autologous B cells, DAUDI, K562 and P815 cell lines. After 10 days’ culture, the T cell count remained unchanged; CD8 cells expanded more than CD4; TCR spectratyping analysis indicated no differences in TCR repertoires. Activation restored the ZAP-70 mRNA (+1.67 fold). The 51chromium release cytotoxicity assay showed an index > 30% in 5/20 patients. The other 15 were partially cytotoxic against P815, K562 and Daudi. Cell line analysis in all 20 confirmed prevalently T cell-mediated cytotoxicity and poor NK/LAK activity. Cytotoxicity did not correlate with B cell mutational status. We tested the cytotoxic activity of autologous activated T cells in NOD/SCID mice co-transplanted with leukaemic B cells. Only activated T cells exerting cytotoxicity vs autologous B-cell CLL prevent CLL in human-mouse chimera, as confirmed by PCR and FACS analysis which visualised only CD3+ cells. In conclusion, in patients with CLL, activating autologous T cells with OKT3 /IL-2 bypasses, at least in part, the T cell immunological deficiencies. These in vitro and in vivo findings might serve to throw light on new mechanisms that could be exploited in immunotherapy designed to exert disease control.
Antigen Activation and Impaired Fas-Induced Death-Inducing Signaling Complex (DISC) Formation in Large Granular Lymphocyte Leukemaia.
Authors: Yang Jun , Epling-Burrbette P.K. , Painter Jeffrey S. , Zou Jian , Bai Fanqi , Wei Sheng , Loughran Thomas P. 
Published: October 2019

Abstract
Clonal T-cell expansion in patients with T-Large-Granular Lymphocyte (LGL) Leukemia occurs by an undefined mechanism that may be related to Fas apoptosis resistance. Here we demonstrate polarized expansion of CD8+ terminal-memory differentiation in such patients, as demonstrated by CD45RA expression and absence of CD62L expression, suggesting repeated stimulation by antigen in vivo. Elimination of antigen-stimulated T-cells normally occurs through Fas-mediated apoptosis. We show that cells from LGL leukemia patients express c-FLIP and display resistance to Fas-mediated apoptosis and abridged recruitment of proteins that comprise the Death Inducing Signaling Complex (DISC) including the Fas-associated death-domain (FADD) protein and caspase-8. Exposure to interleukin-2 (IL-2) for only 24 hours sensitized leukemic LGL to Fas-mediated apoptosis with enhanced formation of the DISC, and increased caspase-8 and caspase-3 activities; a process that required antigen stimulation in normal T-cells. Our results demonstrate that expanded T-cells in patients with LGL leukemia display both functional and phenotypic characteristics of prior antigen activation in vivo and display reduced capacity for Fas-mediated DISC formation.
Early Reduction of Serum Free Light Chain Can Predict Therapeutic Responses in Multiple Myeloma.
Authors: Nakorn Thanyaphong Na , Watanaboonyongcharoen Phandee , Suwannabutra Seksan , Theerasaksilp Sunee , Paritpokee Nara 
Published: October 2019

Abstract
Background M protein as measured by serum protein electrophoresis (SPE) is usually required for the diagnosis and therapeutic monitoring of multiple myeloma. However, SPE may not be able to detect M protein in some cases such as light-chain disease or non-secretory myeloma. Serum free light chain (FLC) assay has been recently developed and shown to be very helpful in such situations. Here, we evaluate the usefulness of this new test in the diagnosis and therapeutic monitoring of myeloma patients treated with bortezomib plus dexamethasone at our institution.
Methods Serial serum FLC assays were performed before and after each cycle of induction therapy. All patients were treated with bortezomib plus dexamethasone every 3 weeks for four cycles. Treatment responses were determined according to the European Group for Blood and Marrow Transplantation criteria.
Results There were total of 25 patients in this analysis. Fifteen were male (60%); the median age was 54 years (range, 35–68 years). Abnormal ratio of serum FLC was detected in 88% of pre-treatment serum samples. After four cycles of induction therapy, nine patients (36%) had achieved complete remission. No baseline clinical and laboratory parameters could correctly predict the therapeutic responses in these patients. When serial FLC assays were taken into account, we found that a more-than-40% reduction in the abnormal serum FLC after the first cycle of treatment was significantly associated with achieving complete response at the end of treatment (Pearson Chi-square, p = 0.041).
Conclusions Serum FLC assay is very useful for the diagnosis of multiple myeloma and allows a more rapid assessment of therapeutic response. Further studies are needed to establish the role of this test in the era of highly effective anti-myeloma therapy.
Genetic Polymorphisms Correlated with Outcome in Multiple Myeloma Patients Receiving High Dose Melphalan.
Authors: Dumontet Charles , Landi Stefano , Reiman Tony , Plesa Adriana , Bellini Ilaria , Barale Roberto , Pilarski Linda M. , Tavtigian Sean , Perry Troy , Gemignani Federica 
Published: October 2019

