bluuebunny/crossref_metadata_2025 huggingface dataset:
Randomized Multicenter Trial of Cladribine Alone (C) or in Combination with Cyclophosphamide (CC), and COP in Previously Untreated Low Grade B-Cell Non-Hodgkin Lymphoma Patients: The First Interim Analysis.
Authors: Kalinka Ewa E. , Wajs Jaroslaw J. , Sulek Kazimierz , Robak Tadeusz , Blasinska-Morawiec Maria , Holowiecki Jerzy , Sawczuk-Chabin Joanna , Ceglarek Bernadeta , Konopka Lech , Mazur Grzegorz , Federowicz Irena , Jerdrzejczak Wieslaw , Watek Marzena , Kisiel Elzbieta , Meder Janusz , Centkowski Piotr , Seferynska Ilona , Czyz Jaroslaw , Warzocha Krzysztof ,   for the Polish Lymphoma Research Group
Published: October 2019

To comparatively assess first-line treatment with cladribine alone (C) or in combination with cyclophosphamide (CC), and COP (cyclophosphamide, vincristine, prednisone) in low grade B-cell non-Hodgkin lymphoma (NHL), previously untreated patients (pts) with Ann Arbor stage II–IV were randomly allocated to receive 6 monthly courses of either C, CC, or COP. End points were treatment response, freedom from progression (FFP) and overall survival (OS), and tolerance. From June 1, 2000, 165 pts were randomized in 17 centers. The first interim analysis performed at July 1, 2004 included 105 out of 165 randomized pts (63.6%) who have completed at least six cycles of the scheduled chemotherapy. Of 105 analyzed pts, 38 (36.2%) were diagnosed as small lymphocytic, lymphoplasmocytoid 8 (7.6%), marginal-zone 22 (21%), follicular 33 (31.4%), and not otherwise specified low grade B-cell NHL 4 (3.8%). