bluuebunny/crossref_metadata_2025 huggingface dataset:
Familial Aggregation of Multiple Myeloma and Its Precursor Monoclonal Gammopathy of Undetermined Significance (MGUS): A Population-Based Study in Sweden.
Authors: Kristinsson Sigurdur Y , Bjorkholm Magnus , Goldin Lynn R. , Blimark Cecilie , Mellqvist Ulf-Henrik , Wahlin Anders , Turesson Ingemar , Landgren Ola
Published: April 2020

Background: Evidence from multiply affected families, case-control and population-based registry studies implicate a role for genetic factors in multiple myeloma (MM). The aim of this large population-based familial case-control study was to quantify risks of MM, monoclonal gammopathy of undetermined significance (MGUS), and other lymphoproliferative disorders among first-degree relatives of MM patients.

Methods: We identified 13,963 MM patients diagnosed in Swedish hospitals 1958–2005, with linkable relatives. Using the population-based central Population- and Multigenerational registries, we obtained 54,610 matched controls and first-degree relatives of MM patients (n=37,838) and controls (n=151,068). Relatives of MM patients and controls were linked with hospital-based outpatient registries and the central Swedish Cancer Registry to define occurrence of MGUS and lymphoproliferative malignancies. Measures of familial aggregation were calculated by a marginal survival model using relatives as the cohort.

Results: First-degree relatives of MM patients had a significantly increased risk of developing MM [relative risk (RR)=2.1; (95% confidence interval (CI), 1.6–2.9)] and MGUS [2.1 (1.5–3.1)]. The risk estimates were very similar when we conducted analyses by gender of proband, by type of first-degree relative (parent, sibling, offspring), and by age at MM diagnosis (below/above 65 yrs) for probands. Among relatives of MM patients, we found no excess risk of chronic lymphocytic leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma, or lymphoplasmacytic lymphoma/Waldenström’s macroglobulinemia.

Conclusions: In this large population-based study, we found relatives of MM patients to have a 2-fold excess risk of developing MM and MGUS compared with relatives of controls. Our findings support the theory that there are common, shared susceptibility genes that predispose to MM and MGUS. Better characterization of early genetic lesions mediating monoclonal plasma-cell proliferation, survival, and migration in the bone marrow microenvironment will potentially provide clues to pathogenesis and allow identification of novel molecular targets.
Regulatory T Cells (Treg) Are Depressed in Patients with Relapsed/Refractory Multiple Myeloma (MM) and Increases towards Normal Range in Responding Patients Treated with Lenalidomide (LEN).
Authors: Quach Hang , Ritchie David , Neeson Paul , Harrison Simon , Tai Tsin , Tainton Kellie , Lynch Kevin , Prince Miles
Published: April 2020

CD4+CD25+Foxp3+ regulatory T cells (Tregs) are a group of immune-suppressive T cells responsible for the maintenance of self-tolerance and autoimmunity. Increased Tregs have been implicated in suppression of tumour-immunity, and are associated with poor prognosis in gastric, oesophageal and ovarian cancers. Conversely, reduced Tregs are associated with aggressive disease and poor prognosis in lymphoproliferative disorders such as follicular lymphoma and Hodgkin lymphoma. Meanwhile, conflicting literature exists with regards to the role of Tregs in the pathogenesis of multiple myeloma (MM). Lenalidomide (LEN) is an immunomodulatory drug (ImiD™) that increases T cell numbers and function in vitro but the impact on Tregs in vivo is unknown. As part of an ongoing clinical trial of low-dose lenalidomide, peripheral blood lymphocyte subsets (LSS) and Treg numbers were examined in patients [n=18, median age 67 years (49–77)] with relapsed/refractory MM at baseline and at various time points during treatment with combination LEN (15mg/o/d d1–21q28)-dexamethasone (dex) (20mg/o/d d1–4,9–12,17–20 q28), in order to measure changes induced by LEN-dex treatment. Results were compared to 10 age matched healthy donors. LSS and Tregs were assessed by flow cytometry, with Tregs strictly defined as CD4+CD25hiCD127dimFOXP3+. At baseline, MM patients had depressed B cells (median 0.76 vs. 3.2 ×105/ml; p=0.02) and CD3+ T cells (median 7.2 vs. 12.1 ×105/ml; p<0.001) compared to healthy donors, with an inverse CD4:CD8 T cell ratio (0.7:1 vs. 2.9:1, p=0.008). Tregs in MM patients were markedly reduced at baseline compared to healthy donors [median 0.40 (range: <0.01–1.9) vs. 4.8 (range 2.4–7.1) ×104/ml; p=0.0005]. In 11 patients completing at least 4 treatment cycles, no recovery of B cells (7.6 vs. 2.3 ×105/ml, p=0.053), NK cells (2.1 vs. 2.7×105/ml, p=0.1) or T cells (5.6 at baseline vs. 7.5 × 105/ml, p=0.45) were seen despite all achieving at least a PR (one with IF negative CR). In contrast, median Treg numbers progressively increased towards normal range, from a baseline of 0.3 to 1.3 × 104/ml (p=0.005) and 2.0 × 104/ml (p=0.009) after 4 (n=11) and 6 (n=7) cycles of LEN-dex respectively (figure 1). Patients with relapsed/refractory MM have profoundly disturbed LSS including marked decreased in Treg numbers. Normalisation of Treg numbers in responding patients treated with LENdex suggest that, as in other tumours, Tregs are actively involved in the pathophysiology of MM and their modulation may be an important component of the immunomodulatory mechanisms of LEN.

