bluuebunny/crossref_metadata_2025 huggingface dataset:
Apolipoprotein E Receptor 2′ Mediates Pathogenic Effects of Dimeric β2glycoprotein I and of Anti- β2glycoprotein I Antibodies in Vivo
Authors: Romay-Penabad Zurina , Urbanus Rolf T , Pappalardo Elizabeth , Hwang Yong , Derksen Ronald H.W.M. , Shilagard Tuya , Vargas Gracie , Pennings Maarten TT , De Groot Philip G , Pierangeli Silvia S
Published: April 2020

Antiphospholipid antibodies (aPL) recognize β2Glycoprotein (β2GPI)-bound to receptor (s) in target cells and trigger a pro-coagulant/pro-inflammatory phenotype [i e.:expression of tissue factor (TF), vascular cell adhesion molecule-1 (VCAM-1)] that lead to thrombosis. The interaction of β2GPI with target cells may involve more than one protein. Investigators have shown that dimeric β2GPI binds to apolipoprotein E receptor 2′ (apoER2′) in platelets, in the absence of anti-β2GPI antibodies, increases their activation and induces enhanced thrombosis and TF activity in mice. However, the role of apoER2′ in vivo in Antiphospholipid Syndrome (APS) is not completely understood. Here, we examined the in vivo effects of dimeric β2GPI and of anti-β2GPI antibodies (IgG-APS) in apoER2′ deficient (−/−) mice and in normal mice pre-treated with recombinant soluble domain 1 of apoER2′ (BD1). In vivo, dynamics of thrombus formation (thrombus sizes), TF activities in carotid artery homogenates and in peritoneal macrophages and ex vivo expression of VCAM-1 in aortas and of TF activity in peritoneal macrophages were examined in the various types of mice after two i.p. injections with 40 μg of recombinant dimeric β2GPI – or with the corresponding monomer control – or with 500 μg IgG-APS (isolated from a patient with APS by protein G Sepharose) or with control IgG (IgG-NHS). Mice injected with IgG-APS had significant titers of anticardiolipin (aCL) and anti-β2GPI antibodies in their sera. In vivo, IgG-APS increased significantly the size of the induced thrombi as well as the TF activities in carotid arteries and in peritoneal macrophages in C57BL/6J (wild type) mice when compared to same type of mice treated with IgG-NHS. Similarly, ex vivo expression of VCAM-1 in mouse aortas and of TF in peritoneal macrophages, detected by two photon excitation laser scanning microscopy were increased in normal mice treated with IgG-APS when compared to control mice. The pre-treatment with 40 μg of BD1 i.p., significantly reduced those effects. Importantly, dimeric β2GPI (in the absence of anti-β2GPI antibodies) or IgGAPS did not increase significantly thrombus size, TF activities in homogenates of carotid arteries or in peritoneal macrophages, or ex vivo expression of VCAM-1 and TF in mice lacking apoER2′.

Conclusions: Altogether these data show that dimers of β2GPI mimic pathogenic effects of anti-β2GPI antibodies in mice. Most importantly, apoER2′ is a mediator of those effects in vivo. These findings may provide insights not only for a better understanding of the pathophysiology of APS but may be important in the development of new targeted therapies, by means of interfering with the binding of β2GPI-aPL complexes with their receptor(s) in target cells in vivo.
Recombinant Factor VIIa Combined with Fibrinogen Corrects the Coagulopathy Associated with Cardiopulmonary Bypass Surgery Ex Vivo
Authors: Onundarson Pall T. , Asvaldsdottir Hanna S. , Gudmundsdottir Brynja R. , Sorensen Benny
Published: April 2020

Major cardiac surgery and use of cardiopulmonary bypass (CPB) is often associated with the development of a severe coagulopathy, hyperfibrinolysis and increased risk of bleeding. The present ex vivo study challenged the hypotheses that whole blood thrombelastometry, activated with minute amounts of tissue factor, can reveal the development of a coagulopathy following cardiac surgery, and that supplementation with fibrinogen and rFVIIa, alone or in combination, can improve the ex vivo clotting pattern. In total, 22 patients with a median age of 68, undergoing coronary artery bypass grafting or valve surgery with use of CPB were included in the study. Dynamic thrombelastometric clotting profiles were recorded using citrated whole blood activated with dilute tissue factor (Innovin®, final dilution 1:17000). Blood samples were collected before surgery (control) and immediately following in vivo neutralization of heparin with protamine sulphate. All blood samples for thrombelastometry were treated with heparinase to ensure neutralization of residual heparin. Standard coagulation laboratory parameters and platelet function confirmed the development of a significant coagulopathy following CPB. The post-operative blood samples were spiked with buffer, rFVIIa (2 μg/mL), fibrinogen (1mg/mL), or the combination of rFVIIa+fibrinogen. The post-operative coagulopathy was evident by thromboelastometry as a statistically significant derangements (Wilcoxon signed rank test). There was prolongation of the onset of clotting (CT, from a median value of 183 seconds pre-op to 385 sec post-op), reduced maximum velocity of clot formation (MaxVel, from 17.5 mm*100/sec pre-op to 15.1 post-op) and reduced maximum clot firmness (MCF, from 6234 mm pre-op to 5527 post-op). Ex vivo spiking with rFVIIa shortened the post-operative clot initiation phase (CT) to a median of 232 sec. Fibrinogen also shortened the post-operative clotting time to a median of 246 sec, and additionally increased the MCF to 5839 mm. Finally, the combination of rFVIIa and fibrinogen together corrected the abnormal thromboelastometric findings associated with CPB-coagulopathy into the pre-operative range, i.e. median CT decreased to 155 sec, MaxVel increased to 16.8 mm*100/sec and MCF increased to 5808 mm.

