bluuebunny/crossref_metadata_2025 huggingface dataset:
The Incidence and Outcome of Acute Lymphoblastic Leukemia in Shanghai, China Over 5 Years (2002–2006)
Authors: Ni Xiong , Shen Zhixiang , Chen Fangyuan , Liang Hui , Lu Fengjuan , Tang Jingyan , Wang Chun , Shao Jingbo , Hou Jian , Zou Shanhua , Wang Jianmin
Published: April 2020

Great advances have been made in the diagnosis, molecular pathogenesis and treatment for acute lymphoblastic leukemia (ALL) in the past decade. Due to the lack of comprehensive population-based studies, the updated trend of incidence and geographic variations of ALL has not been well documented in Shanghai, China. To better understand the incidence and epidemiological features of ALL in Shanghai, we conducted a survey based on the database in the Center for Disease Control and the medical record in all hospitals in Shanghai, especially 28 major hospitals with hematology department. According to the CDC data, 544 patients with median age of 32y (1.2~89y) were diagnosed as de novo ALL during January 1, 2002 and December 31, 2006 and followed-up until December 31, 2007. The yearly incidence of ALL was (0.81±0.11)/100 000, which is slightly higher in men (0.86/100 000) than in women (0.75/100 000). The age-stratified incidence showed 2.31/100 000 in pts ≤17y, 0.54/100 000 in 18~34y, 0.46/100 000 in 35~59y, and 0.94/100 000 in pts > 60y. There were substantial geographic variations in the incidence of ALL, with 0.57±0.14/100 000 in Chongming county, an island eastern of Shanghai city, much lower than that of other districts in the central or around the central city, such as Qingpu, Baoshan, Hongkou, and Jiading districts (>0.9/100 000, P<0.05). Both FAB and WHO classification system were applied in the present study. Eighty-eight pts were diagnosed as L1 (26.2%), 193 L2 (57.4%), and 55 L3 (16.4%), For 302 pts with immunophenotyping data, 242 pts were identified as B cell orgin (80.1%), 59 T cell origin (19.5%), and 1 biphenotype (0.4%). The leukemia cells in 61 pts co-expressed one or two myeloid antigen (20.2%). For 269 pts with cytogenetics data, the incidence of t (9;22) in pts aged <10y, 11–17y, 18–44y, 45–59y and ≥60y were 4.2%, 11.4%, 19.2%,23.1% and 5.3%, respectively. For 473 evaluable pts, the complete remission (CR) rate and 3 year OS was 72.4% and (34.2±2.3)%, respectively. Both CR rates and 3 year OS decreased with age, 88.8% and (75.9±4.1%) in pts aged≤10, 87.3% and (36.8±6.8)% in 11–17y, 78.0% and (27.2±4.5)% in 18–44y, 73.5% and (16.1±4.7)% in 45–59y and 35.1% and (2.7±1.8)% in ≥60y (P<0.01). In this cohort of pts, only 26 received allogeneic hematopoietic stem cell transplantation (Allo-HSCT) in CR1. The probability of 3-year OS was 59.9±9.4%, better than those treated with conventional chemotherapy (36.3±8.0%) or auto-HSCT (33.3±17.2%, P<0.05). The age, WBC count at diagnosis, cytogenetic changes and donor availability are the factors influencing the prognosis.
Clofarabine in Combination with Etoposide and Cyclophosphamide (CLOVE) in Pediatric Patients with Relapsed Acute Leukemia: 4 Italian Pediatric AIEOP Centers’ Experience
Authors: Micalizzi Concetta , Parasole Rosanna , Putti Maria Caterina , Luciani Matteo , Banov Laura , Petruzziello Fara , Messina Chiara , De Rossi Giulio
Published: April 2020

Clofarabine is a new promising chemotherapic agent active in the treatment of pediatric acute leukemia. We planned to utilize the same drugs’ combination currently evaluated in USA as phase II study salvage therapy: clofarabine (40mg/m2/day) in combination with etoposide (100mg/m2/day) and Cyclophosphamide (440mg/m2/day) (CLOVE). We planned to treat the patients (pts) (aged 1–18) with relapsed or refractory acute lymphoblastic leukemia (ALL) or acute myeloblastic leukemia (AML) with 1 or 2 induction cycles (5 days chemotherapy) to reach complete remission (CR) or complete remission without platelet recovery (CRp) and with 1 or 2 consolidation cycles (4 days chemotherapy). ALL patients (pts) received intrathecal methotrexate at day 6 and AML patients intrathecal cytarabine at Day 0. Prednisone was added (0,5 mg/Kg/day) to prevent systemic inflammatory response syndrome (SIRS). Fourteen children (5 AML, 9 ALL) were treated, median age 5,7 years (range 1,75–16,5), 7 pts in first, 3 pts in second and 4 pts in third relapse. Four AML pts had one prior bone marrow transplantation (BMT): 1 autologous, 1 matched familiar donor and 2 matched unrelated donor (MUD) transplants. Five ALL pts had one prior BMT : 2 autologous, 1 syngenic and 2 MUD. The AML pts were in first relapse, and three of them were refractory to FLAG-X protocol: 3/5 (60%) achieved remission (1CRp, 2 CR), response data are currently pending for1 patient (day + 20 first induction), and 1 patient died for disease progression. The remission rate of nine ALL pts was 66% (4 CR, 2CRp). Infection grade 3 was observed in one patient, diarrhea and abdominal pain occurred in 4 pts. Febrile neutropenia was e common toxicity. Severe muscles pain with functional impairment was observed in two pts (1 AML, 1 ALL) 7 days post induction cycle: the gabapentin therapy in association with prednisone was efficient and the symptom quickly disappeared. One ALL patient refractory to first induction cycle, died after second cycle (day + 6) for cardiac failure. One ALL patient relapsed after CLOVE induction, after two more cycles he obtained partial response (BM Blast cells 10% ), and he underwent MUD transplantation and died for infection. The overall survival is 58 %, 7/9 (77 %) responders are alive. The median survival is 3 months (range 20 days –10 months). Four out 9 responding patients proceeded to BMT from unrelated donor, and three of them are alive and in CR. No unexpected transplant related toxicity was encountered. We confirm that the association of Clofarabine with Etoposide and Cyclophosfamide is active in heavily pretreated relapsed or refractory pediatric acute leukemia. patients.
Long-Term Monitoring of Patients with Hematological Malignancies and Hepatitis B and Hepatitis C Virus Infections
Authors: Garmaeva Tatyana , Kulikov Sergei , Michailova Elena , Sudarikov Andrey , Filatov Felix , Savchenko Valeryi
Published: April 2020

INTRODUCTION. Patients (pts) with hematological malignancies are at high risk of infection by Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) due to multiple transfusions and the large number of invasive procedures. The aim of the study was to evaluate the presentation rate of clinical hepatitis, pt survival and how hepatitis infection influenced survival.

RESULTS. The study group was comprised of all pts admitted to the hematology department from Feb 2004 to June 2006. The pts were followed until June 2008. All pts were monitored by testing of HBsAg, anti-HCV, DNA-HBV, RNA-HCV, HBeAg, anti-HBs, anti-HBc, anti-HBe approximately every 3 weeks. Liver biopsy was performed on 64 pts, 23 pts each with HBV and HCV immunohistology. A total of 7800 biological samples were collected for HBV and HCV testing; of these, 4000 were tested by PCR methods. Acute leukemias (AL) and aplastic anemias (AA) constituted 77% (205/265) of all pts. The median age was 38 years (range, 15 to 79), Male; 47% (n=125), female; 53% (n=140). Median transfusion load (the number of donors per pt for the study period) was 45 (range, 0 to 418). When patients new to the hematology dept were examined: HBV was detected in 15% (39/265) of new pts and HCV in 7% (19/265) of new pts respectively. We postulated that the rate of infection is extremely high in the hematology clinic. For the entire study period from Feb 2004 to Jun 2006, 51% (135/265) pts had positive markers for HBV infection, 19% (51/265) pts had positive markers for HCV, and 14% (37/265) pts had positive markers for both HBV+HCV. We have shown that up to 95% of HCV-positive pts and up to 60% of HBV-positive pts developed clinical and biochemical symptoms and signs of viral hepatitis with 3 years of initial detection of HBV and HCV markers. 154 (58%) pts survived, 111 (42%) pts died. Of these, 2 pts developed fulminant liver failure due to severe hepatitis B. The analysis of survival risk factors demonstrates that the expected life duration significantly decreases after HBV infection. For AL pts RR=1.8 (p=0,034), for AA pts RR=4.3 (p=0,022). There was no significant association between the expected life duration and HCV infection. Proportional hazard regression model with time dependent covariates (PHREG SAS) was used for the analysis.

CONCLUSION. The majority of pts infected by HBV and HCV developed clinically recognizable viral hepatitis within 3 years from the first detection of viral markers. Pts with severe immunosupression often do not manifest obvious features of acute viral hepatitis. Pts with hematological malignances should be monitored regularly (at least once in a month) for HBV and HCV markers during all period of treatment and for 1–2 years after completion of therapy. Viral hepatitis B is a risk factor associated with survival risk factor for AL and AA pts.
An Unusual Presentation of Acute Biphenotypic Leukemia without Bone Marrow Involvement
Authors: Bayraktar Ulas D , Escalon Maricer
Published: April 2020

Acute biphenotypic leukemia (ABL) represents a minority of acute leukemia cases (around 5%) in which blasts co-express markers of different lineages (myeloid, T-cell or B-cell). ABL without bone marrow involvement is exceedingly rare. Case report: An 18-year-old man presented with slowly enlarging, non-tender cervical lymph nodes. He had no other symptoms. Family history included Hodgkin’s disease in his mother and maternal great aunt, histiocytosis X in his maternal cousin, and non-Hodgkin’s lymphoma in his paternal grandfather. Physical exam revealed multiple small cervical and axillary lymph nodes. Laboratory analyses including complete blood count, peripheral blood smear, complete metabolic profile, mononucleosis screen, and hepatitis and HIV serologies were unremarkable. Histopathological examination of the left cervical lymph node revealed rare germinal centers with an interfollicular proliferation of generally medium size cells with a fine blastic chromatin pattern and scant cytoplasm. CD34 was positive in more than half of the cells. Between 30% and 80% of the cells were positive for TdT (Figure 1), CD3, CD4, and CD7. Myeloperoxidase (Figure 2) and lysozyme were positive in about 20% of the cells. Flow cytometry revealed an immature myeloid cell population coexpressing CD33, CD13, CD7, and CD34. Chromosomal analysis revealed a complex karyotype. Bone marrow exam was unremarkable and no evidence of leukemia was observed. Flow cytometry of the bone marrow aspirate revealed only 4% of the cells to be CD34+. Whole body PET scan showed extensive uptake in bilateral axillae, neck, and inguinal regions without any extranodal involvement. The patient received hyper-CVAD regimen (alternating cycles of cyclophosphamide, vincristine, adriamycin, dexamethasone and high dose methotrexate, cytarabine) for 8 cycles and remains in remission five months after the end of hyper-CVAD protocol. To our knowledge, this is the third case reported in the literature with acute biphenotypic leukemia not involving the bone marrow. However, our case cannot accurately be referred to as leukemia since the bone marrow is not involved; neither can it be named as lymphoblastic lymphoma because of its myeloid differentiation. Therefore, we will refer to it as extramedullary granulocytic sarcoma with biphenotypic features.

Figure 1 Figure 1. Figure 2 Figure 2.
Inhibition of Long-Term Expansion in a Subgroup of Acute Myeloid Leukemia Samples by Dasatinib
Authors: Han Lina , Schuringa Jan Jacob , Mulder Andre , Vellenga Edo
Published: April 2020

The Src family kinases are frequently constitutively activated in acute myeloid leukemia (AML) due to mutations in receptor tyrosine kinases including Flt3 and c-Kit or to the autocrine or paracrine production of growth factors. Recent studies have demonstrated that dasatinib is an effective inhibitor of Src kinase activity in chronic myeloid leukemia (CML) by blocking the Bcr/Abl-mediated Src activity resulting in inhibition of cellular proliferation. In the present study we questioned whether similar effects can be observed in AML cells exposed to dasatinib and whether differences can be observed in normal cord blood (CB) CD34+ cells and CML cells (K562 cell line). Firstly, we demonstrated that dasatinib was effective in blocking the proliferation of K562 cells at a concentration of 2–10 nM which coincided with a significant reduction in Src, Erk1/2 and Stat5 phosphorylation. Long-term MS5 cocultures initiated with CB CD34+ cells demonstrated a reduced expansion of 50% at 5 nM dasatinib (p=0.005, n=3) after 4 weeks of coculture which was associated with a reduction in progenitor output as determined by colony forming cell (CFC) assays (p=0.031). Moreover, the inhibitory effects of dasatinib were cytokine-specific, eg IL3 and TPO-induced proliferation of both CB and Mo7e cells were not inhibited by dasatinib, while SCF-mediated proliferation was significantly impaired (94% ± 4% inhibition, p<0.001) at 5 nM. These inhibitory effects were associated with a reduced SCF-induced phosphorylation of Erk1/2 and Stat5 in the presence of dasatinib. Subsequently, AML cells (n=19) were studied in the long-term stromal culture assays by using exclusively the sorted CD34+ cell fraction that is enriched for leukemic stem cells. In 79% (15/19) of the tested AML cases long-term expanding AML co-cultures could be generated. In 27% (4/15) of the AML cases a distinct reduction in cell proliferation of AML-CD34+ cells was observed as compared with the untreated group, ranging from 48%–91% inhibition at low concentration of dasatinib (0.5 nM). This concentration of dasatinib did not significantly affect expansion of normal progenitors in the long-term culture assays (36% ± 12% inhibition, p=0.15). The AMLs that responded to dasatinib were characterized by Bcr-Abl (n=1), c-Kit (n=1), Flt3-ITD (n=1) or undetermined (n=1) mutations. The additional Flt3-ITD+ AMLs (n=6) did not show a reduced expansion to dasatinib exposure. No difference in Src expression was observed between the responsive and non-responsive AMLs. In conclusion, our results demonstrate that dasatinib is effective in blocking SCF-mediating signaling in hematopoietic cells which is associated with reduced phosphorylation of Erk1/2 and Stat5, while in the malignant counterpart long-term expansion of a subgroup of AML CD34+ cells can be inhibited at low-doses.
Influence of Genetic Polymorphisms of VKORC1 and CYP2C9 in Patients on Phenprocoumon Steady-State Dose Requirements
Authors: Wu Weidong , Harenberg Job , Weiss Christel , Kirchheiner Julia , Stehle Simone , Fuhr Uwe , Gleiter Christoph
Published: April 2020

The variants of the (Vitamin-K epoxid reductase subcomplex 1) VKORC1 and of cytochrome P450 2C9 (CYP2C9) genes are know to influence the steady state dose of the vitamin-K antagonist (VKA) phenprocoumon during oral anticoagulant therapy. We have analysed in detail the influence of the VCORC1 1173CC and 1173CT variants in combination with the CYP2C9 variants on the steady state dose of phenprocoumon. The weekly phenprocoumon dosage was calculated after a 3 to 6 months of stable anticoagulation by the international normalized ratio (INR) which had to be in a range from 2 to 3 in 114 patients. VKORC1 1173CC, CT, TT and CYP2C9*1/*1, *1/*2, *1/*3, *2/*2, *2/*3 and *3/*3 genotypes were determined using direct DNA sequencing and realtime PCR. Patients carrying VKORC1 1173CT and CYP2C9*1/*1 genotypes required a 33% lower steady state dose of phenprocoumon compared to VKORC1 1173CC and CYP2C9*1/*1 genotypes (p=0.0001). Patients with VKORC1 1173CC in combination with CYP2C9*1/*2, *1/*3 needed 10% and 34% lower steady state dose of phenprocoumon compared to the combination with CYP2C9*1/*1, respectively (p=0.0001). Patients carrying VKORC1 1173CT needed lower doses of phenprocoumon in combination with CYP2C9*1/*1 (−32%), *1/*2 (−29%), or *1/*3 (−33%), and VKORC1 1173TT in combination with CYP2C9*1/*3 (−44%) compared to VKORC1 1173CC combined with CYP2C9*1/*1 (all p<0.001). CYP2C9 variants did not influence the dose of carriers of VKORC1 1173CT gene. Both genotypes together influenced the phenprocoumon maintenance dosage in this naturalistic study design. Genotyping for CYP2C9 and VCORC1 therefore might be a suitable tool for dose individualization in patients at oral anticoagulant drug therapy.
An Assessment of Surgical Thromboprophylaxis in a Tertiary Care Center
Authors: Robinson-Cohen Cassianne , Tagalakis Vicky , Dubois Marie-France , Pilon Danielle
Published: April 2020

Background. Venous thromboembolism (VTE) is a frequent surgical complication. The type of surgery and baseline patient VTE risk factors are important in defining postoperative VTE risk. The 2004 American College of Chest Physicians (ACCP) recommends implementation of pharmacologic thromboprophylaxis according to surgery type and VTE risk factors. We conducted a retrospective cohort study of surgical patients to determine adherence to the 2004 ACCP surgical thromboprophylaxis recommendations and to assess for predictors of non-adherence.