Figure 1: Change in regulatory T cell (Treg) numbers with each cycle of LEN-dex in MM patients Figure 1:. Change in regulatory T cell (Treg) numbers with each cycle of LEN-dex in MM patients
Loss of Chromosome 1p21 Band Is Associated with Disease Progression and Poor Survival in Multiple Myeloma Patients Undergoing Autologous Stem Cell Transplantation.
Authors: Chang Hong , Qi Connie , Jiang Allan , Xu Wei , Trieu Young , Reece Donna E.
Published: April 2020

Amplifications involving chromosome 1q and deletions involving 1p are frequent events in multiple myeloma (MM). The pathogenesis and clinical significance of these anomalies are largely unknown. As karyotyping and SNP based mapping analysis identify a minimal common deletion region involving the 1p21 locus, we investigated the prevalence and prognostic significance of del(1p21) in a cohort of 235 MM patients undergoing highdose therapy and autologous stem cell transplant at our institution. Fluorescence in situ hybridization combined with cytoplasmic light chain detection (cIg-FISH) was used to evaluate clonal plasma cells for 1p21 deletion as well as other myeloma-associated chromosomal abnormalities. In addition, 1p21 status was evaluated in 16 patients with of monoclonal gammopathy of undetermined significance (MGUS) and 41 patients with plasma cell leukemia (PCL). CIg-FISH detected hemizygous 1p21 deletions in 18% of the MM, 34% of PCL but none of the MGUS cases. In MM, the median percentage of clonal plasma cells harboring del(1p21) was 55% (range 20–95%). The presence of 1p21 deletions was strongly correlated with 1q21amplification (p=0.01), and marginally correlated with del(p53) (p=0.05) or t(4;14) (p= 0.06), but not with del(13q) or t(11;14). There was no association between del (1p21) and other biological factors including age, gender, Hb, albumin, C-reactive protein, beta-2 microglobulin level, isotype or bone marrow plasmacytosis. The median follow-up in this cohort was 36 months. Patients with 1p21 deletions had significantly shorter progression-free (median 14.2 vs. 25.4 months, p<0.001) and overall survivals (median 39.4 vs. 82.3 months, p=0.001) than those without such deletions. In multivariate analysis, del(1p21) was an independent risk factor for progression free (p= 0.003) and overall survivals (p=0.013) after adjusting for del(13q), del(p53), t(4;14) and 1q21 amplification. 1q21 amplification was significant risk factor in univariant analysis for either progression-free or overall survivals, but did not remain as an independent risk factor in multivariant model adjusting for other genetic risk factors. Our results indicate that del(1p21) is an independent poor prognostic factor associated with disease progression in MM. We recommend include this genetic aberration as part of a routine work-up in the risk-stratification of MM patients receiving autologous stem cell transplantation.
siRNA, a Potential Therapy for AL Amyloidosis.
Authors: Hovey Beth M. , Ward Jennifer , Seldin David C.
Published: April 2020

AL amyloidosis is a rare, sporadic disorder characterized by the accumulation of misfolded monoclonal immunoglobulin light chains (LC) as systemic amyloid deposits. Current therapies primarily include chemotherapies targeted at killing the plasma cells, often having toxic side effects for the patients. We have designed siRNAs targeted at either the variable (VL) or the constant regions (CL) of the LC to reduce amyloidogenic precursor protein expression in the producing cells. The strategy of targeting the unique VL is to develop sequence-specific siRNAs; alternatively, by targeting shared sequences in CL, siRNAs may be effective for sets of patients sharing the same LC family. Using picomolar concentrations of siRNAs, we have reduced LC protein levels in transfected cells in vitro in a dose-dependent fashion. In an in vivo tumor transplant mouse model of AL amyloidosis we have shown we can decrease the local and circulating LC levels. In this model, SP2/0 plasmacytoma cells stably expressing human amyloidogenic LCs are injected subcutaneously, and allowed to form a tumor. Once the tumor is the appropriate size, In Vivo Electroporation (IVEP) is used to deliver the siRNA oligos into the cells. By examining tumor and serum samples 48 hours post treatment, we have shown the siRNAs can significantly decrease the amount of LC in a sequence-specific manner. This model system highlights the therapeutic potential of siRNA for this disease. In conclusion, siRNA appears to be a viable potential therapy for AL amyloidosis.
Identification of DJ-1/Park-7 as a Determinant of Tnfα-Induced and Stroma-Dependent Apoptosis in Leukemia Using Mass Spectometry and Phosphopetide Analysis.
Authors: Marcondes A. Mario Q. , Xiang Li , Milless Brian P , Deeg H. Joachim
Published: April 2020

