Calr Is up-Regulated in Patients with Essential Thrombocythemia Independently from Calr and JAK2 Mutations

Abstract Somatic mutations in exon 9 of calreticulin (CALR) gene were recently discovered in patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) lacking JAK2 and MPL mutations, and absent in patient with polycythemia vera (PV). Among patients with ET or PMF with un-mutated JAK2 or MPL, CALR mutations were detected in 67% of those with ET and 88% of those with primary PMF. Several types of insertions or deletions were identified and all resulted in a frameshift in exon 9 generating a novel C-terminal peptide in the mutant CALR protein. Over expression of the most frequent CALR deletion caused cytokine-independent growth in vitro owing to the activation of signal transducer and activator of transcription 5 (STAT5) by means of an unknown mechanism. Patients with myeloproliferative neoplasms carrying CALR mutations present with higher platelet counts and lower haemoglobin levels than patients with mutated JAK2. Studies suggest these patients have a lower risk of thrombosis and longer overall survival than patients with mutated JAK2. We analysed by Real Time PCR, CALR expression in peripheral blood (PB) of 38 patients affected by ET, 17 JAK2 mutated (45%), 4 CALR (10.5%) mutated, 1 MPL mutated (3%) and 14 with no apparent molecular abnormalities. These were compared with a cohort of healthy volunteers. We found a significant over expression of CALR (median 5.15; range 1.13-270.08) comparing with controls (median 0.38, range 0.18-1). CALR mRNA expression is independent from the CALR mutational status. No significant difference was found comparing CALR expression in CALR mutated (median 4.9, range 1.51-37.14) and CALR/JAK2 un-mutated patients (4.68, range 1.51-28.71). CALR up-regulation is not mutually exclusive with JAK2 mutations; no difference was seen in CALR mRNA between JAK2 mutated (median 5.09, range 1.13-270) and wild type ET patients (median 5.08, range 1.51-37). There was no significant difference when we correlated CALR expression with PLT counts, spleen size or type of cytoreductive therapy. A larger cohort of patients is required to confirm these preliminary findings. Disclosures No relevant conflicts of interest to declare.

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A Case Report on Coexisting JAK2 V617F and Calr exon 9 Mutation in Essential Thrombocythemia

Blood ◽

10.1182/blood.v126.23.5215.5215 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5215-5215

Author(s):

Munazza Rashid ◽

Rifat Zubair Ahmed ◽

Shariq Ahmed ◽

Muhammad Nadeem ◽

Nuzhat Ahmed ◽

...

Keyword(s):

Essential Thrombocythemia ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Diagnostic Algorithm ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Common Mutation ◽

Hypochondriac Region ◽

Exon 9 ◽

Calr Mutations

Abstract Myeloproliferative Neoplasms (MPNs) are a heterogeneous group of clonal disorders derived from multipotent hematopoietic myeloid progenitors. Classic "BCR-ABL1-negative" MPNs is an operational sub-category of MPNs that includes polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). These three disorders are characterized by stem cell-derived clonal myeloproliferation. The most common mutation in the MPNs PV, ET and PMF is JAK2 V617F. JAK2 V617F can be detected in about 95% of patients with PV while remaining 5% of PV patients carry a somatic mutation of JAK2 exon 12. Approximately one third of patients with ET or PMF do not carryany mutation in JAK2 or MPL. In December 2013 mutations were described in calreticulin (CALR) gene in 67-71% and 56-88% of JAK2 V617F and MPL negative patients with ET and PMF, respectively. Since this discovery, CALR mutations have not only been recommended to be included in the diagnostic algorithm for MPNs, but also CALR exon 9 mutations have been recognised to have clinical utility as mutated patients have a better outcome than JAK2 V617F positive patients.CALR mutations have also been reported to be mutually exclusive with JAK2 V617F or MPL mutations. According to our knowledge so farthere have been only six reports published,which described patients harbouring concurrent JAK2 V617F and CALR exon 9 mutations; seven ET, three PMF, one PV and one MPN-U. In the present study we are reporting ET patient with coexisting JAK2 V617F and CALR exon 9 mutations from our center. In July 2011, 55-years-old female patient was referred to our hospital with a history of gradual elevation of platelet counts accompanied with pain in right hypochondriac region and feet. Bone Marrow aspirate consisted of 'Stag-horn' appearance Megakarocytes. Multiple platelets aggregates and islands were seen throughout the aspirate smear. ARMS-PCR for JAK2 V617F mutation was positive whereas bidirectional Sanger sequencing for CALR exon 9 exhibited c.1214_1225del12 (p.E405_D408del) mutation pattern. Disclosures No relevant conflicts of interest to declare.