In conclusion, the experiments suggest a potential role of fibrinogen supplementation during control of bleeding following CPB, either alone or in combination with rFVIIa, since the combination corrected the CPB-associated coagulopathy remaining following neutralization of heparin.
Involvement of Pkcε And Stat3 in Apoptosis of Hematopoietic Cells from Patients with Aplastic Anemia
Authors: Liu Hong , Lin Zenghua , Ruan Changgeng
Published: April 2020

Aplastic anemia (AA) is characterized by peripheral cytopenias in the setting of marrow hypoplasia. Recent reports have indicated that apoptosis plays an important role in injure to the stem and progenitor compartments in these patients. We previously revealed that impaired stem cells could be caused by excess IFN-γ in the serum or hypersensitivity to IFN-γ in the patients. To investigate the molecular mechanism of the apoptotic function of the serum from the patients against hematopoietic stem and/or progenitors, the apoptosis of CD34+ cells was assayed with flow cytometry 24 hr after incubation with the serum of normal controls, patients with non severe AA and severe AA. The CD34+ progenitors from the cord blood sample of healthy donors contained a significantly greater proportion of apoptotic cells after incubation with serum from SAA patients than incubated with serum from normal controls and NSAA. Moreover, the percent apoptosis of CD34+ cells after 24hr incubation with serum from NSAA was slight higher than that of normal controls. To further explore the mechanism of increased apoptosis induced by the serum of AA patients, the expression of phosphorylated protein kinase Cε(pPKCε) and phosphorylated signal transducer and activator of transcription 3 (pstat3) were measured after incubation with serum from the normal controls and AA patients using western blot. After incubation with serum from the AA patients, both pPKCε and pstat3 were overexpressed, correlating with the increased apoptosis induced by the same serum from AA patients. In addition, RT-PCR results shown that the stats gene expression had no difference after incubation with serum from AA patient. Together, these results implicate pPKCε and pstat3 may be involved in the apoptosis of CD34+ cells induced by sera from patients with aplastic anemia. Our findings may contribute to understanding the decreased number of stem cells characteristic of AA.
Successful Treatment of Refractory Idiopathic Aplastic Anemia with Rituximab
Authors: Katragadda Sreedhar , Nelson John C
Published: April 2020

We report the first case of refractory idiopathic aplastic anemia who responded to Rituximab (anti-CD 20 monoclonal antibody). The patient is a 22 year old Hispanic male construction worker who presented with a two week history of weakness, dyspnea on exertion and gum bleeding was found to have a platelet count of 11 × 109/L, hemoglobin of 7.4 g/dL, and white blood cell count of 1.6 × 109/L with 30% neutrophils. A bone marrow biopsy showed hypocellular marrow with relative lymphocytosis of mixed B and T cells and a normal chromosome analysis. A paroxysmal nocturnal hemoglobinuria (PNH) panel was negative. Serological studies did not show any evidence of HBV, HCV, CMV or EBV infection, but did show previous infection with parvo B19. He was initially treated with horse antithymocyte globulin (ATG) at a dose of 40 mg/kg for four days and cyclosporine. After ten weeks of treatment he was still requiring weekly packed red blood cell and platelet transfusions and G-CSF support. At that time he was treated with rabbit antithymocyte globulin and cyclosporine was continued. He had a partial recovery with absolute neutrophil count remaining mostly above 1 × 109/L, but he was still requiring red blood cell and platelet transfusions with baseline hemoglobin of 6 gm/dL and platelet count of less than 20 × 109/L. A bone marrow biopsy done ten weeks after the rabbit ATG treatment showed hypocellular marrow (20% cellularity) with trilineage hematopoiesis, with no evidence of dysplasia. Due to his transfusion dependence after eight months from his rabbit ATG treatment, he received Rituximab at a dose of 375 mg/m2 weekly for four weeks. He had a dramatic improvement of hemoglobin to 11.5 gm/dL and white blood cell count to 3.5 × 109/L, with an absolute neutrophil count of 2 × 109/L, although his platelet count remained at 20 × 109/L. He remains transfusion independent for a follow-up period of 8 months after the Rituximab treatment. Review of literature showed partial to good responses with Rituximab in

aplastic anemia patient who refused treatment with ATG and cyclosporine (Hansen PB et al), aplastic anemia associated with CLL (Bharwani L et al), severe aplastic anemia induced by fludarabine and cyclophosphamide in a patient with B-CLL (Castiglioni MG et al) and, refractory Diamond-Blackfan anemia (Morimoto A et al).
Results of Immunosupresive Therapy in Children with Aquired Severe Aplastic Anaemia (SAA). Report Polish Pediatric Hematology Group
Authors: Pawelec Katarzyna , Niewiadomska Edyta , Matysiak Michal , Kowalczyk Jerzy , Stefaniak Jolanta , Balwierz Walentyna , Zaleska-Czepko Ewa , Chybicka Alicja , Szmyd Krzysztof , Sonta-Jakimczyk Danuta , Bubala Halina , Wysocki Mariusz , Kurylak Andrzej , Wachowiak Jacek , Grund Grzegorz , Mlynarski Wojciech , Bulas Monika , Krawczuk-Rybak Maryna , Leszczynska Elzbieta , Urasinski Tomasz , Peregud-Pogorzelski Jaroslaw
Published: April 2020