The bone marrow microenvironment provides essential signals for the fate of normal hematopoietic and for leukemic cells. Contact with marrow stroma, which is part of the microenvironment, is generally thought to convey anti-apoptotic signals to (clonal) leukemia cells. Patients with low-grade myelodysplastic syndrome (MDS) early in the disease course show high rates of apoptosis in normal and clonal marrow cells, mediated by tumor necrosis factor alpha (TNFα) and other cytokines. As MDS advances and evolves to leukemia, clonal cells tend to become apoptosis resistant. We showed previously that the leukemia-derived cell line KG1a was resistant to TNFα-mediated apoptosis, but TNFα did induce caspase-3 activation and apoptosis in KG1a cells when co-cultured with the human marrow stroma cell line HS5 (derived from healthy marrow). Apoptosis was contact dependent and required expression of TNF receptor 1 on KG1a cells. Identical results were obtained in co-cultures with primary stroma cells. Gene expression profiling of KG1a cells showed that stroma contact resulted in significant upregulation of genes involved in apoptosis, including PYCARD and p53. To further dissect the relevant signaling pathways, we used a PhosphoScan proteomic LC-MS (Liquid chromatography-mass spectrometry) method to identify proteins that were phosphorylated in response to stroma contact. In parallel to KG1a we examined the parent cell line KG1, which is sensitive to TNFα mediated apoptosis. We determined the phosphorylation sites in proteins within the leukemic cell lines using MS2 and MS3 scans. Database searches were performed with X! Tandem and Mascot and results analyzed by PeptideProphet using data from a synthetic doubly-phosphorylated peptide as control. In KG1a cells cultured without stroma support, the peptide DJ-1/Park-7 was highly phosphorylated, and expression of p53 was inhibited as indicated by decreased levels of p53 mRNA and protein. In co-culture with stroma, KG1a cells expressed higher levels of p53 protein, and levels of phosphorylated DJ-1/ Park-7 were undetectable over a time course of 30 min to 24 hours. In apoptosis-sensitive KG1 cells constitutive DJ-1/Park-7 phosphorylation (in the absence of stroma contact) was undetectable, and p53 was expressed at higher levels than in KG1a cells, consistent with the observed activation of caspase-3 and induction of apoptosis in KG1 cells. Taken together, these data suggest that phosphorylation of DJ-1/Park-7, originally identified as an oncogene product involved in cellular transformation, oxidative stress responses, and transcriptional regulation, was associated with repression of p53 and resistance to TNFα-mediated apoptosis. The relevance of DJ-1/Park-7 (and other genes identified by the PhosphoScan proteomic method) in primary MDS cells is currently being investigated at the molecular and functional levels.
Smad4 as a Therapeutic Target in Nup98-HoxA9-Induced Leukemia.
Authors: Quere Ronan , Karlsson Goran , Andradottir Silja , Rissler Marianne , Olsson Karin , Hertwig Falk , Reckzeh Kristian , Cammenga Jorg , Karlsson Stefan
Published: April 2020

Hematopoietic stem cells (HSC) are subject to complex regulation by many signal transduction pathways and transcription factors. In leukemia, these regulatory circuits are abnormal and generate dysregulated growth of HSC and their progeny. Among the prominent transcription factors that are dysregulated in leukemia are the homeobox (Hox) transcription factors or fusion-proteins derived from Hox proteins. The Nup98- HoxA9 oncogenic fusion protein is found to enforce expression of HoxA9 target genes that immortalize and block the differentiation of HSC. TGFβ inhibits Nup98-HoxA9 transformation of bone marrow cells, through direct interaction between Smad4 and the fusion protein, in a fashion which is surprisingly independent of active Smad signaling (EMBO J, 2006). Our hypothesis is that an interaction between the TGFβ and the Hox pathways is involved in leukemogenesis. To directly address this issue, we used our knockout mouse model (J.Exp Med, 2007) to determine HSC behavior in Smad4-/- cells and analyze interconnection between the TGFβ signaling pathway and the HoxA9 oncogenic activity. Using retroviral vectors to generate enforced expression of Nup98- HoxA9 in Smad4 null HSC, the transforming function was found increased approximately 4-fold in vitro. Transplantation of these transduced cells into recipient mice confirms that a block of the TGFβ pathway leads to a fast development of leukemia. At the molecular level, a 5-fold accumulation of Smad4 protein is found in wt HSC transduced by the Nup98-HoxA9, suggesting that the oncogenic potential involves a sequestration of Smad4 and a block of the TGFβ pathway. To reactivate the TGFβ pathway, one therapeutic strategy would be to induce a re-allocation of the Smad4 protein accumulated in these cells. To directly address this issue, we generated retroviral constructs that enable expression of different portions of the MH1 part of the Smad4 protein, which has been described to interact physically with the Nup98-HoxA9 oncoprotein. The retroviral transduction of one of this portions encoding a 20aa-peptide, allows release of the endogenous sequestrated- Smad4 by competition, which leads to reactivation of the TGFβ pathway and the expansion of leukemic cells is prevented in secondary recipient mice. While, administration of TGFβ remains inconceivable, because of secondary effects, the findings from these studies might consequently be used to design novel therapies for leukemia induced by abnormal Hox proteins.
Transgenic Expression of Wild Type JAK2 Suppresses Myeloproliferative Disorder Phenotypes Induced by Mutant JAK2V617F in Mice
Authors: Xing Shu , Zhao Wanming , Ho Wanting Tina , Zhao Zhizhuang Joe
Published: April 2020

JAK2V617F, a mutant form of tyrosine kinase JAK2, is found in the majority of patients with myeloproliferative disorders (MPDs). It displays increased kinase activity and causes MPD phenotypes in transgenic mice in a transgence dosage-dependent manner. Following our initial generation and characterization of JAK2V617F transgenic mice, we further generated transgenic mice expressing wild type JAK2 by using the same vav promoter employed for JAK2V617F. Three lines of JAK2 transgenic mice were generated. Real time PCR analyses revealed transgene copy numbers of 38, 2, and 1. All these mice are viable and fertile, and they displayed normal blood cell counts. This proves that the V617F mutation but not gene overexpression per se caused MPD phenotypes in JAK2V617F transgenic mice. We then crossed the JAK2 and JAK2V617F transgenic mice to generate JAK2/JAK2V617F double transgenic hybrids. Interestingly, these hybrid mice developed no or mild MPD phenotypes with only a slight increase in blood counts in contrast to the striking elevation observed in JAK2V617F transgenic mice. Expression of wild type JAK2 also blocked the constitutive activation of signal transduction components caused by JAK2V617F. Our data indicates that over-expression of wild type JAK2 suppresses the pathogenic function of mutant JAK2V617F. Therefore, JAK2V617F is not a typical dominant oncogene. Homozygous mutation or in the case of heterozygous mutation, its amplification with concurrent deletion or suppression of wild type JAK2, is required to produce MPD phenotypes. Our transgenic mouse models will serve as an invaluable tool to study the interplay of JAK2 and JAK2V617F and the mechanism by which specific MPD phenotypes develop.
Identification of the Leukemia-Specific Domains of the CALM/AF10 Fusion Gene, a Product of the Leukemia Associated T(10;11) Translocation.
Authors: Deshpande Aniruddha J , Arseni Natalia , Lin Yihui , Xu Guoliang , Hiddemann Wolfgang , Bohlander Stefan , Feuring-Buske Michaela , Buske Christian
Published: April 2020