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Calreticulin Mutants Induce an Early Clonal Dominance and a Megakaryocytic Phenotype through the Activation of MPL/JAK2 Pathway in Human Primary Cells

Blood ◽

10.1182/blood.v128.22.1959.1959 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1959-1959

Author(s):

Mira EL Khoury ◽

Gaelle Vertenoeil ◽

Caroline Marty ◽

Christophe Marzac ◽

Matthieu Mosca ◽

...

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Primary Myelofibrosis ◽

Lymphoid Cells ◽

Reading Frame ◽

Spontaneous Growth ◽

Constitutive Activation ◽

Calr Mutations ◽

Cell Fractions

Abstract Myeloproliferative neoplasms (MPNs) are clonal malignant disorders characterized by the increased production of mature myeloid cells in blood. The classical MPNs include Polycythemia Vera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF). Those pathologies are due to the acquisition of gain-of-function mutations leading to the constitutive activation of the cytokine receptor / JAK2 signaling pathway: JAK2V617F in 70% of cases and mutations in the thrombopoietin receptor (MPL) gene in 5% of cases. More recently, around fifty different mutations in the calreticulin (CALR) gene have been described in 30% of ET and PMF with two more frequent mutations called del52 (type 1) and ins5 (type 2). All the CALR mutations induce a frameshiflt to an alternative reading frame in the exon 9 leading to a new C-term tail of the protein with hydrophobic features, and the loss of the KDEL sequence, which is involved in its endoplasmic reticulum retention. The goal of this work was to understand the role of CALR mutants (del52, del46, del34, ins5, del19, del13) in human hematopoiesis. By studying the variant allele frequency (VAF) in 20 patients, we have shown that the CALR mutations are present in all blood mature cells not only in granulocytes and monocytes (CD14+) with a VAF >30% but also in B cells (CD19+), NK cells (CD56+) and in some cases in T cells (CD3+). Moreover, we have observed that CALR mutations are present in all hematopoietic progenitors including CD34+CD38-CD90+ (HSC), CD34+CD38-CD90- (immature progenitors) and CD34+CD38+ (committed progenitors) cell fractions after investigating the clonal architecture of the progenitors. CALR mutation was detectable in more than 40% of progenitor cells except in 2 patients (15 patients studied) and with, in some cases, no detectable wild type CALR progenitors. Homozygous CALR mutations were rare except in one case associated with disease progression. Whatever the VAF, there was no significant differences among the different progenitor types and granulocytes. Finally, we observed that all the associated mutations studied (TET2, PHF6, SYNE1, SCARA5, PIK3CD, SETD1B) in 6 patients postdated CALR mutations. We could also show in 15 patients samples that CALR mutants give a specific megakaryocytic progenitor (CFU-MK) spontaneous growth mediated both by MPL and JAK2 activation using specific inhibitors and short hairpin RNAs. The CFU-MK spontaneous growth correlated with a constitutive activation of JAK2/STAT3/5 pathway in megakaryocytes derived from in vitro cultures of CD34+ progenitors. In aggregate, these results show that all CALR mutants studied are present in all human hematopoietic cells including myeloid and lymphoid cells, give an early clonal advantage at the level of the HSC compartment and a specific increased growth of the megakaryocytic lineage via MPL/JAK2 activation. Disclosures No relevant conflicts of interest to declare.

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A Novel Pathogenic CALR Exon 9 Mutation in a Patient with Essential Thrombocythemia

Laboratory Medicine ◽

10.1093/labmed/lmz064 ◽

2019 ◽

Vol 51 (3) ◽

pp. 306-309

Author(s):

Jee-Soo Lee ◽

Ho Young Kim ◽

Miyoung Kim ◽

Young Kyung Lee

Keyword(s):

Essential Thrombocythemia ◽

Frameshift Mutation ◽

Myeloproliferative Neoplasms ◽

Case Reports ◽

Individual Case ◽

First Case ◽

Exon 9 ◽

Calr Mutations

Abstract The clinical phenotypes and prognoses of CALR-mutant myeloproliferative neoplasms depend on the mutation type. The 2 most common mutations, type 1 (52-bp deletion) and type 2 (5-bp insertion), account for 85% of CALR-mutated neoplasms. The former confers a myelofibrotic phenotype, and the latter is associated with a low risk of thrombosis and an indolent clinical course. Individual case reports for patients with novel pathogenic CALR mutations are rare. Herein, we present the first case in the literature, to our knowledge, of a 63-year old ethnic Korean man with essential thrombocythemia who was diagnosed with a novel +1-bp frameshift mutation in CALR, which was predicted to exhibit a type 2–like phenotype.