Introduction: Bone marrow transplantation (BMT) from HLA identical family donors is the treatment chosen, according indication of Polish Pediatric Hematology Group and Working Party SAA of the EBMT, for children with severe aplastic anemia (SAA). When no donor is available, combined immunosuppressive therapy (IST) is given. Material and method: SAA was recognized in 116 children (45 girls, 71 boys) aged 2–18 years in ten pediatric hematological center in Poland between 1993–2007 years. All patients received IST according Polish Pediatric Hematology and Working Party SAA of the EBMT protocol: antithymocyte globulin horse or rabbit, cyclosporine A (CSA), prenisolon and in 90 patients G-CSF or GM-CSF was additionally administered. Hematological response was evaluated on day 112 and 180 of therapy. Results and conclusion: On day 112 remission was obtained in 61children from 116, complete remission (CR) in 47 (40,5%), partial remission (PR) in 24(20,8%). There was no response (NR) to treatment in 45 (38,7). On day 180 remission was obtained in 88 from 116 patients (75,8%), CR in 57 patients (49,1%), PR in 31 (26,7%), NR in 29 patients (24,2%). Period of observation was from 8 months to 14 years. During this time relapse occurred in 32 patients (27.5%). Patients with NR and relapse were treated second course of IST or BMT from matched bone marrow donors. Unrelated BMT was performed in 18 patients with NR or relapsed after IST. We observed 27 deaths (8 early during the first 3 months of IST) in all group. During the 14 year of observation we notice one case with late clonally complication (PNH), one case of transformation to AML and one to MDS. Probably of 14year survival was 76,7% in our group. Our results are effective and compared to other hematological centers.
Israeli Fanconi Anemia Registry
Authors: Tamary Hannah , Alter Blanche P. , Nishri Daniella , Rosenberg Philip S
Published: April 2020

Using epidemiological data from retrospective cohorts of patients with Fanconi Anemia (FA) in North America and Germany a quantitative model to estimate bone marrow failure (BMF) and cancer risk was previously generated. To evaluate generalizability to another population, and to determine the risks for adverse outcomes in Israel, we created an Israeli FA registry and used the model to evaluate complications. We reviewed patient charts of 66 patients with FA diagnosed in Israel between 1964–2005. The data base included demographic information, as well as data describing the congenital abnormalities, FA complementation groups, BMT course and malignancies. Thirty six (36) patients were of Jewish origin [Ashkenzi 7, Sephardic 23, mixed 6] and 30 of Arabic origin. The first adverse event was bone marrow failure (BMF) in 35 patients (53%), hematological malignancy in 7 (11%) and 2 solid tumors in each of 3 patients (5%). The cause-specific hazard of BMF peaked at 10.5%/year at age 10 years (95% CI: 6.7–14.1%/year). The hazard of AML/ALL and MDS were stable at 0.9%/year (95% CI: 0.42–1.85%/year) and 1.4%/year (95% CI: 0.76–2.49%/year) respectively. The cumulative incidence of each outcome to age 32 was 70% for BMF, 13% for AML/ALL, and 17% for solid tumor. A five item congenital abnormality score was significantly associated with the risk of BMF (P = 0.009). The ratio of observed to expected cancer was 71 for all cancers [50 for solid tumors, 175 for leukemia] and >11,000 for myelodysplastic syndrome. Significantly elevated ratios of observed to expected cancers were observed for head and neck squamous cell carcinoma in 2 patients (986-fold), tumor of larynx (13,238-fold), vulva (3,701-fold), cervix (244-fold) and breast (88-fold). The complementation group was known in 41 patients [A 25 (63%), C 9 (22%), G 6 (15%), and D1 1 (2%)]. However, associations between complementation groups and specific outcomes were not significant. Despite the different ethnic background and the smaller number of FA patients in the Israeli cohort the risk estimates compared with the US and German cohorts were similar. As previously suggested the congenital abnormality score was significantly associated with the risk of BMF; an extraordinary risk of developing AML/MDS and later specific solid tumors was also found.
A Novel BACH2-BCL2L1 Fusion Gene in the Burkitt’s Lymphoma Derived Cell Line BLUE-1
Authors: Türkmen Seval , Riehn Mathias , Mundlos Stefan , Thiel Eckhard , Burmeister Thomas
Published: April 2020

Abnormalities of the long arm of chromosome 6 are a common feature in various B-cell malignancies. However, in most cases the involved genes have not yet clearly been identified. We have molecularly characterized the recently established cell line BLUE-1 that has been derived from a relapsed sporadic Burkitt lymphoma. This cell lines carries a t(6;20)(q15;q11.2) rearrangement in addition to the typical t(8;14) with MYC-IgH fusion. The involved gene loci on chromosomes 6 and 20 were up to now unknown.

To identify the involved gene loci on both chromosomes we applied a sequential BAC clone mapping strategy. BAC RP11-243J16 clone (20q11.21, bp 29,756,679–29,925,538) was found to hybridize with der(6) as well as der(20). BAC RP1-104D1 (6q15, bp 91,001,943–91,064,406) also covered the breakpoint region. Two of the involved genes in this region are the transcription factor BACH2 (basic leucine zipper transcription factor 2) on 6q15 and BCL2L1 (BCL-X) on 20q11, a member of the BCL2 anti-apoptosis gene family. We hypothesized that these two genes could be involved and by testing different primer combinations were able to amplify a BACH2-BCL2 fusion mRNA transcript using RT-PCR. In this fusion transcript the first (non-coding) exon of BACH2 was fused to the second (partially coding) exon of BCL2L1 thus effectively placing the BCL2L1 gene under the control of the BACH2 promotor. Western blot analysis showed a strong expression of BCL2L1.

This is the first report of a fusion gene involving the genes BACH2 and BCL2L1. The prototype of the BCL2 family, BCL-2 on 18q21.3 is known to play a crucial role in various lymphomas but a clear role for the closely related BCL2L1 gene in lymphomas has not yet been established. BACH2 is known to be expressed in B-cells at various maturation stages and is believed to be involved in the machinery of class switch recombination (CSR). Bach2 −/− mice show increased IgM but decreased IgG and IgA levels and a deficient T cell-independent and T cell-dependent IgG response associated with defective CSR.