Abstract
High dose melphalan is an essential component in the treatment of patients with advanced multiple myeloma. However few data are available regarding genetic polymorphisms possibly correlated with patient outcome or toxicity in this setting. To identify polymorphisms predictive of survival and/or toxicity we performed a retrospective single nucleotide polymorphism (SNP) analysis using APEX technology in 169 patients having received high dose melphalan for multiple myeloma. All patients had received one or two courses of high dose melphalan with stem cell reinfusion. DNA was obtained from peripheral blood mononuclear cells and analyzed for 230 SNPs in genes selected for their role in drug metabolism, DNA repair, cell cycling or apoptosis. There were 108 males and 61 females and the median age was 55 years. The average number of VAD courses received before high dose melphalan was 4 (3–6). 58 patients (34%) received two high dose melphalan courses. A complete/very good response was observed in 43 of 103 evaluable patients (42%). Forty patients among 107 evaluable (37%) experienced high grade (III–IV) mucositis after high dose melphalan. The median freedom from progression was 45 months and the overall survival was 91 months. SNPs in SULT1A1, MDR, ERCC5, GSTP1, NQO1, CYP1A2, CCND1, PARP, RAD51, DRD4, P53 or associated proteins were found to be correlated with response to VAD therapy. SNPs in GRPR, ERCC5, GSTM3, GSTP1, XRCC1, ERCC4, CYP1B1, BARD1, MDR1, P53BP1, TERT, DRD4 were found to be correlated with response to high dose melphalan. Polymorphisms in CYP2E1, CYP2C9, CYP1A1, CYP1B1, TMPT, OGG1, PCNA, DRD2, XPC were correlated with disease progression while polymorphisms in ERCC1, DRD4, CYP2D6, CYP2C9, CYP1A1, CYP1A2, CYP1B1, ALDH, BARD1, BRCA1, MYH, TP53BP1 were correlated with death. Polymorphisms in XRCC1, SNP103, CYP2C9, CYP2C19, BRCA1, BRCA2, NBS1, CDKN1A were correlated with severe mucositis. These results suggest that SNPs of genes involved in drug metabolism or repair could be used to distinguish patients subgroups with different toxicity/efficacy profiles after high dose melphalan.
Elevated Normal Immunoglobulins and Stable M-Protein Levels in Multiple Myeloma Patients on Erythropoietin Treatment.
Authors: Gadassi Noa , Sagiv Sari Prutchi , Oster Howard S. , Newmann Drorit , Mittelman Moshe 
Published: October 2019

Abstract
Recombinant human erythropoietin (rHuEPO) is a well-known treatment for anemia in multiple myeloma (MM) patients. We have previously reported that rHuEPO treatment was associated with prolonged survival of several patients suffering from advanced disease (Mittelman et al., 1997). Recently we have demonstrated that treatment of MM patients with rHuEPO is associated with significant improvements of certain immunological parameters and functions (Prutchi-Sagiv et al., 2006), mainly related to the cellular compartment. The objective of the present retrospective study was to determine whether rHuEPO treatment, in addition to its effects on the cellular immune compartment, also modulates the humoral arm of the immune system in MM patients. Medical charts of eighteen consecutive IgG and IgA MM patients were analyzed and the levels of normal immunoglobulins (Ig) and M-protein before and during rHuEPO treatment were recorded. We have found a significant increase in the levels of normal Ig (IgG, IgA and IgM) in response to rHuEPO, during the 3–9 months fromtreatment initiation. Importantly, the levels of M-protein remained stable for a period of 10–12 months from treatment initiation. These results are in line with previous studies, including our study in murine models (Katz et al., 2007), demonstrating that EPO improves humoral immune responses. The current study highlights the concept that EPO’s immunomodulatory actions on MM patients might also involve the humoral compartment of the immune system.
MN1 Overexpression Promotes Proliferation and Self-Renewal of Primitive Normal Human Hematopoietic Cells
Authors: Imren Suzan , Heuser Michael , Chen Ling-Yi , Edin Glen , Eaves Connie J. , Humphries R. Keith 
Published: October 2019