We have demonstrated that the expression of the CALM/AF10 (C/A) fusion gene in murine BM cells results in an aggressive acute myeloid leukemia (AML). We sought to identify the domains involved in leukemogenesis mediated by the CALM/AF10 fusion gene. For this purpose we employed the CFU-S assay in which it was observed that C/A transduced BM cells generated an average of 75±23 day 12 CFU-S colonies per input 10^5 cells as compared to an average of 2 ±3 colonies with cells expressing the empty GFP vector (~ 37 fold increase; P < 0.0005) in the number of day-12 CFU-S content. Expression of the CALM gene truncated to the breakpoint of CALM/AF10 (CALMdelta3’) or the CALM/ AF10 fusion gene with a deleted octapeptide motif - leucine zipper domain (CALM/AF10 delta OM-LZ) gave 10 (±11) and 12 (±11) day12 CFU-S respectively per input 10^5 bone marrow cells showing that the AF10 portion of C/A, especially the OM-LZ region is necessary for the observed enhancement of d-12 CFU-S. BM cells transduced with a construct harboring the CALM gene fused only to the OM-LZ domain of AF10 (CALM+ OM-LZ) showed 68(±5) d-12 CFUS per input 10^5 cells (~34 fold Vs MIG; P=0.0006) comparable to the C/A fusion gene.

We observed that the C/A fusion gene fails to show leukemic transformation of BM progenitors in CFC or proliferation assays in vitro in contrast to its striking leukemogenic potential in vivo. We tested different mutants of the C/A fusion gene using these assays and observed that the expression of a mutant of the CALM/AF10 fusion gene with a C-terminal portion of CALM (amino acids 400 – 648) fused to the OM-LZ motif of AF10, showed a significant increase in the number of secondary CFCs (32 fold Vs MIG; 15.38 fold Vs C/A), with the appearance of predominantly blast-like colonies. Interestingly, this mutant could also initiate leukemias in mice (n=5) with a latency and phenotype similar to mice injected with C/A transduced BM cells.

Taken together, we demonstrate that the OM-LZ domain of AF10 is necessary for the expansion of early hematopoietic progenitors by CALM/AF10 and also sufficient for in vivo leukemic transformation. We also demonstrate that the deletion of amino acids 1 to 410 of CALM confers in vitro transformation potential to the C/A fusion gene. Our data identify the domains of C/A that are crucial for leukemogenesis and provide insights into the mechanism of transformation in t(10;11) (p13;q14) positive leukemia.
Free Protein S Levels on the Risk of First Venous Thrombosis and Recurrence. Results from a Retrospective Family Cohort Study in 1143 Relatives.
Authors: Lijfering Willem M , Mulder René , Kate Min Ki ten , Mulder Andre , van der Meer Jan
Published: April 2020

Conflicting data have been reported on the risk for venous thrombosis in subjects with protein S type III deficiency. As some studies included subjects with protein S type I deficiency, combined with an inadequate cut-off level for defining protein S type III deficiency (i.e. lower limit in healthy volunteers, generally < 65 IU/dL) might explain this discrepancy. We performed a single center retrospective study in a large cohort of families wherein probands had thrombosis and a thrombophilic defect, to define an optimal free protein S level that can identify subjects at risk for venous thrombosis. Probands were excluded as were relatives with protein S type I deficiency. Free protein S antigen levels were measured after precipitation of protein S complexed with C4b-binding protein with polyethylene glycol. 1143 relatives were analyzed. Mean age at onset of the first episode of venous thrombosis was 39 years. Relatives with venous thrombosis had lower free protein S levels than relatives without venous thrombosis (P< 0.001), which was most pronounced in the lowest quartile (Figure). Only relatives with free protein S levels below the 5th percentile (< 41 IU/dL) or below the 2.5th percentile (< 33 IU/dL) were at higher risk of first venous thrombosis compared to relatives with free protein S levels in the upper quartile (³ 91 IU/dL); annual incidence 1.20% (95% CI, 0.72–1.87), and 1.81% (95% CI, 1.01–2.99), respectively; hazard ratio, adjusted for age, sex and thrombophilic defects 6.7, (95% CI, 3.3–13.5) and 11.3 (95% CI, 5.2–22.1). Recurrence rates in these subgroups were 12.12% (95 CI, 5.23–23.88) and 12.73% (95% CI, 5.12–26.22) per year. Relatives with free protein S levels lower than the 5th or 2.5th percentile had an adjusted 2.8 (95% CI, 0.8–8.6) and 3.1 (95% CI, 0.9–10.5) fold higher risk of recurrence compared to relatives with free protein S levels in the upper quartile. We conclude that decreased free protein S levels are a risk factor for venous thrombosis. However, the free protein S level to identify subjects at risk for venous thrombosis due to protein S type III deficiency lies far below the lower limit of the laboratory reference range obtained from healthy volunteers.

Figure. Cumulative distribution curve of free protein S levels in relatives of probands with a thrombophilic defect Figure. Cumulative distribution curve of free protein S levels in relatives of probands with a thrombophilic defect

Gray dashed, dashed dotted and dotted lines display free protein S level for relatives with and without venous thrombosis at cumulative frequency of 25%, 50% and 75%, respectively.
Decreased beta2-Glycoprotein I Plasma Levels as a Risk Factor for Myocardial Infarction in Men.
Authors: De Laat Bas , de Groot Philip G. , Derksen Ronald H.W.M. , Urbanus Rolf T. , Mertens Koen , Rosendaal Frits , Doggen Carine J.M.
Published: April 2020

Background: Several factors influence the occurrence of acute myocardial infarction. One of these factors is thought to be Von Willebrand Factor which serves as adhesive surface for platelets to adhere to the vessel wall. We have recently found that beta2- glycoprotein I is able to inhibit platelet binding to von Willebrand Factor by binding to the A1 domain of Von Willebrand Factor 1. This could indicate that beta2-glycoprotein I possesses antithrombotic properties with respect to arterial thrombosis. In the present study we investigated whether differences in beta2-glycoprotein I plasma levels influence the risk of myocardial infarction.