Methods. Using data from the administrative healthcare database CIRESSS (Centre Informatisé de Recherche Évaluative en Services en Soins de Santé), in patients admitted for surgery between January 1st and December 31st 2006 at the Centre Hospitalier Universitaire de Sherbrooke (CHUS), a tertiary care center in Sherbrooke, Quebec, Canada, we retrospectively assembled a cohort of all consecutively admitted surgical patients who met ACCP criteria for pharmacologic thromboprophylaxis. We assessed the proportion of patients who received in the 24 hours pre- and post-surgery a prescription for thromboprophylaxis. We used conditional logistic regression to determine clinical characteristics associated with an absent prescription. The incidence of objectively-defined symptomatic postoperative VTE was assessed at three months.

Results. Of 2286 surgical admissions that met ACCP recommendations for pharmacologic thromboprophylaxis, 1852 (81%) received thromboprophylaxis and 434 (19%) did not. Male sex (odds ratio (OR): 1.9, 95% confidence interval (CI) (1.4–2.4)), age <40 years (OR: 1.8, 95% CI (1.2–2.9)), absent varicose veins (OR: 3.2, 95% CI (1.1–10.0)), pregnancy within 3 months of surgery (OR: 8.5, 95% CI (3.5–20.8)), a moderate risk for post-operative VTE as per ACCP criteria (OR: 4.4, 95% CI (2.9–6.52)), non-major surgery (OR: 4.5, 95% CI (2.3–8.8)), short-duration hospitalization (2–3 days vs. 13 days or more) (OR: 15.3, 95% CI (9.6–24.4)), absence of active cancer (OR: 1.6, 95% CI (1.2–2.1)), absence of heart failure (OR: 1.8, 95% CI (1.1–2.8)), and non-orthopaedic surgery (OR: 28.2, 95% CI (15.9–49.9)) were associated with lack of pharmacologic thromboprophylaxis. At three months following surgery, 16 patients (0.7%) developed VTE, and the adjusted relative risk of VTE in patients without thromboprophylaxis was 2.1 (95% CI:0.5–7.9).

Conclusions. Though surgical thromboprophylaxis was observed in 81% of surgical patients, 19% of patients who met criteria for ACCP thromboprophylaxis did not receive prophylaxis. Targeted recommendations in particular toward pregnant women undergoing non-obstetrical surgery, patients with short duration hospitalization, and patients undergoing non-orthopaedic surgery may ameliorate thromboprophylaxis compliance rates, which in turn may impact on post-operative VTE risk.
Anticoagulant Effects of the Low-Molecular-Weight Heparin Certoparin in Combination with Phenprocoumon in Patients with Recent Atrial Fibrillation Undergoing Cardioversion
Authors: Harenberg Job , Wierdack Jolanta , Weiss Christel , Giese Christina , Abletshauser Claudia , Tebbe Ulrich
Published: April 2020

Patients with recent onset of atrial fibrillation (AF) receive anticoagulation with low-molecular-weight heparin (LMWH) and coumarin targeted at an INR of 2 to 3. Recently, LMWH is given alone before cardioversion followed by coumarin. At present, no method is available to determine the combined effect of LMWH and coumarin in order to get information on the in vivo anticoagulant status of these patients. Patients with recent onset of AF (n=200) received 8.000 IU LMWH certoparin bid upon entry into the study. Phenprocoumon was started after electrical cardioversion. LMWH was stopped after 2 days within therapeutic INR range of phenprocoumon. The clincial data have been published (Clin Res Cardiol 2008). Blood samples were drawn from patients before LMWH administration (visit 1, V1), before start of phenprocoumon (V4) and after reaching the desired INR range and before stopping LMWH (V5) to determine a set of anticoagulant parameters. The study was accepted by the local ethics committees and patients gave written informed consent. Activated partial thromboplastin time, chromogenic factor Xa assay, heptest time values, prothrombinase-induced clotting time and tissue factor pathway inhibitor demonstrated the anticoagulant effect of certoparin at V4 and V5. Results of these parameters differed between V1 and V4 as well as between V1 and V5 (all p<0.001) but not between V4 and V5. Prothrombin time was prolonged at V5 versus V1 and V4 (both p<0.001). D-Dimer and prothrombinfragment F1+2 significantly decreased from V1 to V4 and from V4 to V5 (all p<0.001). Endogenous thrombin potential assay (ETP) was inhibited stronger at V4 compared to V1, and at V5 compared to V4 (both p<0.001). D-Dimer, F1+2 and ETP are the parameters reflecting the combined anticoagulant effect of certoparin and phenprocoumon while overlapping anticoagulation with these compounds in patients with recent AF for electrical cardioversion. The clinical effects on thromboembolic or bleeding incidences remain to be determined.
Favorable Immunologic Response Profile to Recombinant Thrombin Application in Surgery: An Integrated Analysis of 8 Clinical Trials
Authors: Chapman William , Renkens Kenneth , Gamelli Richard , Randleman Carlton , Ballard Jeffrey , Alexander W. Allan
Published: April 2020