Abstract
Overexpression of MN1 is a negative prognostic factor in patients with acute myeloid leukemia (AML) with normal cytogenetics. We have previously demonstrated that overexpression of MN1 is sufficient as a single genetic event to induce AML in mice with very short latency (Blood110:1639, 2007). To investigate the effects of MN1 on the functional activity of primitive human hematopoietic cells, we transduced CD34+ human cord blood cells with an oncoretrovirus (3 experiments) or lentivirus (3 experiments) encoding GFP ± MN1. Immediately post-transduction primary colony-forming cell (CFC) assays showed no significant differences in the number or type of colonies generated from the MN1 and control-transduced cells. However, the number of CFCs detectable in secondary assays of the MN1-transduced cells was 17-fold higher than from the GFP-transduced cells and this difference was found to increase a further 234-fold as detected by tertiary CFC assays. To assess the effect of MN1 on cells that can be propagated for more prolonged periods on mouse fibroblast feeders engineered to produce human Steel factor, IL-3 and G-CSF, 104 unsorted cord blood cells were plated into such cultures and the number and types of cells present was then determined 8 weeks later. The remarkable potency of MN1 in stimulating the output of primitive cells under these long-term culture (LTC) conditions was evident from the 1700 greater increase in total cell output and 277-fold higher output of CFCs as compared to control cultures and an accompanying large expansion of CD34+ cells (representing up to 18 % of the final population) whereas CD34+ cells were no longer detectable in the control cultures. Further analysis by limit dilution assay (2 experiments) showed that the frequency of cells able to produce CFCs for at least 6 weeks in these stromal based cultures was increased 10-fold (1/7656 control cells vs 1/718 MN1-transduced cells). In addition the CFC output of each of these was enhanced, on average, 6-fold. Remarkably, these effects of MN1 were sustained for a further 16 weeks as shown by the analysis of the cells they produced in secondary and tertiary LTCs. Overall this resulted in a cumulative 6250-fold increase in total cells over 24 weeks, whereas control cells declined to undetectable levels within 16 weeks. Cells harvested from the secondary and tertiary LTCs initiated with MN1-transduced cells also showed a continuing output of 878,967 ± 332,300 and 264,458 ± 120,600 CFCs after 16 and 24 weeks, respectively. This study sets the stage for further investigation of the effect of MN1 on human NOD/SCID/γcnull mouse repopulating cells and the molecular mechanisms by which MN1 blocks the differentiation fate of primitive human hematopoietic cells and how this may be related to its contribution to the genesis of poor prognosis AML.
Ott1 (Rbm15) Is Required for the Large to Small Pre B-Cell Transition
Authors: Kharas Michael , Manis John , Gilliland D. Gary , Raffel Glen D. 
Published: October 2019

Abstract
The OTT1 gene is fused with the MAL gene in t(1;22) infant-associated acute megakaryocytic leukemia generating a chimeric protein, OTT1-MAL. OTT1 is a transcriptional activator/repressor related to the spen/SHARP/Mint family. Mint was shown to regulate follicular versus marginal zone B-cell development in the spleen; however, Ott1’s physiologic role in B cell development is not fully understood. Recent data utilizing conditional deletion of a targeted Ott1 allele in mice delineated multiple regulatory roles for Ott1 in myeloid and lymphoid differentiation. Previous work by our laboratory showed that loss of Ott1 in addition to causing a myeloid and megakaryocytic expansion, results in a block in B development prior to the B220+CD43-IgM+ stage. We have characterized Ott1-deficient B-cell progenitors to identify stage- and process-specific requirements for Ott1 in pre B development. Ott1-deleted bone marrow and fetal liver could not generate pre B colonies in methylcellulose, however, we were able to establish IL-7-dependent pro B cell lines in vitro and observed no significant differences in proliferation, apoptosis or the ability to form v-Abl-transformed cell lines. Activated Ras or overexpression of Bcl2 failed to rescue pre B colony formation. In vivo, Ott1 null fetal liver pre B-cells expressed Ig heavy chain but failed to express the B-cell receptor (BCR) on their surface even though kappa rearrangement was detectable in vitro. In comparison to wildtype cells, B220+CD43-IgH+ Ott1 null cells were larger in size, had lower levels of IL-7R, but proliferated at higher levels and with an associated increase in apoptosis. Moreover, these cells had normal pre-BCR proximal signaling as judged by phospho-Blnk and phospho-Erk, but increased phosphorylation of S6 after IL7 and Ig stimulation. Ott1 null large pre B-cells had normal expression of Myc, but higher levels of expression of Cyclin D1. Taken together, these data indicate that loss of Ott1 results in enhanced proliferation and apoptosis in the pre B-cell compartment causing a developmental block at the large to small pre B-cell transition. Differentiation blocks at the pro and pre B stage through mutations in B cell regulatory genes, such as PAX5, BTK and BLNK, have been recently demonstrated in acute lymphocytic leukemias. It is plausible that mutations in OTT1, given its position in the tightly regulated process of B cell development, may likewise contribute to pre B-leukemogenesis.