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Association Between EZH2 and Other Acquired Mutations In Myelofibrosis and Myelodysplastic/Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v116.21.625.625 ◽

2010 ◽

Vol 116 (21) ◽

pp. 625-625

Author(s):

Thomas Ernst ◽

Joannah Score ◽

Claire E Hidalgo-Curtis ◽

Amy V Jones ◽

Andreas Hochhaus ◽

...

Keyword(s):

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Uniparental Disomy ◽

Myeloproliferative Neoplasm ◽

Jak2 V617f ◽

Primary Myelofibrosis ◽

Mutational Status ◽

Significant Difference ◽

Chromosome 7Q ◽

Secondary Myelofibrosis

Abstract Abstract 625 We recently identified EZH2 as the major target of chromosome 7q acquired uniparental disomy (aUPD) in myeloproliferative neoplasm (MPN) and myelodysplastic syndromes (MDS). To determine the prevalence of EZH2 mutations we screened all coding exons for mutations in total of 624 cases with myeloid disorders (MPN, n=157; MDS, n=154; MDS/MPN, n=219; AML, n=54, CML in transformation, n=40) and found 49 monoallelic or biallelic EZH2 mutations in 42 individuals, most commonly MDS/MPN (27/219; 12%), primary or secondary myelofibrosis (4/30; 13%) and MDS (9/154; 6%). To determine if EZH2 mutations might co-operate with other known abnormalities or whether they might be mutually exclusive, we tested the mutational status of TET2, ASXL1, CBL, RUNX1, CEBPA, FLT3, NPM1, and WT1 in 187 of the 219 MDS/MPN cases that were screened for EZH2. We also tested an additional cohort of 52 primary myelofibrosis cases for both EZH2 and JAK2 V617F mutations. Of the 187 MDS/MPN cases (CMML, n=97; atypical CML, n=68; MDS/MPN-U, n=22), mutations were seen most frequently in TET2 (67/187; 36%), followed by ASXL1 (38/187, 20%; not including cases with the controversial c.1934dupG variant), RUNX1 (27/187; 14%), EZH2 (25/187; 13%), CBL (22/175; 13%), FLT3 (8/187; 4%), CEBPA (7/187; 4%), NPM1 (6/187; 3%) and WT1 (2/187; 1%). Sixty six (35%) cases tested negative for mutations in all 9 genes. Of the 25 cases with EZH2 mutations, 22 (88%) had mutations in at least one other gene, most frequently TET2 (n=11) and ASXL1 (n=10). EZH2 mutations were also seen in combination with mutations in CBL (n=5), CEBPA (n=4), RUNX1 (n=3) and FLT3 (n=2), however there was no significant difference in the frequency of other mutations on comparison of EZH2 mutated and EZH2 unmutated cases. When the analysis was restricted to the 10 cases with homozygous EZH2 mutations, a similar heterogeneity was observed with mutations in CBL, RUNX1, CEPBA and TET2 only (n=1 for each gene), ASXL1 only (n=2), TET2+ASXL1 (n=1), TET2+ASXL1+RUNX1 (n=1) or no other mutation (n=2). Analysis of CFU-GM from one case that tested positive for both EZH2 and TET2 mutations revealed a complex pattern with an EZH2 mutation clearly preceding the sequential acquisition of two TET2 mutations. Of the 82 primary and secondary myelofibrosis cases, 9 (11%) tested positive for an EZH2 mutation. Of these, 5 were positive for JAK2 V617F and 4 were negative. In 2 cases both EZH2 and JAK2 V617F were homozygous indicating that the predominant clone must harbor both mutations. Overall, these data indicate a complex interaction between different abnormalities with little indication of co-operativity or functional redundancy. Whilst these observations will need to be refined by detailed analysis of single clones, they do suggest that the development of both myelofibrosis and MDS/MPN requires functional alterations in multiple pathways. Disclosures: No relevant conflicts of interest to declare.

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Crispr/Cas9 Engineered 61bp Deletion in the Calr Gene of Mice Leads to Development of Thrombocytosis

Blood ◽

10.1182/blood.v128.22.4274.4274 ◽

2016 ◽

Vol 128 (22) ◽

pp. 4274-4274 ◽

Cited By ~ 5

Author(s):

Thomas Balligand ◽

Younes Achouri ◽

Ilyas Chachoua ◽

Christian Pecquet ◽

Jean-Philippe Defour ◽

...