In summary, this molecularly characterized translocation provides a new tool for studying recurrent 6q aberrations in lymphomas and for the action of the BCL2L1 antiapoptosis gene.
Serum BAFF (B-CELL Activating Factor Of The TNF Family) predicts time to First Treatment in Early B-CELL Chronic Lymphocytic Leukemia
Authors: Molica Stefano , Vitelli Gaetano , Cutrona Giovanna , Digiesi Giovanna , Mirabelli Rosanna , Giannarelli Diana , Sperduti Isabella , Ferrarini Manlio
Published: April 2020

We analyzed the correlation between well-established biological parameters of prognostic relevance in B-cell chronic lymphocytic leukemia [CLL] (i.e, mutational status of the immunoglobulin heavy chain variable region [IgVH], ZAP-70− and CD38− expression) and serum levels of BAFF (B-cell activating factor of the TNF family) by evaluating the impact of these variables on the time to first treatment [TFT] in a series of 125 previously untreated Binet stage A B-cell CLL patients. By using a commercial ELISA (R & D Systems, USA) we found that higher levels of BAFF characterized more frequently females (P=0.01), patients with Rai stage 0 (P=0.03), mutated IgVH disease (P=0.008) and low ZAP-70 expression (P=0.04). In contrast, age (P=0.35), peripheral blood lymphocytosis (PBL)(P=0.09), hemoglobin (Hb) level (P=0.64), platelet (PLT) count (P=0.12), serum β2-m (P=0.49), LDH (P=0.85) and percentage of CD38-positive B-CLL cells (P=0.63) did not reflect circulating levels of BAFF. We used an optimal cut-point search to determine how to best split soluble BAFF data. Maximally selected log-rank statistics plots identified a BAFF serum concentration of 311 ng/mL as the best cut-off (P<0.0001). Accordingly, patients who had BAFF levels higher than 311 ng/mL experienced a longer TFT (median 108 months) in comparison to patients whose BAFF levels were lower than 311 ng/mL (median 30 months; P<0.0001). Along with serum concentration of BAFF, the univariate Cox proportional hazard model identified Rai substage I–II (P=0.003), lower PLT count (P=0.04), higher PBL count (P=0.01), increased LDH (P=0.01), ZAP-70 expression > 20% (P=0.02) and absence of mutation of IgVH (P<0.0001) as predictor of shorter TFT. In multivariate analysis only soluble BAFF (Hazard ratio [HR], 6.13; CI 95%, 2.31–16.25) and mutational status of IgVH, (HR= 2.99; CI 95% 1.33–6.76, P=0.008) maintained their discriminating power. The effects of BAFF on TFT were masked by mutational status of IgVH in patients with unmutated IgVH. However, serum levels of BAFF and mutational status of IgVH had a joint effect on TFT in patients with mutated IgVH which translates into a segregation of patients with mutated IgVH in two groups with different TFT according to BAFF levels (HR= 8.9; P<0.0001). Our results indicate that in early B-cell CLL biological profile including among other parameters soluble BAFF may provide a useful insight into the complex interrelationship of prognostic variables. Furthermore, BAFF along with mutational status of IgVH can be adequately used to predict clinical behaviour of patients with low biological risk.
Epidemiology and 15-Year Follow up after Cladribine Treatment of Patients with Hairy Cell Leukemia (HCL): Population-Based Swedish Data and Long-Term Follow-up of Scandinavian Study Patients Identify Older Patients with Poorer Outcome
Authors: Samuelsson Henrik , Heldal Dag , Jerkeman Mats , Juliusson Gunnar
Published: April 2020

HCL has been considered to be a disease of predominantly young males with, after the introduction of effective treatment, excellent prognosis. However, although very effective, these therapies introduced in the 1980’s are not curative. The Swedish Lymphoma Registry records all patients diagnosed since year 2000, with a 96 % coverage compared to the Swedish Cancer Registry, which is compulsory since 60 years. In contrast, previously published studies include patients highly selected for inclusion in treatment studies or referral to major medical centers. The Swedish registry data confirms the high male to female ratio, with yearly incidences of 5.3 males and 1.1 females per million. However, the median age at diagnosis was 62 years (range 30–92). The incidence in males rise with age as follows: 3 (30–49 yrs), 11 (50–69 yrs), 16 (70–79 yrs), and 20/million (80–95yrs). The 5-year overall survival of these unselected patients was 81%. Age and performance status had strong influence on survival. If HCL was diagnosed before age 60 the 5-year survival was 94%, whereas it was 70% for patients over 60 years. We also performed a 15-year follow-up of patients included in our first cladribine trials in the early 1990’s. The progression-free survival at 10 years was 60% and at 15 years 55%, irrespective of age. However, the 10 year and 15 year overall survivals were 91% and 82% for patients <60 yrs, in contrast to 65% and 42% for those with diagnosis after age 60 years. We conclude that there is a proportion of older patients with HCL with a similar progressionfree survival after cladribine treatment as the younger, but poorer overall survival. Further analyses, including epidemiology of unselected total populations diagnosed since 1987, age-adjusted survival estimates, and incidence of secondary cancers among the HCL patients will be presented.
Quantitative Gene Expression Analysis of Surrogate Markers for Genetic Risk Groups and Survival in CLL
Authors: Kienle Dirk , Benner Axel , Winkler Dirk , Hensel Manfred , Dalla-Favera Riccardo , Lichter Peter , Stilgenbauer Stephan , Döhner Hartmut
Published: April 2020