Recombinant thrombin (rThrombin) is an active topical hemostat approved by the FDA as an aid to hemostasis. Treatment with thrombin derived from bovine sources has occasionally been associated with adverse events which appear to be related to the formation of antibodies against bovine thrombin and/or Factor V. The safety and immunogenicity of rThrombin have been evaluated in 8 clinical trials: 1 Phase 1 study, 5 Phase 2 studies, 1 randomized, double-blind Phase 3 study, and 1 open-label, single-group Phase 3b study. Pooled results from these trials provide a consistent and clinically relevant summary of safety information on the use of rThrombin in surgery. These trials utilized standardized data collection methods; more than 100 investigators from over 60 institutions participated. With 514 subjects treated with rThrombin in clinical trials to date (cut-off date July 2008), these results represent the largest safety database available for any commercially-available thrombin product. Standard safety measures were collected prior to surgery (baseline) through Day 29 post-surgery. Immunogenicity was evaluated at baseline and Day 29 using enzyme-linked immunosorbent assays and a time-to-clot assay to measure neutralizing potential of any detected anti-rThrombin antibodies. Of the subjects for whom data are currently available (N=514), 54% were male and 46% were female. Mean age was 57.0 years (SD, 15.7; range, 12–89 years). The following surgery types were represented: spine, 32% of patients; liver, 14%; peripheral arterial bypass, 23%; arteriovenous graft, 18%; burn, 12%. The median dose of rThrombin was 10,000 IU (range, 1000–50,000 IU). Topical application of rThrombin was well tolerated by study subjects. Common adverse events (AEs; reported by ≥10% of subjects) were similar regardless of surgery type and were consistent with the post-surgical setting. Common AEs included incision site complication, procedural pain, nausea, constipation, pyrexia, anemia, vomiting, insomnia, incision site pain, and pruritus. No single serious adverse event type occurred in >1% of patients. Twelve of 514 subjects (2.3%) had anti-rThrombin product antibodies at baseline, a finding that is consistent with reports of antibodies to other recombinant forms of endogenous proteins. None of the 12 subjects with anti-rThrombin product antibodies at baseline had a change in titer ≥1.0 unit at Day 29. Among subjects who did not have anti-rThrombin product antibodies at baseline, 5/502 (1.0%) had detectable antibodies at Day 29. In total, 5/514 subjects (1.0%) were antibody positive at Day 29. None of the antibodies neutralized native human thrombin. These clinical studies, enrolling 514 subjects to date, collectively demonstrate that rThrombin is well-tolerated and has a favorable immunologic response profile when used in numerous surgical settings.
The Overall Haemostatic Potential (OHP) Assay Detects Abnormalities in Fibrinolytic Factors
Authors: Curnow Jennifer L , Morel-Kopp Marie-Christine , Ward Christopher
Published: April 2020

No routine assays are available which quantify abnormalities in the fibrinolytic system. The Overall Haemostatic Potential (OHP) assay is a global assay of fibrin generation and fibrinolysis. Our aim in this study was to assess changes in fibrinolysis measured by the OHP assay in samples with variable levels of the different components of the fibrinolytic system: plasminogen, a2-antiplasmin, PAI-1 and TAFI. Plasmas deficient in these factors were obtained commercially. Samples were spiked with Lys-plasminogen, Glu-plasminogen, PAI-1, TAFI or pooled normal plasma to produce serial increases in these factors. Potato tuberose carboxypeptidase inhibitor (PTCI) was also used to inhibit TAFI in PNP and patient samples. OHP assays were performed utilizing either thrombin (0.03 IU/ml) or tissue factor (5 pM) to trigger fibrin generation, and rt-PA (350 ng/ml) to trigger fibrinolysis. Data analysis involved calculation of Spearman correlations for OHP assay parameters and concentrations of the various fibrinolytic factors tested. The OHP assay demonstrated markedly reduced fibrinolysis in plasminogen deficient plasmas, which normalised when plasminogen levels were increased to the normal range. At elevated Lys-plasminogen levels fibrin generation was impaired. Overall fibrinolytic potential was mildly increased in a2-antiplasmin deficiency. PAI-1 depleted plasma had mildly increased fibrinolysis compared with PNP but with addition of PAI-1, to concentrations of 300 ng/ml or higher, fibrinolysis was markedly impaired. TAFI deficient plasma had reduced fibrin generation but no change in fibrinolysis was seen in spiked samples, even with elevated TAFI levels. Representative hypofibrinolytic patient samples showed normalisation of fibrinolysis with TAFI inhibition by PTCI.

The OHP assay shows appropriate changes in OFP with variations in the concentrations of activators and inhibitors of the fibrinolytic system. The assay provides a simple means of quantifying overall fibrinolysis and is sensitive to abnormalities in individual components of the fibrinolytic system. It should be useful for investigating fibrinolytic abnormalities in patients, where no routine assay is currently available.
Biological Activity of the First Epidermal Growth Factor-Like Domain of Rat Coagulation Factor VII
Authors: Mei Heng , Hu Yu , Wang Huafang , Zhang Xiaoping , Guo Tao , Huang Shiang
Published: April 2020

Abstract: The first epidermal growth factor-like (EGF1) domain of coagulation factorVII (FVII) is essential for binding with tissue factor (TF) in human. We hypothesized that the function of rat FVII (rFVII) EGF1 domain might be the same as the human being’s. The clone and expression of rFVII EGF1 domain would be helpful in studying the anticoagulant drugs based on FVII/TF interaction. Accordingly, the model of rat TF expression in vitro was established by lipopolysaccharide(LPS) induction. We amplified the EGF1 domain from a rat liver by reverse transcriptase PCR (RT-PCR), and then a fusion expression vector named pET28a-EGFP-EGF1 was constructed. After expression in Ecoli BL21 cells, the EGFP-EGF1 fusion proteins were analyzed for binding affinity to rat TF by confocal microscope and flow cytometry. Prothrombin time assay was employed to test biological activity of the fusion protein by using FVII depleted human plasma. The resulting plasmid expressed fusion protein EGFP-EGF1 in the soluble form in E.coli BL21, and the recombinant protein was purified successfully with a major band at 36KD in SDS-PAGE(Figure 1). The purified EGFP-EGF1 fusion proteins could definitely depress recombinant mouse FVII((rmFVII) binding with rat TF(rTF)(70.06%VS 53.80%, P< 0.01)(Figure 2 &3). However, the proteins lost the activity of coagulation when compared to that of blood serum of normal SD rats (56.8sVS 17.8s, P< 0.01). (Table 1)We conclude that the rat EGF1 region could specifically bind to TF without the ability of coagulation, and it might facilitate the development of molecular target study in anti-thrombosis treatment.

Table 1. Prothrombin Time test of Fusion proteins

Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Figure 4 Figure 4.
Combined Coagulation Factor XI and Factor XII Deficiency in An Asymptomatic Female
Authors: Herrada Juan , Adcock Dorothy M , Casillas Arturo , Jobe Jeff T. , Noble Luis S. , Landeros Mark M.
Published: April 2020

Background: Multiple combined coagulation factor deficiencies have been reported, although the genetic defects associated with these conditions remain largely unknown. We present an asymptomatic female with a persistent deficiency of both coagulation Factor XI (FXI) and Factor XII (FXII). To our knowledge, this particular combination of coagulation factor deficiency has not been previously reported.

Case Report: In February 2004 a healthy 33 year-old white female without personal or family medical history presented with an elevated partial thromboplastin time (PTT) incidentally found during a routine pre-operative evaluation. Her prothrombin time (PT) was normal. Her PTT was 45 seconds (reference interval 23–37 seconds), and normalized with 1:1 mixing studies of patient and normal plasma. Preliminary laboratory evaluation included negative tests for the presence of a Lupus Anticoagulant, a normal serum albumin level, and a normal urinalysis. Additional laboratory testing of coagulation and included FXI and XII activities, measured by means of a one stage APTT-based clotting assay using APTT reagent (Automated APTT, Trinity Biotech, Bray Ireland), congenitally depleted deficient plasma (HRF, Inc, Raleigh NC) using the MDA II Trinity Biotech, Bray Ireland). FXI was 46% (reference range 60–150) and FXII was 42% (reference range 50–150). In March 2004, a repeated the laboratory evaluation showed a FXI of 41% and a FXII of 46%. In April 2006, FXI was 49%, and FXII was 40%. Is to be noted that the patient has never had a documented thromboembolic episode or excessive bleeding, despite the exposure, respectively, to estrogen supplementation for seven years, to a full-term pregnancy, and to an abdominal surgical event.

Conclusion: Although modern laboratory methods are able to identify novel combinations of coagulation defects, their clinical significance requires additional investigation.
Nramp1 Promotes Efficient Macrophage Recycling of Iron Following Phenylhydrazine-Induced Hemolytic Anemia
Authors: Soe-Lin Shan , Andriopoulos Bill , Andrews Marc , Schranzhofer Matthias , Kahawita Tanya , Santos Daniel Garcia , Azad Nabila , Apte Sameer Sadashiv , Ponka Prem
Published: April 2020

Natural resistance-associated macrophage protein 1 (Nramp1) is a divalent metal transporter expressed exclusively in phagocytic cells such as macrophages and neutrophils. Based on our earlier in vitro study (Soe-Lin et al. Exp Hematol.2008;36:929–937), we hypothesize that Nramp1 may participate in the recycling of iron acquired through the phagocytosis of senescent red blood cells by macrophages. In order to examine the effect of Nramp1 on iron recycling in vivo, the iron parameters of wildtype (Nramp1+/+) and Nramp1 knockout mice (Nramp1−/−) were analyzed following both acute and chronic induction of hemolytic anemia by phenylhydrazine treatment. We observe that untreated Nramp1−/− mice exhibited greater serum transferrin saturation and splenic iron content, higher duodenal ferroportin (Fpn) and divalent metal transporter 1 (DMT1) expression, and dramatically lower hepcidin mRNA levels than untreated Nramp1+/+ mice. Significant iron loading of the reticuloendothelial organs was found to increase with age in knockout mice. Following acute treatment with the hemolytic agent phenylhydrazine, Nramp1−/− mice experienced a significant decrease in serum iron levels and hematocrit, while their Nramp1+/+ counterparts were relatively unaffected. Following a month-long phenylhydrazine regimen, Nramp1−/− mice retained markedly increased quantities of iron within the liver and spleen, and exhibited greater splenomegaly and reticulocytosis than wild-type mice. Furthermore, while hepcidin mRNA levels decreased following chronic phenylhydrazine treatment in both Nramp1+/+ and Nramp1−/− mice, this effect was significantly more pronounced in Nramp1−/− mice. The data presented in this report suggest that in the absence of Nramp1, iron accumulates to a greater degree within reticuloendothelial organs such as the liver and spleen following acute and chronic hemolytic anemia. We hypothesize that the low hepcidin mRNA levels seen in Nramp1−/− mice are a response to a diminished availability of iron for erythropoiesis resulting from the aberrant increase in iron retention within their splenic reticuloendothelial macrophages. Our observation of increased DMT1 and ferroportin within the duodenums of the Nramp1−/− animals imply that the increase in transferrin saturation despite the impaired iron release from erythrophagocytosing macrophages occurs due to a compensatory increase in iron absorption from the diet. These findings are consistent with our hypothesis that Nramp1 promotes the efficient recycling of iron in erythrophagocytosing macrophages.
The Use of Tetradenoylphorbol Acetate Stimulated Peripheral Blood Cells for Interphase Fluorescence In-Situ Hybridization Analysis May Enhance Its Prognostic Value for Patients with Chronic Lymphocytic Leukemia
Authors: Delgado Julio , Aventin Anna , Briones Javier , Sanchez Jana , Nomdedeu Josep , Sierra Jorge
Published: April 2020

Chronic lymphocytic leukemia (CLL) is a mature B-cell malignancy characterized by a variable clinical course. Genomic aberrations, as identified by interphase fluorescent in situ hybridization (I-FISH), have a remarkable predictive power in terms of response to therapy, time to first treatment and overall survival of these patients. I-FISH studies can be performed either on unstimulated peripheral blood mononuclear cells (I-FISH-PBMC) or on tetradecanoylphorbol acetate (TPA) stimulated peripheral blood cells (I-FISH-TPA). We have previously observed that I-FISH-TPA could identify genomic abnormaties that might be overlooked with I-FISH-PBMC.

The aim of the study was to evaluate whether this increased detection of genomic abnormalities, as identified by I-FISH-TPA, was clinically relevant for a group of consecutive CLL patients.

Blood samples from 47 CLL patients were stimulated with TPA and cultured in RPMI medium supplemented with fetal calf serum. Peripheral blood mononuclear cells were isolated using density-gradient centrifugation and fixed according to standard methods. I-FISH was performed on both TPA stimulated and unstimulated cells using 11q22.3 (ATM), 13q14 (13S272), 17p13.1 (p53), and centromeric 12 (D12Z3) probes. 200 nuclei were evaluated for each probe, and cut-off points were set at 6%, 7%, 5% and 2% for del(11q), del(13q), del(17p) and trisomy 12, respectively. Metaphase FISH and conventional cytogenetics were also performed in selected cases. For all patients, chemotherapy was initiated according to Cheson criteria and treatment-free and overall survival curves were plotted using SPSS software.

Following a modified version of Dohner’s hierarchical model, patients were divided in those with del(17p) and/or del(11q) and those with other or no genomic abnormalities. Fourteen cases with negative or bordeline results with I-FISH-PBMC became positive with I-FISH-TPA for del(11q) (2 cases), del(13q) (9 cases) and trisomy 12 (3 cases). In all but one patient, either conventional karyotyping or metaphase FISH confirmed these abnormalities. I-FISH-TPA provided a better prediction of treatment-free interval compared to I-FISH-PBMC (P= 0.002 vs 0.019, see Figures 1 and 2). In particular, two patients with no cytogenetic abnormalities detected by I-FISH-PBMC required chemotherapy 3 and 11 months after diagnosis, more in keeping with the presence of del(11q) found using I-FISHTPA. Furthermore, I-FISH-TPA also improved the overall survival prediction compared to I-FISH-PBMC (P= 0.036 vs 0.042).

In summary, I-FISH-TPA increased the detection rate and had an improved prognostic value compared to I-FISH-PBMC. Further studies with larger numbers of patients are warranted.

Figure Figure Figure Figure
PKCβ Is Essential for the Development of CLL in the TCL1 Transgenic Mouse Model: Validation of PKCβ as a Therapeutic Target in CLL
Authors: Egle Alexander , Holler Claudia , Pinon Josefina D. , Denk Ursula , Heyder Christoph , Greil Richard
Published: April 2020

The development and the propagation of chronic lymphocytic leukemia (CLL) has been linked to signalling via the B-cell receptor (BCR). Protein kinase C (PKC) beta is an essential signalling element of the BCR and was recently shown to be overexpressed in human CLL. We used the TCL1 transgenic mouse model to directly target PKC beta in the development of murine CLL. While loss of PKC beta in wild type mice leads to a complete loss of CD5+ B cells in these mice, the targeted disruption of the PKC beta gene in Tcl1 transgenic mice did not abrogate the accumulation of CD5 positive B cells in the pretumor phase in these mice. This suggests that the basic survival signal transmitted by PKC beta in B1 cells can be substituted by the augmented Akt signalling in the Tcl1 transgenic cells. Yet despite this, PKC beta deficient TCL1 transgenic mice did not develop a CLL disease, suggesting a role of PKC beta in the establishment of the malignant clone. In fact PKC beta is likely to be essential for transformation in limiting the BCR signal via a negative feedback loop, suggesting that BCR signalling strength is an important component of the transformation process. In order to provide support for the potential of PKC beta as a drug target in CLL we targeted PKC beta with the specific inhibitor Enzastaurin. This led to killing of human CLL samples in vitro and this was independent of risk factors in the samples analysed. We thus propose that PKC beta may be a relevant target for the treatment of CLL.
Reactive Oxygen Species Are Differentially Regulated in Chronic Myeloid Leukemia Versus Philadelphia Negative Myeloproliferative Neoplasms
Authors: Guerin Estelle , Belloc Francis , Etienne Gabriel , Duffau Pierre , Mahon Francois-Xavier , Lippert Eric
Published: April 2020