Keyword(s):

Bone Marrow ◽

Board Of Directors ◽

Advisory Committees ◽

Mutational Status ◽

Thrombopoietin Receptor ◽

Exon 9 ◽

Calr Mutations

Abstract In a subset of patients suffering from myeloproliferative neoplasms (MPNs), calreticulin (CALR) exon 9 frameshift mutations are known to be responsible for the development of either essential thrombocythemia (ET) or primary myelofibrosis (PMF) (1, 2). The most prevalent mutations are a 52-bp deletion (del52, type-1 mutation) and a 5-bp TTGTC insertion (ins5, type-2 mutation). In these patients, the mutational status is almost always heterozygous. Our group and collaborators have recently shown that the pathogenic mutant CALR proteins require interaction with and activation of the thrombopoietin receptor (TpoR) for activation of the JAK-STAT pathway (3, 4). Until now, no knock-in mouse model of these diseases has been published. In this abstract, we show how we succeeded in creating such a model. We had shown that the murine CALR mutant proteins behave just like their human counterparts (5). Specifically, the del52, ins5 and del61 (61bp deletion, type-1) Calr mutations were able to transform Ba/F3 cells (murine pro-B lymphocytic cells normally dependent on IL-3 for growth) expressing the thrombopoietin receptor (TpoR) and render them cytokine-independent. Importantly, we also mutated the Ba/F3 genome using the widely adopted CRISPR/Cas9 system in order to create a 61-bp deletion of the exon 9 of Calr. This too successfully transformed the Ba/F3 cells, showing that endogenous levels of expression of a mutant CALR protein are sufficient to induce phenotype in vitro. Now, using the same approach, we injected C57BL/6J mouse zygotes with the same CRISPR/Cas9 constructs to create the same 61-bp deletion in the murine Calr gene. Out of 46 pups born from the procedure, one male pup was heterozygous for the 61-bp deletion. By in vitro fertilization, we subsequently obtained heterozygous Calr del61/WT pups. After inter-breeding the mice, we analyzed the blood of 12 Calr del61/WT males and 12 Calr WT/WT males (littermates) at three different timepoints (15, 18 and 22 weeks old) and found that the Calr del61/WT mice showed significantly higher levels of circulating platelets. Conversely, red blood and white blood cell numbers were the same between both groups at all time points. We further show that expression of a mutant CALR protein, in a heterozygous state, is sufficient to induce abnormal proliferation of megakaryocytes and develop an ET phenotype in vivo in mice. Follow-up in dynamics of the phenotype and bone marrow and spleen pathology (examination of myeloproliferation and fibrosis) allow comparison with the retroviral murine models of CALR-mutant MPNs and with the known features of the human disease. The only limitation of our model is the fact that the Calr del61 mutation is parentally acquired and widespread throughout the organism. With this new model, we aim to test the efficiency of various drugs to prevent or cure the MPN phenotype, such as ruxolitinib, a JAK2 type-1 inhibitor that is already used in clinics in patients suffering from CALR-mutated MPNs. We also now have a means to generate a high number of Calr del61/WT bone marrow cells to extensively study the oncogenic properties of the Calr mutations at different stages of the hematopoeisis. It will also be of great interest to study, if generated, a homozygous mutational status of Calr del61 in vivo. Thus, our system will shed light on the importance of the negatively charged tail of CALR and on the effects of the novel positively charged tail on myeloproliferation. References 1. Klampfl T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD, et al. N Engl J Med. 2013 Dec 10;369(25):2379-90. 2. Nangalia J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC, et al. N Engl J Med. 2013 Dec 10;369(25):2391-405. 3. Chachoua I, Pecquet C, El-Khoury M, Nivarthi H, Albu RI, Marty C, et al. Blood. 2015 Dec 14;10.1182/blood-2015-11-681932. 4. Marty C, Pecquet C, Nivarthi H, Elkhoury M, Chachoua I, Tulliez M, et al. Blood. 2015 Nov 25;10.1182/blood-2015-11-679571. 5. Balligand T, Achouri Y, Pecquet C, Chachoua I, Nivarthi H, Marty C, et al. Leukemia. 2016 Feb 29;10.1038/leu.2016.47. Disclosures Constantinescu: Teva: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Personal Genetics: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

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Metalloproteases Could Be Involved in Erythroid Differenciation in MPN

Blood ◽

10.1182/blood.v118.21.5161.5161 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5161-5161

Author(s):

Miguel Gallardo ◽

Santiago Barrio ◽

Ana Jimenez ◽

Oscar Toldos ◽

Rosa-Maria Garcia-Martin ◽

...