Methods and Results: We have measured beta2-glycoprotein I and Von Willebrand Factor antigen levels in 539 men with a first myocardial infarction and in 611 control subjects who participated in the case-control Study of Myocardial Infarction Leiden (SMILE). Although we did not find a profound effect of beta2-glycoprotein I plasma levels on myocardial infarction in the overall population (odds ratio 0.93, 95% confidence interval 0.65–1.33), there appeared to be a dose-dependent protective effect of increasing beta2-glycoprotein I plasma levels on myocardial infarction in men of 60 years and older. In this age group we found an odds Ratio of 0.44 (95% confidence interval 0.25–0.77) for high beta2-glycoprotein I levels compared to low levels. Furthermore, high plasma levels of beta2-glycoprotein I remained protective for myocardial infarction despite high levels of Von Willebrand Factor. In addition, we studied a possible association between age and Von Willebrand Factor and beta2-glycoprotein I plasma levels. It appeared that both Von Willebrand Factor and beta2-glycoprotein I plasma levels increased with age, but a larger increase in Von Willebrand Factor plasma levels was observed than in beta2-glycoprotein I plasma levels (13.7 % every 10 years versus 5.7% every 10 years).

Conclusions: In this study high circulating levels of beta2-glycoprotein I appeared to be associated with a lower risk of myocardial infarction in men over 60 years. In addition we observed a larger increase in Von Willebrand Factor levels with age than beta2- glycoprotein I levels. As beta2-glycoprotein I possesses antithrombotic properties by inhibiting the activity of Von Willebrand Factor in-vitro, this might indicate that during aging the haemostatic balance slowly shifts to a more prothrombotic state 1. Future in-vivo experiments are needed to investigate the exact contribution of beta2-glycoprotein I on the pathophysiology of myocardial infarction and arterial thrombosis in general.
Efficacy of Dasatinib in Patients (pts) with Previously Untreated Chronic Myelogenous Leukemia (CML) in Early Chronic Phase (CML-CP)
Authors: Cortes Jorge , O’Brien Susan , Borthakur Gautam , Jones Dan , Ravandi Farhad , Koller Charles , Mesina Ofelia , Ferrajoli Alessandra , Shan Jianqin , Kantarjian Hagop
Published: April 2020

Background: Dasatinib (BMS-354825) is a multi-targeted kinase inhibitor of BCRABL and SRC with significant activity in pts with CML-CP resistant to or intolerant of imatinib (IM). We initiated a phase II trial to study efficacy and safety of dasatinib in pts with previously untreated CML-CP.

Aims: To investigate the efficacy and safety of dasatinib as initial therapy for patients with CML-CP.

Methods: The primary objective was to estimate the proportion of pts attaining major molecular response (MMR) at 12 months (mo). Pts with previously untreated CML-CP were eligible and received dasatinib 100 mg/day, randomized to either 50 mg-twice-daily (BID) or a 100 mg-once-daily (QD).

Results: Fifty pts have been enrolled (25 on the QD schedule, 25 BID). Median age was 45 years (yrs) (range 18–76 yrs); 75% are Sokal low risk. Median follow-up is 24 months (mo). Overall, 44/45 (98%) evaluable patients achieved complete cytogenetic response [CCyR]. The CCyR rate at 3, 6 and 12 mo compares favorably to that observed in historical controls treated with imatinib 400mg or 800 mg daily:

Percent with CCyR (No. evaluable) Mo on therapy Dasatinib Imatinib 400mg Imatinib 800mg P value 3 78 (45) 37 (49) 62 (202) 0.0003 6 93 (41) 54 (48) 82 (199) <0.0001 12 97 (35) 65 (48) 86 (197) 0.0001 18 88 (33) 68 (38) 89 (179) 0.004 24 80 (25) 70 (40) 88 (173) 0.006

MMR was achieved in 12/35 (34%) at 12 mo and 12/25 (48%) at 18 mo. One of 46 (2%) evaluable pts have achieved confirmed complete molecular response, and 1 other unconfirmed (ie, only achieved on their last assessment). Grade 3–4 non-hematologic toxicity (regardless of causality) included pruritus (13%), fatigue (6%), neuropathy (4%), and memory impairment (4%). Pleural effusion occurred in 21% evaluable pts (grade 3–4 in 2%). Grade 3–4 hematologic toxicity (transient) was thrombocytopenia in 11%, neutropenia in 21%, and anemia in 9%. Twenty-seven (54%) pts required transient treatment interruption. The actual median daily dose for all pts was 100mg. There is no significant difference in grade 3–4 toxicity by treatment schedule. Four pts came off study: 1 pts choice after 1 dose, 1 for toxicity (pleural effusion, QD schedule), and 2 lost response after multiple treatment interruptions (1 myelosuppression, 1 pleural effusion, both BID schedule). Two other pts have lost response because of non-compliance. 24 month EFS (event = loss of CHR, loss of MCyR, AP/BP, death, or off because of toxicity) is 81%.

Conclusion: Rapid CCyR occurs in most patients with previously untreated CML-CP treated with dasatinib frontline therapy with a favorable toxicity profile. Accrual to this trial continues.
Adamts-13 Gene Mutations and Transcription Analysis in Primary Antiphospholipid Syndrome.
Authors: Hancer Veysel sabri , Diz-Kucukkaya Reyhan , topal-Sarikaya Aysegul
Published: April 2020

Von Willebrand factor (vWF) is a large multimeric protein produced by endothelial cells and megakaryocytes. vWF mediates adhesion of plateletes at sites of vascular injury, and transports factor VIII. After secretion, vWF multimers are cleaved by ADAMTS-13 (A disintegrin –like and metalloprotease with thrombospondin type 1 motif 13) enzyme to smaller multimers. Decreased activity of ADAMTS-13 by autoantibodies, mutations and other etiologies causes thrombotic microangiopathy (intraluminal platelet thrombosis in the microvasculature, thrombocytopenia, and red blood cell fragmentation). Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by recurrent arterial and venous thrombosis and fetal losses associated with elevated levels of antiphospholipid antibodies. The pathology of small vessel thrombosis in APS patients has been shown to be related with thrombotic microangiopathy. Recently, Austin et al reported that ADAMTS-13 activity was reduced in patients with APS. They also showed an increase of large vWF multimers in plasma of those patients. In this study, we aimed to investigate ADAMTS-13 mutations which decrease the activity of enzyme and the quantitative gene transcrition rates in patients with primary APS and healthy controls. We evaluated 70 primary APS patients (male to female ratio was 25/45), and age and sex matched healthy controls. Exons 10+11,12,13+14 of ADAMTS-13 gene were amplified with the PCR assay for 365del, Q449stop C508Y mutations and P475S polymorphism analysis. PCR products were cleaved with restriction digestion enzymes and genotyped. C508Y mutation was screened by a DNA sequencer system. Total RNA samples were extracted from peripheral blood and cDNA samples were synthesized with reverse transcriptase. Original primers and probes were designed for the target gene ADAMTS-13 and the reference gene HPRT- 1. cDNA amounts of target and reference genes in patient and healthy control groups were determined by real-time PCR. We did not find C365del, Q449stop mutations and P475S polymorphism in the control group and the patients with APS (Table1.) We used Pfaffl method for transcription analysis and found that mRNA amounts of ADAMTS-13 was significantly decreased in APS group compared to helathy controls (Table 1). Our results may suggest that ADAMTS-13 gene is down regulated at the transcription stage in patients with APS. Transcription factors or existence of other mutation(s) at promotor region may explain this effect. This study demonstrated that ADAMTS 13- vWF related mechanisms may have important role in the development of thrombosis in APS patients.

Table 1. Data of transcription analysis and mutations which affect the activity of ADAMTS13.

C365del Q449stop P475S C508Y ADAMTS13 CP* HPRT1 CP* ADAMTS13 mRNA ratio (PAPS/ Control) * Cross Point APS n=70 0 0 0 0 33,71 30,43 0,5 Control n=100 0 0 0 0 32,23 30,05
Thrombin Generation Is Normal in Most Patients with Cirrhosis despite a Prolonged INR.
Authors: Gatt Alexander , Riddell Anne , Calvaruso Vincenza , Makris Michael , Tuddenham Edward , Burroughs Andrew
Published: April 2020

Introduction: Advanced liver disease is associated with prolongation of the prothrombin time (PT). In order to decrease the inter-laboratory variability of PT measurement, the international normalised ratio (INR) calculated as the ratio of patient’s PT to a normal (control) sample, raised to the power of the international sensitivity index (ISI) of the particular thromboplastin used was developed. However, the ISI is derived from PT results of patients on warfarin and results cannot be extrapolated to liver patients. Despite this, the INR is still commonly performed to assess bleeding risk in patients with liver disease worldwide. Furthermore the INR is only affected by factors I, II, V, VII, and X and is not influenced by other factors such as factor VIII which is usually raised in hepatic cirrhosis. Recently it has been reportd that thrombomodulin addition (to take into account the protein C pathway) normalises thrombin generation (TG)1 despite these patients having a low TG if thrombomodulin is not used.

Aim: We speculated that TG, which is a global assay of coagulation and sensitive to all coagulation factors, when triggered by a low tissue factor (TF) concentration might not correlate with the INR in patients with liver disease and that contact inhibition with corn trypsin inhibitor (CTI) might better reflect the coagulation potential in this patient group.

Results: 73 unselected patients with liver cirrhosis due to various diseases and 25 normal subjects were studied. INR and TG using the calibrated automated thrombogram (CAT) at 1pM tissue factor (TF) with CTI, 5pM without CTI and with and without Protac (a Protein C activator) were performed using platelet poor plasma (PPP). The INR range was 0.8–4.0 (mean 1.6). At 5pM TF without Protac, the patient group had a significantly lower endogenous thrombin potential (ETP) than the controls (mean ETP difference 752nM/min; P <0.0001). With Protac, no significant differences could be detected between the 2 groups. However, if the ETP without Protac was divided by the ETP with Protac x 100, the liver group showed more resistance to PC activation (mean % difference 25.4; P 0.0002). At 1pM TF, the mean ETP in the cirrhosis cohort was slightly lower than the normal group (difference between means 216nM/min; P 0.03). However, only 7 (9.6%) patients had ETP values less than the normal range (mean±2SD). No correlation was found between the ETP at 1pM and the INR. The mean FVIII:C was raised at 185.6 (78–420U/dl).

Conclusion: TG measured at low TF with CTI is normal in the majority of patients with cirrhosis. These patients are also more resistant to PC activation and have supranormal FVIII:C. Thus most patients have a normal or high thrombin potential despite an abnormal INR. These findings have important implications as in the absence of bleeding, “prophylactic” plasma and clotting factors are unnecessary.
Clinical and Laboratory Risk Factors for Venous Thromboembolism in Patients with High-Grade Gliomas: Results of a Prospective Study of 107 Newly-Diagnosed Patients.
Authors: Streiff Michael B. , Ye Xiaobu , Desideri Serena , Kickler Thomas , Jani Jayesh , Grossman Stuart
Published: April 2020

Introduction: Patients with malignant gliomas are at high risk for venous thromboembolism (VTE). The reason for this association is unclear. We sought to identify clinical and laboratory risk factors for VTE in adult patients with high-grade gliomas.