In CLL, a variety of surrogate markers for individual genetic features, mostly the VH mutation status, were proposed from gene expression analyses. However, their detailed relation to specific genetic subsets such as V3-21 usage, del11q22-q23 (11q−), and del17p13 (17p−), and their prognostic value in relation to established factors is not elucidated yet. Gene expression markers (ADAM29, ATM, CLLU1, DMD, GLO1, HS1, KIAA0977, LPL, MGC9913, PCDH9, PEG10, SEPT10, TCF7, TP53, Vimentin, ZAP-70, ZNF2) were evaluated using real-time quantitative RT-PCR (RQ-PCR) in purified samples of 151 patients. VH sequencing and FISH screening for genomic aberrations were carried out for all cases, survival information was available for 133 cases. Logistic regression was performed to test the predictive value of gene expression for genetic risk groups, Cox proportional hazards statistics for survival analysis. VH mutation status was best assigned by LPL and ZAP70, followed by TCF7, a marker with a characteristic overexpression in VH mutated CLL patients (correct VH prediction in 83%, 83%, and 75% of the patients, respectively). A similar rate of correct VH assignments was achieved in the subgroup of patients with 11q− or 17p− when using these markers (88%, 86%, and 79%, respectively). In contrast to LPL and TCF7, most of the patients with V3-21 usage were recognized as risk patients by ZAP70 independently of the VH status. Therefore, ZAP70 yielded the best results for the overall recognition of patients with a genetic risk constellation (VH unmutated or V3-21 usage or 11q− or 17p−). Comparison of ZAP-70 determination by RQPCR and flow cytometry was performed for 72 patients and revealed 30% of discordant cases. Thereof, the majority was VH unmutated (including several cases with 11q− or 17p−) showing ZAP-70 negativity by FACS and positivity by RQ-PCR. In multivariate analysis of time to first treatment (TFT), ADAM29 was an independent prognostic factor besides the VH status and Binet stage. In overall survival analysis including the gene expression variables only, LPL was the strongest predictor for overall survival. When genetic and clinical factors were added to this analysis, V3-21 usage, 17p−, age, binet stage, and expression of ATM, ADAM29, SEPT10, and TCL1 were identified as significant prognostic factors. In conclusion, novel gene expression markers allow screening for patients at risk but can not fully substitute for the genetic factors, which should therefore at present remain the basis for risk stratification approaches. Some of the novel markers appear to have a prognostic relevance independently of the established factors, which points to relevant biologic and clinical implications demanding further investigation.
Elevated Serum Level of Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor Correlates with Stage of Chronic Lymphocytic Leukemia and Resistance to Treatment
Authors: Bakirov Bulat , Sgibneva Anastasiya , Bakirov Akhat
Published: April 2020

Background: B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in western world with an incidence of 3,36/100,000 in European males. It is characterized by a clonal growth of long lived, slowly proliferating mature B lymphoid cells in the bone marrow (BM), peripheral blood (PB), and lymphoid tissues. Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) is a pleiotropic cytokine that plays a role in hematopoiesis and apoptosis. Angiogenesis may have a role in a pathophysiology of leukemias and antiangiogenesis therapy could have an anticancer effect.

Aim: analyze the clinical significance of serum levels of angiogenic factors and how it’s correlate with clinical stage and survival in patients with B-CLL

Methods: the enzyme-linked immunosorbent assays (ELISAs) for VEGF and bFGF were performed in 78 CLL patients and 29 healthy person as a control group. The patients was divided on low-risk disease (44 patients) and high-risk disease (34 patients).

Results: VEGF and bFGF serum levels were significantly increased in patient with high-risk disease, the median serum VEGF level was 185.66 pg/ml, compared with 72.67 pg/ml in patient with a low-risk and in control, it was 48.62 pg/ml. The difference in the VEGF levels was significant for the comparison between low- and high-risk disease (p<0.001). VEGF levels correlate with high white blood cell/lymphocyte counts, short period of time to begin treatment, disease progression, lymphocyte doubling time and worse answer to chemotherapy. No significant increase was found in bFGF serum level between low- and high-risk disease (34.06 pg/ml and 35.84 pg/ml, respectevely), but between patient and control group it was differences in serum level of bFGF (34.85 pg/ml and 7.77 pg/ml, respectively) (p<0.001).

Conclusion: as we found, serum levels of bFGF and VEGF were significantly higher in the patients with B-CLL than in controls. High serum level of VEGF and bFGF associated with poor prognosis and worse answer on treatment. In summary, our data suggest that angiogenic factors play a significant role in the leukemic process and may suggest novel therapeutic approaches in B-CLL.
Formin-Like Gene Expression in Chronic Lymphocytic Leukemia Is Associated with Unfavorable Prognostic Factors and Clinical Outcome
Authors: Calpe Eva , Martinez Antonio , Favaro Patricia , Crespo Marta , Codony Carles , Baptista Ma.ÂJoao Gomes , Gine Eva , Montserrat Emili , Bosch Francesc
Published: April 2020

Formin proteins are large proteins evolutionarily conserved that govern cell shape, adhesion, cytokinesis, and morphogenesis by remodeling the actin and microtubule cytoskeletons. The human leukocyte formin, named as FMNL1, corresponds to an extended cDNA of the 5′-truncated KW-13 which was reported as a tumor-associated antigen in CLL, using a serologic identification by recombinant expression cloning (SEREX). In addition, FMNL1 co-immunoprecipitates with P-Akt in CLL cells, suggests that this protein may contribute to regulate cell survival. The aim of this study was to analyze the expression of FMNL1 in normal B-cell subsets and in a series of 73 patients (median age, 59 years; male/female 40/33; Binet A: 90.2%) with CLL. FMNL1 expression was analyzed by Western Blot, Immunohistochemistry and by Real-Time RT-PCR using expression in Jurkat as baseline. In normal lymphocytes subsets, FMNL1 was only expressed in memory B-cells, and in T-cells, whereas germinal center lymphocytes were negative. Among lymphoid B-cell malignancies, FMNL1 was expressed in mantle-cell lymphomas, Burkitt’s lymphomas and in DLBCL of GCB-type. In CLL cases mean of FMNL1 expression by QRT-PCR was 2.18 AU (SD, 1.01 AU). Using an arbitrary cut-off of 3.2 AU, cases with increased expression of FMNL1 were associated with a younger age at diagnosis (< 50 yrs), elevated lymphocyte count, high serum β2-microglobulin (β2-m) levels, increased ZAP-70 and CD38 expression, shorter time to progression, and shorter survival as compared to cases with low FMNL1 expression. No relationship was observed with genetic abnormalities. In summary, increased expression of FMNL1 gene couples with adverse clinical and biological parameters in patients with CLL. Finally, the interaction between FMNL1 and AKT protein in lymhoproliferative disorders is under investigation.
Angiopoietin-2 mRNA Expression Is Elevated in Chronic Lymphocytic Leukemia Patients with Poor Prognostic Features
Authors: Vrbacky Filip , Smolej Lukas , Vroblova Vladimira , Pekova Sona , Hrudkova Monika , Pecka Miroslav , Maly Jaroslav
Published: April 2020