Keyword(s):

Mononuclear Cells ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Hypothesis Test ◽

Statistical Significance ◽

Statistical Hypothesis ◽

Healthy Controls ◽

Bone Marrow Biopsies ◽

Over Expression

Abstract Abstract 5161 Background Several hypotheses have been developed to explain how a unique mutation is related to three different phenotypes in Philadephia negative myeloproliferative neoplasms (MPN). Metalloproteases are a group of proteins involved in matrix remodelling, migration and differentiation processes. Although their role has never been studied in MPN, our group has found a differential expression of Matrix metalloproteinase-14 (MMP14) and CD44 between PV and ET JAK2V617F patients (E. Albizua et al, Ann Hematol. 2011Feb). Aims The purpose of the present study is to validate MMP14 and CD44 differential gene expression profile between PV and ET using a proteomic-based approach and an in vitro model, with the aim of confirming MMP14 and CD44 as MPN biomarkers as well as novel targeted therapy. Methods Seventy MPN patients diagnosed by WHO criteria were included in the study: 24 Polycythemia Vera (PV) 24 Essential Thrombocythemia (ET) JAK2V617F, 11 ET JAK2 wild type (JAK2WT) and 11 Primary Myelofibrosis (PMF). In addition 24 healthy donors were used as controls. Peripheral blood was analyzed by flow cytometry (FCM) using anti-CD44 and anti-CD45 antibodies in 10 PV and 10 ET. Fifty-five bone marrow biopsies (11 PV, 11 ET JAK2V617F, 11 ET JAK2WT, 11 PMF and 11 healthy controls) were processed to perform immunohistochemistry (IHC) with anti-MMP14 and anti-CD44 antibodies (R&D). Finally an in vitro MPN model was performed. Mononuclear cells from 10 MPN (5 PV and 5 ET) and 5 healthy controls were extracted and seeded in Methocult with IL-3, SCF and EPO. MMP14 was inhibited by Marimastat at 50μM, 25μM and 10μM; and CD44, with anti-CD44 antibody at 10mg/ml, 1mg/ml and 0.1mg/ml. Results were analyzed by BFU-E count, viability study by trypan blue and FCM employing anti-CD45, anti-CD41, anti-CD34, anti-CD71, anti-CD44, and Annexin antibodies. BFU-E DNA was extracted to analyze JAK2V617F allele burden by RT-PCR. Intracellular proteins, including phospho-proteins p38, P-p38, MEK, P-MEK, STAT1, P-STAT1, AKT and P-AKT were studied by cytometric bead array multiplexed bead-based immunoassay (CBAs) technique. The Mann-Whitney non-parametrical statistical hypothesis test was used to assess the statistical significance of our results. Results First of all, FCM of the granulocytes showed a slight over-expression of CD44 in PV population versus ET. Secondly, IHC also showed MMP14 over-expression in megakaryocites of PV (100% positive patients, 60% positive megakaryocites), and ET (100% positive patients 73% positive megakaryocites) compared to controls (36% positive subjects, 2% positive megakaryocites). Additionally 45% ET JAK2WT were MMP14 positive (20% positive megakaryocites). Significant differences in inhibition of BFU-E growth and cell proliferation were found comparing cultures either under anti-MMP14, Marimastat (50 μM and 25 μM) or under anti-CD44 (10 mg/ml and 1 mg/ml-) to cultures under no inhibitory treatment (P=0.05 and P=0.037 respectively). FCM of BFU-E cultures pointed to a significant decrease of CD71 (erythroid population) with any of the inhibitors. Over-expression of CD44 in PV compared to ET was observed and confirmed in BFU-E by FCM. All phospho-proteins studied halved after anti-CD44 (1 mg/ml) and Marimastat treatments (25 μM). Conclusions Our results suggest that MMP14 and CD44 could play a role in erythroid differentiation by JAK-STAT, MAPK and AKT pathways. Differences between ET and PV may be caused by both molecules, which may contribute to phenotypic divergence. Additionally, MMP14 IHC could become a useful diagnostic tool in, at least, 45% of ET JAK2WT. Our results suggest that MMP14 and CD44 could become future therapeutic targets and could help to MPN diagnosis. Disclosures: No relevant conflicts of interest to declare.