Methods: The NABTT CNS Consortium prospectively enrolled patients with newly-diagnosed grade 3 or 4 malignant glioma prior to anti-neoplastic therapy. Patients with a previous history of VTE, anti-neoplastic therapy or on chronic anticoagulation were excluded. At enrollment, we collected demographic and clinical information (age, gender, ethnicity, tumor histopathology and grade, Karnofsky Performance Status (KPS), and ABO blood group) and blood samples for measurement of factor VIII activity (FVIII), fibrinogen, and quantitative D dimer using standard laboratory assays. Endogenous thrombin potential (ETP) was measured using Innovin® and a synthetic chromagenic thrombin substrate on a BCS® coagulation analyzer (Dade Behring Inc. Newark, DE). Fisher’s exact test and the Student’s-T test were used for individual comparison of categorical data and continuous data, respectively. Cox regression modeling was used to examine the association of factors with VTE. The probability of thrombosis-free survival was estimated using the product-limit method of Kaplan and Meier.

Results: One hundred seven patients (49% male) with a median age 57 years (range 28–85) were enrolled between 6/05 and 4/08. Ninety patients (84%) had glioblastoma multiforme. Median KPS at enrollment was 90. After a median follow up of 324 days, twenty two patients (21%) have suffered VTE and 45 patients (42%) have died. Median time to VTE was 67 days post-operation (95% Confidence Interval 33–128). No fatal VTE have occurred. VTE was associated with a lower KPS (p=0.008), higher baseline FVIII (178% versus 151%, p=0.04) and greater ETP (473nmol/L versus 438nmol/L, p=0.04) but not with ABO blood group. Patients suffering VTE were more likely to die than patients without VTE (68% versus 35%, p<0.005).

Conclusions: In a prospective cohort study of newly-diagnosed patients with high-grade gliomas, we have identified a substantial incidence of VTE. Patients with VTE had a lower initial KPS, higher factor VIII activity, greater ETP and were more likely to die than patients without VTE. With additional follow up, we hope to identify additional clinical and laboratory risk factors for VTE. These data should lead to greater insight into the pathogenesis of VTE in patients with high-grade gliomas and facilitate identification of patients in whom primary long-term VTE prophylaxis may be beneficial.
Lactadherin and Clearance of Platelet-Derived Microvesicles.
Authors: Dasgupta Swapan Kumar , Abdel-Monem Hanan , Niravath Polly , Le Anhquyen , Bellera Ricardo , Langlois Kimberly , Nagata Shigekazu , Rumbaut Rolando , Thiagarajan Perumal
Published: April 2020

Objective— In platelets, as in most mammalian cells, the anionic phospholipids such as phosphatidylserine are present only in the inner leaflet of the membrane bilayer. During platelet activation, phosphatidylserine moves from the inner to the outer leaflet of the membrane bilayer. The transbilayer movement of phosphatidylserine is responsible for platelet procoagulant activity as the exposed phosphatidylserine provides high affinity binding sites for the assembly of the prothrombinase and tenase complex. Externalization of anionic phospholipids in platelet is accompanied by the release of phosphatidylserine-rich microvesicles. These microvesicles account for the procoagulant activity of plasma by providing an efficient catalytic surface.

Lactadherin, also known milk fat globule-EGF 8, is a 45 kDa glycoprotein secreted by macrophages. Lactadherin contains EGF-like domains at the amino terminus and two C-domains at the carboxy terminus that share homology to the phosphatidylserinebinding domains of blood coagulation factors V and VIII. Lactadherin binds to apoptotic lymphocytes and phosphatidylserine-expressing red blood cells via the C-domains and anchors them to macrophage integrins via its RGD sequence in the EGF domain. We have examined the role of lactadherin in clearance of phosphatidylserine-rich platelet-derived microvesicles.

Methods and Results—Platelet-derived microvesicles were labeled with the fluorophore BODIPY-maleimide and incubated with THP-1 cell derived macrophages. The extent of phagocytosis was quantified by measuring the intracellular fluorescence by flow cytometry. Lactadherin promoted phagocytosis in a concentration-dependent manner with a half-maximal effect at ~ 5 ng/ml. A monoclonal antibody to lactadherin and a carboxy terminal fragment of lactadherin inhibited lactadherin-dependent phagocytosis. Lactadherin-deficient mice had increased number of microvesicles in their plasma and generated more thrombin compared to their wild type littermates. In addition, splenic macrophages from lactadherin-deficient mice showed decreased capacity to phagocytose platelet microvesicles. Finally, in a in vivo model of light/dye-induced endothelial injury/ thrombosis model, lactadherin-deficient mice, showed enhanced thrombus formation (5.93 ± 0.43 min) compared to their wild-type littermates ( 9.80 ± 1.14 min; P=0.01, n=9 in each group) in the cremastric venules.