Several studies have demonstrated potential prognostic importance of angiogenesis in chronic lymphocytic leukemia (CLL). Elevated expression of angiogenic activator angiopoietin-2 (Ang-2) was recently reported in CLL but data regarding prognostic significance of Ang-2 in CLL is limited. Therefore, we evaluated potential relationship between Ang-2 expression and other prognostic factors in patients with CLL. Ang-2 mRNA was quantified by real-time quantitative PCR in purified mononuclear cells of 33 untreated CLL patients. Ang-2 transcript levels were further analyzed with regard to clinical stage, disease course, IgVH mutation status, CD38 and ZAP-70 expression. Elevated Ang-2 mRNA was detected in 12 cases; twenty-one patients had very low or undetectable levels of Ang-2 transcript. High Ang-2 mRNA levels were significantly associated with unmutated IgVH genes (p=0.010) and high CD38 expression (p=0.011) but not with ZAP-70 expression (p=0.784), Rai stage (p=0.305) or stable vs. progressive clinical course (p=0.443). Interestingly, there was a trend towards shorter progression-free survival in patients with high Ang-2 expression, but it did not reach statistical significance (median 15 months vs. not reached; p=0.090). In conclusion, our data show that Ang-2 mRNA is differentially expressed in patients with CLL and its increased expression appears to be associated with poor prognostic features.
Serum CD26 (Dipeptidyl Peptidase IV, DPP IV) compared with Immunoglobulin Heavy-Chain Mutation Status, ZAP-70 and CD38 as a Predictor of Time to First Treatment in Early B-CELL Chronic Lymphocytic Leukemia
Authors: Molica Stefano , Vitelli Gaetano , Cutrona Giovanna , Mirabelli Rosanna , Digiesi Giovanna , Giannarelli Diana , Sperduti Isabella , Morabito Fortunato , Ferrarini Manlio
Published: April 2020

We analyzed the correlation between well-established biological parameters of prognostic relevance in B-chronic lymphocytic leukemia [CLL] (i.e, mutational status of the immunoglobulin heavy chain variable region [IgVH], ZAP-70- and CD38-expression) and serum levels of CD26 (dipeptidyl peptidase IV, DPP IV) by evaluating the impact of these variables on the time to first treatment [TFT] in a series of 69 previously untreated Binet stage A B-CLL patients. By using a commercial ELISA (Human DPPIV/CD26 Quantikine R D Systems, USA) we found that circulating levels of CD26 did not reflect Rai sub-stages (P=0.52), β2-microglobulin (P=0.68), LDH (P=0.06), mutational status of IgVH (P=0.28), ZAP-70 (P=0.52) and CD38 (P=0.48). We used an optimal cut-point search to determine how to best split soluble CD26 data. Maximally selected logrank statistics plots identified a CD26 serum concentration of 371 ng/mL as the best cut-off. Accordingly, patients who had CD26 levels higher than 371 ng/mL experienced a shorter TFT (median 40 months) in comparison to patients whose CD26 levels were lower than 371 ng/mL (median 120 months; P=0.003). The univariate Cox proportional hazard model identified higher serum concentration of CD26 and absence of mutation in IgVH as predictor of shorter TFT. Again in multivariate analysis all these parameters maintained their discriminating power: mutational status of IgVH (HR= 0.23; CI 95% 0.10−0.51, P<0.0001), soluble CD26 (HR= 0.38; CI 95% 0.17−0.85, P=0.02). The effects of CD26 on TFT were masked by mutational status of IgVH in patients with unmutated IgVH. In contrast, serum levels of CD26 and mutational status of IgVH had a joint effect on TFT in patients with mutated IgVH which translates into a segregation of patients with mutated IgVH in two sub-groups with different TFT according to CD26 levels (HR= 0.18; CI 95% 0.05−0.60, P=0.005). Our results indicate that in early B-cell CLL biological profile including among other parameters soluble CD26 may provide a useful insight into the complex interrelationship of prognostic variables. Furthermore, CD26 along with mutational status of IgVH can be adequately used to predict clinical behaviour of patients with low biological risk.
Alemtuzumab Administered by Subcutaneous Route Is Less Effective Than Intravenous Route for First Line Therapy of T-Cell Prolymphocytic Leukaemia: Results of a Pilot Study (UKCLL05)
Authors: Khot Ami t S , Matutes Estella , Kaczmarek Pawel A , Hamblin Mike , Roy Ashok , Grand Effie , Ethell Mark E , Dearden Claire E
Published: April 2020