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Unraveling the Connections between Calreticulin and Myeloproliferative Neoplasms via Calcium Signalling

10.1101/2021.08.05.455248 ◽

2021 ◽

Author(s):

Amit Jaiswal ◽

Zhiguo Wang ◽

Xudong Zhu ◽

Zhenyu Ju

Keyword(s):

Essential Thrombocythemia ◽

Calcium Ions ◽

Calcium Binding ◽

Structural Integrity ◽

Myeloproliferative Neoplasms ◽

Calcium Signalling ◽

Dynamics Simulation ◽

Structural Variations ◽

Exon 9

Mutations in the form of insertions and deletions (INDEL) in the calreticulin gene lead to essential thrombocythemia which is characterized by the formation of thrombosis. However, the connection between calreticulin INDEL and essential thrombocythemia remains largely elusive. Through combined molecular dynamics simulation and in-vitro studies on the wild type and mutated isoforms of calreticulin, the mechanism underlying the calreticulin INDEL induced essential thrombocythemia was investigated at the molecular level. Our results demonstrate that mutations in exon-9 could lead to significant conformational variations of calreticulin structure and thereby reducing its interaction with calcium ions due to decreased electrostatic contributions. The consequence of mutations on calreticulins structural integrity was revealed by identifying the key residues and their roles in calcium binding. Furthermore, mutations implemented by CRISPR-Cas9 in exon-9 showed diminished calcium signaling in HEK-293T cells, which agree well with our in-silico findings. The current study might help in understanding of the interactions between calreticulin exon-9 INDEL and calcium ions mediated by the structural variations of calreticulin. The results provide useful information for designing novel therapeutic approaches targeting essential thrombocythemia.

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Inhibition of Plasmin Generation in Plasma By Heparin, Low Molecular Weight Heparin and a Covalent Antithrombin-Heparin Complex (ATH)

Blood ◽

10.1182/blood.v124.21.4217.4217 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4217-4217

Author(s):

Gabriela Chang ◽

Helen M. Atkinson ◽

Leslie R. Berry ◽

Anthony K.C. Chan

Keyword(s):

Molecular Weight ◽

Plasminogen Activator ◽

Low Molecular Weight Heparin ◽

Conflicts Of Interest ◽

Area Under The Curve ◽

Low Molecular Weight ◽

Plasminogen Activator Inhibitor 1 ◽

Significant Difference

Abstract Introduction: Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) are widely used anticoagulants for thrombosis treatment. However, these anticoagulants have limitations such as increased bleeding, variable dose response, required frequent monitoring, and, in the case of LMWH, inability to inhibit thrombin. This has led to the development of a covalent complex of antithrombin and heparin (ATH), which has been shown to overcome many of these shortcomings. ATH has faster rates of inhibition of many coagulation factors, is able to inhibit clot-bound thrombin, and is a more effective inhibitor of both venous and arterial thrombosis in animal models. Moreover, in a rabbit thrombosis model, ATH has been shown to decrease clot mass and fibrin accretion, while the contrary was observed for UFH. From these observations, it was suggested that ATH may enhance fibrin breakdown and thus led to investigations into the effects of UFH and ATH on fibrinolysis. In vitro studies have shown that UFH enhances antithrombin inhibition of plasmin. In addition, ATH displays a slightly greater inhibition of plasmin generation and activity. Such studies were conducted in purified systems, in the absence of other plasmin inhibitors naturally present in plasma. Therefore, the aim of the present study was to compare the effects of UFH, LMWH, and ATH on plasmin generation in plasma. Methods: At 37°C tissue plasminogen activator (tPA) and soluble fibrin fragments (fib) were added to normal adult pooled platelet poor plasma supplemented with 0.35, 0.7, 1.4, or 2.1 U anti-Xa/ml UFH, LMWH, or ATH, to initiate plasmin generation (8.93nM tPA and 300µg/ml fib). At various time points, subsamples were mixed with excess plasminogen activator inhibitor 1 (PAI-1) (55.12nM) to stop further plasmin generation. The plasmin concentration at each time point was determined using a plasmin-specific chromogenic substrate and a standard curve produced from purified plasmin. Results: Comparisons of mean area under the curve (AUC) for plasmin generation displayed a significant decrease in plasmin generation in the presence of all three anticoagulants at all doses tested (p<0.05). Comparing the anticoagulants at similar doses, plasmin generation was significantly decreased in the presence of ATH (15384.66±1930.23nM/min) compared to LMWH (23892.28±3090.54nM/min) at 0.7 U/ml (p<0.05). At a dose of 1.4 U/ml, there was significantly less plasmin generated, over time, in the presence of UFH (20089.49±3022.1623nM/min) and ATH (19273.86±1805.7323nM/min) when compared to LMWH (24743.18±1265.1023nM/min) (p<0.05). There was no significant difference in plasmin inhibition between UFH and ATH at any of the doses tested. Conclusion: The present study supports previous findings that UFH and ATH can facilitate antithrombin inhibition of plasmin. It is also observed that LMWH catalyzes the inhibition of plasmin by antithrombin but possibly to a lesser extent. These findings suggest that ATH has a similar inhibitory effect on plasmin generation and activity in plasma compared to UFH, despite its overall superior anticoagulant properties. Therefore, previous in vivo observations displaying decrease in clot mass with administration of ATH was due to its enhanced anticoagulant abilities and not fibrinolysis enhancement. These findings add to our understanding of ATH mechanisms of action and aid in its development for clinical use. Disclosures No relevant conflicts of interest to declare.