Conclusion— Our studies show that lactadherin mediates the clearance of PS expressing platelet-derived microvesicles from the circulation and that a defective clearance can induce a hypercoagulable state.
Hepcidin Suppression Relative to Iron Status in Patients with Chronic Hepatitis C.
Authors: Girelli Domenico , Pasino Michela , Goodnough Julia , Nemeth Elizabeta , Castagna Annalisa , Busti Fabiana , Campostrini Natascia , Martinelli Nicola , Guido Maria , Vantini Italo , Corrocher Roberto , Ganz Tomas , Fattovich Giovanna
Published: April 2020

Patients with chronic hepatitis C (CHC) often have increased liver iron deposits, a condition associated with reduced sustained response to antiviral therapy, more rapid progression to cirrhosis, and development of hepatocellular carcinoma. The hepatic hormone hepcidin is a major regulator of iron metabolism that inhibits iron absorption and recycling from erythrophagocytosis. Hepcidin decrease is a possible pathophysiological mechanism of iron overload in CHC, but studies in humans have been hampered so far by the lack of reliable quantitative assays for the 25-amino acid bioactive peptide in serum (s-hepcidin). Using a recently validated immunoassay (Ganz T, Blood 2008, epub Aug 8), we measured s-hepcidin levels in 82 CHC naïve patients and 57 sex-matched healthy controls with rigorous definition of normal iron status. All CHC patients underwent liver biopsy with histological iron score (according to Deugnier YM, Gastroenterology 1992). S-hepcidin was much lower in CHC than in controls (geometric means with 95% CIs: 33.7, 21.5–52.9 versus 90.9, 76.1–108.4 ng/ml, respectively; P<0.001), while both serum ferritin and transferrin saturation were significantly higher in CHC than in controls, as expected. S-hepcidin strongly correlated with serum ferritin in both controls (r=0.741; P<0.001) and CHC patients (r = 0.718; P<0.001). In CHC patients, s-hepcidin also correlated with histological total iron score (r = 0.46; P<0.001), but not with serum interleukin-6 (r = −0.042; P = n.s.). After stratification for serum ferritin quartiles, s-hepcidin levels increased significantly across quartiles in both controls and CHC patients (chi for trend, P<0.001). However, in the latter group s-hepcidin levels were significantly lower than in controls for each corresponding quartile (ANOVA, P<0.001). In the lowest ferritin quartile, s-hepcidin was significantly inversely correlated with viral loading (e.g. serum HCV-RNA IU/ml; r = −0.526; P=0.036), while this association gradually disappeared with increasing ferritin quartiles. These results, together with very recent studies in animal and cellular models (Nishina S, Gastroenterology 2008; Miura K, Hepatology 2008), indicate that though hepcidin regulation by iron stores is maintained in CHC, the suppression of this hormone by HCV is likely an important factor in liver iron accumulation in this condition.
Epigenetic Analysis of Beta-Globin Gene Cluster during Hematopoiesis.
Authors: Ekwattanakit Supachai , Riolueang Suchada , Viprakasit Vip
Published: April 2020

Hemoglobin (Hb) switching is described as temporal, tissue- and stage-specific patterns of globin gene expression; from embryonic to fetal and adult Hb in parallel to developmental stages of erythropoiesis. DNA methylation, one of the epigenetic mechanisms, was associated with inactivated chromatin domain and repressive transcription. To study the role of the DNA methylation on the beta (β)-globin genes, we analyzed CpG dinucleotides in 87 kb regions around β-globin gene cluster, including 5’upstream locus control regions (LCR; DNAse I Hypersensitive site (HS) 1–5), 3’HS1, the promoter regions of the G-and A-gamma (Gγ and Aγ), and β-globin genes, in several representative cells. These cells were

primary adult erythroid cells culture (three different stages: early, intermediate, and late), fetal cord blood DNA, and neutrophil cell line (non-erythroid).

Using bisulphite modification, followed by nested PCR and in vitro translation, the cleavage products were analysed by MALDI-TOF Mass Spectrometry to quantify the DNA methylation level. The results were consistent with bisulphite sequencing. We found that the promoters of Gγ and Aγ-globin genes were significantly hypomethylated in fetal cells (44% and 47% global methylation), when γ-globin genes were fully expressed, while they were heavily methylated in non-erythroid (86% and 95%). There was also a decreasing trend of the DNA methylation level at Gγ and Aγ-globin genes during adult erythroid differentiation from 80% and 82%, in early stage, to 67% and 66% in late stage (p=0.12 and 0.04). At β-globin promoter, the global methylation level changed from 90% in non-erythroid to 81%, 42%, and 26% in fetal, early and late adult erythroid cells, respectively. Moreover, we found the significant changes at 5’HS4, 3, and 1 as all erythroid cells were hypomethylated compare to non-erythroid. While at the insulators, 5’HS5 and 3’HS1, all tested CpG dinucleotides were heavily methylated in all cells. This is the first report that demonstrates the differences in DNA methylation at β-globin LCR between erythroid and non-erythroid cells. These epigenetic marks were associated with globin genes expression and might be useful to predict clinical severity in patients with β-thalassemia intermedia.
Regulation of Chromatin Looping and Transcription by PRMT1 Mediated H4R3 Methylation.
Authors: Li Xingguo , Hu Xin , Zhou Zhuo , Qiu Yi , Felsenfeld Gary , Bungert Jorg , Huang Suming
Published: April 2020

Communication between distal enhancers and proximal promoters is critical in controlling proper transcription of genes. However, the functional link between certain histone modifications and the formation of long-range chromatin interactions involved in transcriptional activation remains unknown. In the globin locus, the b-globin genes are regulated by highly organized chromatin structure that juxtaposes the locus control region (LCR) located far upstream of the genes with the proximal b-major globin promoter (bmajpromoter). We report here that the localized asymmetric dimethylation of Arg3 at histone H4 tails (dimethyl H4R3) catalyzed by the methyltransferase PRMT1 is essential for establishing the long-range chromatin interactions between the LCR and the bmaj-promoter and strongly correlates with the activation of adult b-globin gene transcription. In addition, dimethyl H4R3 potentiates the recruitment of histone acetyltransferases (HATs), CBP and PCAF, and is required for the establishment of subsequent histone acetylation at the globin locus. Suppression of PRMT1 activity disrupts the recruitment of transcription complexes, TBP and RNA polymerase II (RNA Pol II), at the active b-globin promoter, but not at the LCR. Taken together, our data implicate PRMT1-mediated dimethylation of H4R3 in the regulation of long-range enhancer/promoter communications, which are required for the efficient recruitment of transcription complexes to the active gene promoter.