T-cell prolymphocytic leukaemia (T-PLL) is a rare aggressive post thymic lymphoid disorder with distinctive clinical and pathological features. It is resistant to conventional chemotherapy and has a short median survival of 7 months. Intravenous (IV) alemtuzumab is an effective and well tolerated therapy for this disease, inducing remission in 76% of patients in the relapsed and refractory setting and increasing median survival to 24 months in responders. The aim of this study was to determine the efficacy of subcutaneous (SC) administration of alemtuzumab in previously untreated T-PLL patients. After an initial week of dose escalation, patients were treated with a dose of 30 mg 3 times weekly. Patients could be switched to the IV route if they had local side effects or showed a lack of response. Nine patients were enrolled. Male: female ratio was 2:1; median age was 61 years. All cases were centrally reviewed and confirmed to have a diagnosis of T-PLL. Eight had an immunophenotype typical of T-PLL (CD2, CD3, CD4, CD5 and CD 7 positive). One patient was unusually CD7 negative and also did not have an abnormality of chromosome 14. Seven had a complex karyotype and 8 had chromosome 14 abnormalities. Patients were treated on trial for a median of 7 weeks (range 3–13 weeks); 55% required a change to IV treatment due to a lack of response. This resulted in an increase in overall response (OR) from 33% (1 complete response [CR], 2 partial response [PR]) to 44 %.(2 CR, 2 PR). Five patients required addition of pentostatin to alemtuzumab at a dose of 4 mg/m2 IV weekly to augment response. This increased OR to 77% (1 additional CR and 2 PR). 5 patients had haemopoietic progenitor cell transplants (HPCT) as consolidation; 1 was autologous and 4 were allogeneic. Median overall survival was 20 months (range 1–26 months) and disease free survival was 12 months (range 0–20 months). The treatment was well tolerated; however 2 patients had skin reactions to the SC injections. One patient required treatment for CMV reactivation and 2 patients had grade 4 haematologic toxicity which was related to disease and not treatment. Four patients remain alive and well in CR with a follow up of between 12 and 23 months, of whom 3 had allogeneic HPCT. The study was terminated early as a data safety monitoring review felt that SC treatment was not efficacious and patients should not be offered SC treatment. This pilot study suggests that the SC route of administration of alemtuzumab is not as effective as IV in the treatment of T-PLL; our series of 16 patients receiving first line therapy with IV alemtuzumab has shown an OR of 94% with 88% CR. It is therefore advisable that IV treatment should be used. Pentostatin is an effective agent to augment response. Allogeneic HPCT should be used as consolidation therapy when possible.
Assessment of Targeted Systemic Exposure Based on Ex-Vivo Concentrations of Bendamustine and Chlorambucil and Related Clinical Outcomes
Authors: Darwish Mona , Brown Peter , Bensen-Kennedy Debra M , Young Lauren
Published: April 2020

The alkylating agent bendamustine (100 mg/m2 iv) was recently shown to be superior to chlorambucil (0.8 mg/kg po) in the treatment of patients with chronic lymphocytic leukemia, in respect to both overall response rate and progression-free survival (Knauf et al 2007). In order to better understand the greater activity exhibited by bendamustine, the ex-vivo potencies of these two drugs against primary CLL cells were compared with the peak plasma concentrations obtained in patients. Published studies indicate that ex-vivo bendamustine treatment results in a mean LD50 (dose causing 50% cell death) of 7.4 and 4.3 μg/ml in CLL cells from chemo-naive and previously-treated patients respectively (Schwanen et al 2002). This is comparable to the Cmax attained in patients: a 120 mg/m2 dose of bendamustine infused over 60 min resulted in a mean Cmax of 5.6 μg/ml in a study of lymphoma patients. In contrast, the corresponding LD50 values for chlorambucil were reported to be 12.3 and 26.2 μg/ml for chemo-naive and previously-treated CLL patients (Silber et al 1994). These concentrations are considerably higher than the plasma concentrations attained with oral administration of chlorambucil: an oral dose of 60 mg (~0.8 mg/kg) resulted in a mean Cmax of 2.2 μg/ml of parent compound and 1.1 μg/ml of the active phenylacetic acid metabolite. A higher dose of 70 mg did not result in higher plasma exposure (Hartvig et al 1988). The ability to achieve adequate therapeutic concentrations of bendamustine in CLL patients may explain, in part, the greater activity observed with this novel chemotherapy as compared to chlorambucil.
The Effects of BCR/ABL on DNA Damage and Repair Are Dependent on Apoptosis Competence
Authors: Burke Beth A , Carroll Martin
Published: April 2020

Chronic myeloid leukemia (CML) is a two-stage disease associated with the t(9;22) translocation. This translocation fuses the BCR gene with the ABL tyrosine kinase, forming the BCR/ABL oncogene. BCR/ABL is a constitutively activated tyrosine kinase, which causes the first stage of CML, chronic phase. However, it is still unknown why patients progress to the second phase, blast crisis, a phase marked by increased chromosomal abnormalities. Previous data from our lab suggests that cells expressing BCR/ABL have an increase in chromosomal translocations after DNA repair compared to control cells, as assessed by spectral karyotyping. We hypothesized that BCR/ABL alters the apoptotic threshold in response to DNA damage, and we sought to determine if cells lacking BCR/ABL that were unable to undergo apoptosis would show similar responses in DNA damage and repair assays to BCR/ABL expressing cells. In order to study this question we have used an interleukin 3 (IL3) dependent cell line that lacks expression of the pro-apoptotic proteins, Bax and Bak. These cells grow in the presence of IL3 but do not undergo apoptosis after cytokine withdrawal or genotoxic stress. We have generated a subclone of these cells that expresses BCR/ABL. This cell line, designated DBA, grows in the absence of IL3. As expected, growth of the cells in the absence of IL3 is suppressed by the ABL kinase inhibitor, dasatinib. BCR/ABL expressing and control cells proliferate at similar rates following DNA damage. After damage, both control cells and DBA cells have a delay in the G2/M phase of the cell cycle, which is modestly prolonged in BCR/ABL expressing cells. Both control and BCR/ABL expressing cells have a similar amount of DNA damage after irradiation as assessed by pulsed field gel electrophoresis. Additionally, the rate of repair of DNA double strand breaks is not significantly different in the two cell types. Spectral karyotype analysis is ongoing to determine whether error prone DNA repair in BCR/ABL cells is secondary to the effects of BCR/ABL on inhibition of apoptosis or reflects other proposed mechanisms of action of the oncogene.
Hereditary Deficiencies of Protein C or Protein S Confer Increased Risk of Arterial Thromboembolic Events at Young Age. Results from a Large Family Cohort Study
Authors: Mahmoodi Bakhtawar Khan , Brouwer Jan-Leendert P. , Veeger Nic J.G.M. , van der Meer Jan
Published: April 2020

Introduction: Hereditary deficiencies of protein S, protein C or antithrombin are strong risk factors for venous thromboembolism (VTE). Whether these deficiencies are associated with arterial thromboembolism (ATE) and whether history of VTE in these subjects predisposes to subsequent ATE has yet to be determined.