Deregulation of tyrosine-kinases is a characteristic of most Myeloproliferative Neoplasms (MPN); evolution from chronic phase to acute leukemia depends on the acquisition of additional mutations. Reactive Oxygen Species (ROS), the production of which is increased by tyrosine-kinase activation, can be responsible for additional mutations. The role of ROS in generating genetic aberrations has been mainly studied in BCR-ABL-positive cell lines. Little is known of ROS metabolism in primary cells from CML or Philadelphia-negative MPN (Ph-MPN). After informed consent, cells from blood or bone marrow were obtained from patients diagnosed with CML (12 bone marrow (BM), 8 peripheral blood (PB)), or Ph-MPN (4 Polycythemia Vera, 6 Essential Thrombocythemia, 3 Primary Myelofibroses, 2 atypical CML) and from healthy donors (bone marrow donors) or patients devoid of hematological disease undergoing thoracotomy. Cells were incubated with DCFDA, a fluorogenic marker of ROS production, labelled with an anti-CD45 antibody, stimulated with either the oxidant hydrogen peroxide (H2O2) or the PKC activator Phorbol Myristate Acetate (PMA), and analysed for ROS production by flow cytometry. CD45/SSC gating allowed separate analysis of granulocytes, monocytes or lymphocytes. The basal level of ROS was not higher in CML cells as compared to normal BM or PB leukocytes. It was even significantly lower in CML lymphocytes, either from the BM (2.35 Arbitrary Units vs 8.3 AU, p=5.5 10−5) or PB (2.47 AU vs 7.4 AU, p=3.10−5) and in CML granulocytes from peripheral blood (14 AU vs 45 AU, p =10 −5), but not bone marrow. The ROS levels of Ph-MPN cells were similar or slightly higher than control cells. Upon H2O2 stimulation however, ROS production increased significantly more in CML cells as compared to normal cells (6 fold increase), whatever the cell type (granulocytes, monocytes and lymphocytes) or their origin (PB or BM). In contrast, for Ph-MPN cells, H2O2-stimulated ROS production was close to that of normal cells, with only BM lymphocytes showing ROS generation four fold higher than control BM lymphocytes. After PMA stimulation, which yielded a more modest ROS production than H2O2, CML cells behaved similarly to normal cells, whereas ROS production was four fold higher in Ph-MPN cells, whatever their type and origin. In conclusion, ROS levels at the basal stage are not higher in MPN cells, whether they are Philadelphia positive or negative, as compared to normal cells. Various kinds of stimulation induce different patterns of response, CML cells being more sensitive to oxidants whereas Ph-MPN cells respond more to the cytokine-mimicking agent PMA. These results suggest that the mechanisms of ROS generation and thus of genetic instability are different in CML and Ph-MPN.
Chronic Myeloid Leukemia Patients Present Elevation of the Circulating Endothelial Cells
Authors: Godoy Carla Rosa Teixeira , Meireles Ana Luiza Langanke Pedroso , Brocardo Graciela Aparecida , Chamone Dalton , Pereira Juliana
Published: April 2020

The increase of the angiogenesis is a prognostic factor in solid tumors. In oncho-hemathology, it is associated to the Myelodysplastic syndromes, Acute Leukaemia and Multiple myeloma. The impact of the raise in the expression of the growing endothelial (VEGF) in the course of chronic myeloid leukaemia (CML) is yet unknown. Yet, there are reports that those patients have presented a higher vascular density in bone marrow (BM) than healthy individuals, specially in blastic phase. Some authors suggest that abnormalities in the angiogenesis may be involved in the phatogenesis in the CML, as well as to its transformation in blastic phase. The increase of the circulating endothelial cells (CEC) is associated to the phatogenesis and to the prognostic of breast cancer and Non-Hodgkin lymphoma, but there are no studies in CML. Thus, we have elaborated a study in order to quantify the CEC in patients with CML in accelerated, chronic and blast phases for flow cytometry. Up to the moment, samples of 46 patients with CML (15 in accelerated phase, 24 in chronic phase and 7 in blastic phase) have been evaluated. Thirty six platelet donors for apheresis with negative sorology were used as a control group. The samples were processed at the same day of the collection, using monoclonal antibodies (MoAb) previously validated in cell of lineage HUV-EC. For the research of the CEC, we used the MoAb CD146 FITC, CD34 PE, CD133 APC e CD62E PE. O AcMo CD45 PC5 was used as a gate strategy for the analysis. The samples were acquired in flow cytometry FACS-Calibur G5 (100.000 events) and analysed in software CellQuest Pro. We noticed an increase statistically significant of CEC in the chronic (p< 0,01), accelerated (p = 0,02) and blastic (p = 0,01) phases of the CML in comparison to the control group, given that these endothelial cells were found in a mature stage. The precursor endothelial cells did not present any statistically significant difference. Patients with LMC present an increment in the amount of CEC in all the phases. The quantification of these cells can be used in the evaluation of therapeutic response, of prognostics and as potential therapeutic target in LMC. The mechanisms involved in the increase of the CEC must be evaluated in future studies. Financial Support: FAPESP
Atypical Chronic Myeloid Leukemia: An Analysis of 9 Cases
Authors: Ma Yan , Xu Xiaoping , Wang Xiaoqin , Chen Bobin , Lin Guowei
Published: April 2020

Objective FTo investigate the clinical characteristics of atypical chronic myeloid leukemia (aCML) and characterize the aCML.

Methods FFrom 2003 to 2007, the consecutive samples of 54 MDS/MPD patients in Sino-US Shanghai Leukemia Cooperative Group were collected by prospective methods and diagnosed with WHO classification. We identified 9 aCML cases and analyzed the clinical and laboratory data of these cases.

Results FThe median age of 9 cases was 66 years old(range 38~78y). The ratio of male and female is 1:1.25. Median hemoglobin concentration was 76.5g/L(range 43.3~116), median white blood cell count 27.5×109/L(range 7.55 ~317.2), median neutrophil percentage 66.4%(range 43.3~83.5), median basophil percentage 2.86%(range 0.24~5.79), median monocyte percentage 7.59%(range 0.92~16.3), median platelet 77.6×109/L(range 20.7 ~504)and median marrow blast percentage 2.5 %(range 0.5~6). Abnormal karyotype occurred in 20% of all patients. Median survival was 11.5 months(2.5 `44months).

Conclusions FaCML occurs rarely and is associated with a poor prognosis. The major distinguishing features of aCML are Ph(−)or BCR/ABL(−)and severe dysgranulopoiesis. In addition, hepatosplenomegaly and leukocytosis are less prominent in aCML than chronic myeloid leukemia(CML) and chronic myelomonocytic leukemia (CMML). Anemia is much severe than CML and CMML.