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Treatment of B-CLL Cells with Bortezomib and Rituximab Reduces Cell Viability In Vitro..

Blood ◽

10.1182/blood.v104.11.2798.2798 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2798-2798 ◽

Cited By ~ 1

Author(s):

Eloisa Jantus Lewintre ◽

Elena Sarsotti ◽

Maria Jose Terol ◽

Isabel Benet ◽

Jose A. Martinez-Climent ◽

...

Keyword(s):

Gradient Centrifugation ◽

Combined Treatment ◽

Biological Significance ◽

Vitro Activity ◽

Rabbit Serum ◽

Dependent Manner ◽

In Vitro Activity ◽

Mutational Status ◽

Significant Difference

Abstract Introduction: In B-CLL, somatic mutation of IgVH genes defines a group of patients with favorable prognosis, whereas the absence of IgVH mutations is correlated with a worse outcome. Our previous data suggested that BCL-6 mutations identify a subgroup of patients with high risk of progression despite the presence of mutated IgVH gene, but the clinical significance of this molecular alteration remains uncertain. New approaches are now being tested for the treatment of B-CLL. Proteasome inhibitor, Bortezomib (Btz), and monoclonal antibodies specific for surface antigens, Rituximab (Rtx), represent potential therapeutic strategy. Objetives: To study the effects of Btz and Rtx on viability of CLL cells in culture, and to correlate the responses to the mutational status of the samples. Material and Methods: PBMC from 29 B-CLL patients (Binet stage A) were isolated by Ficoll-Hypaque gradient centrifugation. Samples were culture in RPMI supplemented with 20% FCS. Complement rabbit serum was added to the cultures in Rtx treated cells. Cells were exposed to various concentrations of Btz (0.1, 1 and 10 uM), Rtx (10 ug/ml) or both together during 24 hours. Then, cells were stained with Propidium Iodide and subjected to flow cytometry analysis. Results: Btz and Rtx reduce viability of B-CLL cells in a dose and time dependent manner. The kinetic of both drugs were different, whereas Rtx reaches its maximun effect within 3 hours after treatment, Btz do the same after 48 hours. Combined treatment (Btz + Rtx) increases the effect of each one separately. All differences between treatments with increased doses of Btz + Rtx vs Btz alone were statistically significant. In the group of IgVH mutated patients, there were a significant difference in the response to Btz and Rtx in BCL-6 mutated (mut) and unmutated (unmut) groups (n= 10 and 8 respectively), whereas no significant differences were observed in the number of lymphocytes, lymphocyte doubling time, CD38 expression and cytogenetic alterations. Effects of Btz and Rtx on IgVH mutated samples: correlation with BCL-6 mutations BCL-6 Mean viability (%) SD p * * U. Mann Whitney test Control unmut 82.13 8.70 0.42 mut 86.36 6.01 Btz 0.1 uM unmut 49.05 10.39 0.006 mut 64.28 12.16 Btz 1 uM unmut 39.86 7.19 0.040 mut 53.27 13.35 Btz 10 uM unmut 30.21 7.41 0.041 mut 43.99 14.73 Rtx 10 ug/ml unmut 48.31 14.51 0.050 mut 34.62 12.51 Btz 0.1 uM + Rtx unmut 32.96 14.15 0.153 mut 24.50 10.01 Btz 1 uM + Rtx unmut 25.99 10.88 0.315 mut 19.89 7.53 Btz 10 uM + Rtx unmut 19.27 9.04 0.491 mut 16.31 7.52 Conclusion: Our data show that Btz and Rtx have in vitro activity on B-CLL cells, being used alone or in combination. Interesting, in IgVH mutated cells, Btz and Rtx have statistically significant differences in their in vitro activity on B-CLL cells according to their BCL-6 mutational status, but the biological significance of this differential responses remains unclear

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Monocytes/Macrophages Are the Major Targets of the CCL3 Chemokine Produced by CD38+CD49d+ Chronic Lymphocytic Leukemia Cells.