Methods: Based on pedigree analysis we enrolled a total 552 subjects (52% women; mean age, 46±17 years), belonging to 84 different kindreds, in this retrospective family-cohort study. Detailed information on previous episodes of VTE, ATE, anticoagulants use and atherosclerosis risk factors (i.e. diabetes, hypertension, hyperlipidemia, and smoking) were collected. In addition to the index deficiencies participants were also tested for other thrombophilic defects; including factor V Leiden, prothrombin G20210A, increased FVIII and lupus anticoagulants. Primary study outcome was objectively verified symptomatic ATE. As the assumption for proportional hazards for the final model was not met over the entire observation period, we opted for a piecewise Cox model with a cut off point set at 55 years of age.

Results: Of 552 subjects (mean age±SD, 46±17 years; 52% women), 308 had either protein S (35%), protein C (39%) or antithrombin deficiency (26%). Age, atherosclerosis risk factors and other thrombophilic defects were similar (P>0.23) between deficient and non-deficient subjects. A total of 44 arterial thromboembolic events had occurred, corresponding to an overall annual incidences of 0.34% (95% CI, 0.23–0.49) in deficient and 0.17% (0.09–0.28) in non-deficient subjects, hazard ratio 2.3 (1.2–4.5; P=0.01). However, the risk hazards varied over lifetime; while risk of ATE conferred by these deficiencies was 5.4 (1.6–18.4; P=0.006) before age 55 years, it was 1.3 (0.6–2.9; P=0.51) thereafter. After adjusting for atherosclerosis risk factors and clustering of ATE within families, deficient subjects had 4.7-fold (1.5–14.2; P=0.007) higher risk of ATE before age 55 years, versus 1.1 (0.5–2.6; P=0.84) thereafter, compared to non-deficient family members. For separate deficiencies these were 4.6 (1.1 – 18.3), 6.9 (2.1 – 22.2) and 1.1 (0.1 – 10.9) in protein S-, protein C- and antithrombin-deficient subjects, respectively, before age 55 years. History of VTE was not related to subsequent ATE, hazard ratio 1.1 (0.5 – 2.2).

Conclusions: Compared to non-deficient family members, subjects with protein S or protein C deficiencies but not antithrombin deficiency have an increased risk for ATE before age 55 years, independent of prior VTE. After age 55 years conventional atherosclerosis risk factors accounted for ATE. In thrombophilic families, deficiencies of protein S and protein C should be considered in atherothrombotic risk assessment before age 55 years.
Growth Factor Deprivation Combined with Prolonged Inhibition of BCR-ABL Does Not Eradicate Functional CML Stem Cells
Authors: Hamilton Ashley , Helgason Gudmundur V , Allan Elaine , Alcorn Michael J. , Nicolini Franck E , Blair Allison , Schemionek Mirle , Koschmieder Steffen , Brunton Valerie G , Holyoake Tessa L
Published: April 2020

Chronic myeloid leukaemia (CML) is a myeloproliferative disorder that originates in a haemopoietic stem cell (HSC) and is characterised by the BCR-ABL oncogene. We have previously shown that cancer stem cells exist in all patients with newly diagnosed CML. These cells are primitive, quiescent, transplantable in NOD/SCID mice and insensitive to the tyrosine kinase inhibitor (TKI) imatinib. This intrinsic resistance may in part be explained by high expression of BCR-ABL in the most primitive cells. Our overall aim was to achieve complete and prolonged inhibition of BCR-ABL within CML CD34+ cells in vitro, using the highest clinically achievable concentration of a novel dual Src/ABL TKI, dasatinib, in serum free medium in the absence of growth factors. The drug was replenished every 3 days and cells remaining at 12 days were analysed. Despite growth factor deprivation combined with 12 days continuous treatment with dasatinib 150nM, 10% of the starting CD34+ cells were recovered. CFSE stained CML cells showed that treatment with dasatinib resulted in a “backing up” of cells within the undivided cell population and earlier cell divisions, demonstrating that dasatinib, like imatinib, is anti-proliferative. Inhibition of P-CrkL and hence BCR-ABL was maximal at the earliest time-point (day 4) and in the cells that were able to proliferate through several divisions, likely the most mature cells. Cells which remained undivided/quiescent and maintained expression of stem cell markers, CD34 and CD133, demonstrated significantly elevated levels of P-CrkL, as compared to the mature cycling populations. Following drug washout, the remaining viable cells achieved 5-fold expansion when cultured in growth factors for 7 days. Moreover, these cells were able to form colonies following culture in LTCIC assays, which were BCR-ABL positive by FISH and did not show gene mutation. Retrospective studies which assessed the efficacy of TKI treatment times and concentrations also showed that transient dasatinib exposure for 1 hour at 150nM, resulted in a ~80% reduction of viable CML cells when assessed 72 hours later and was as effective as 72 hours continuous exposure at concentrations as high as 1000nM. Our next approach will be to combine BCR-ABL knock-down using shRNA with dasatinib to fully inhibit BCR-ABL in the stem cell population in an effort to determine whether these cells are dependent on BCR-ABL for survival and proliferation.