Blood ◽

10.1182/blood.v114.22.2350.2350 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2350-2350

Author(s):

Antonella Zucchetto ◽

Dania Benedetti ◽

Claudio Tripodo ◽

Riccardo Bomben ◽

Fleur Bossi ◽

...

Keyword(s):

Endothelial Cells ◽

Chronic Lymphocytic Leukemia ◽

T Lymphocytes ◽

Conflicts Of Interest ◽

Cell Types ◽

Lymphocytic Leukemia ◽

Bone Marrow Biopsies ◽

Tnf Α ◽

Significant Difference

Abstract Abstract 2350 Poster Board II-327 Introduction: CD38 and CD49d are associated negative prognosticators in chronic lymphocytic leukemia (CLL). Recent gene expression profiling studies comparing CLL cases expressing low versus high levels of CD38 and CD49d, identified CCL3 as a gene upregulated by CD38+CD49d+ CLL. The release of CCL3 by cultured CLL cells was also demonstrated upon CD38 triggering, and CCL3 protein was found in CLL cells from bone marrow biopsies (BMB) of CD38+ cases (Zucchetto et al., Cancer Res, 2009; 69:4001-9). Given the role of CCL3 as potent chemoattractant for different cell types, we aimed at identifying the major targets of CCL3, as produced by CD38+CD49d+ CLL cells. Methods: CLL infiltrates of BMB were characterized by immunohistochemistry (IHC). Expression of the CCL3 receptors CCR1 and CCR5 by PB CLL subpopulations was evaluated by flow cytometry. T lymphocyte and monocyte migrations were performed by in-vitro transwell chemotaxis assays. Results: IHC analysis of BMB from 16 CLL cases revealed a higher number of infiltrating CD68+ cells in the context of CLL-involved areas of BMB from CD38+CD49d+CCL3+, compared to CD38−CD49d−CCL3− cases (p=0.01). CD3+ lymphocytes were interspersed in the CLL aggregates, but with no significant difference between the two subgroups. Evaluation of CCR1 and CCR5 in PB cell subpopulations from 40 CLL cases expressing or not surface CD38 and CD49d, showed the highest mean fluorescence intensity (MFI) levels for both CCR1 (624±60) and CCR5 (64±9) in the monocytic component, irrespective of CD38 and CD49d expression by CLL cells. Conversely, both CLL cells and residual T lymphocytes showed low MFI levels for CCR1 (19±4 and 14±3) and CCR5 (21±2 and 20±2). High CCR1 and CCR5 expression levels were detected in in-vitro differentiated monocytes from purified PB cells of four CD38+CD49d+ CLL. Accordingly, CCR1 expression was documented in macrophage-like cells in BMB from CD38+CD49d+ CLL. Next, we evaluated the capability of purified monocytes and T lymphocytes from 10 CLL cases to migrate in response to CCL3. In keeping with the strong expression of CCR1, monocytes migrated toward CCL3 at a concentration of 3 ng/mL (migration index, MI= 8.8±0.9, p=0.03), whereas T lymphocytes required a higher CCL3 concentration (100 ng/mL) to display slight migration capability (MI= 1.6±0.2, p=ns). The increased infiltration of macrophages in BMB from CCL3-producing CD38+CD49d+ CLL, prompted us to verify the capability of CCL3-stimulated macrophages to induce the expression by endothelial cells (EC) of the CD49d specific ligand VCAM-1. By using two different EC models (HUVEC and ADMEC), we documented a significant up-regulation of VCAM-1 by EC exposed to conditioned media (CM) collected from cultures of macrophages challenged in-vitro with CCL3 (p=0.002). Notably, increased levels of the pro-inflammatory cytokine TNF-α were detected in CCL3-CM (p=0.006), and neutralization of TNF-α by specific antibodies reverted the capability of CCL3-CM to induce VCAM-1 by EC models. In agreement with these in-vitro data, we found a more prominent meshwork of VCAM-1+ stromal/endothelial cells in lymphoid infiltrates from CD38+CD49d+ CLL compared to CD38−CD49d− cases (p=0.002), and engagement of CD49d by VCAM-1 was able to significantly delay the spontaneous apoptosis observed in cultured CLL cells. Conclusions: CD68+ monocytes/macrophages are likely the main targets for the CLL3 chemokine produced by CD38+CD49d+ CLL cells, and are active in determining, through the release of TNF-α and other yet unidentified cytokines, the overexpression of VCAM-1 by endothelial cells. Experiments aimed at investigating further roles of CD68+ monocytes/macrophage in CLL are currently matter of study. Disclosures: No relevant conflicts of interest to declare.

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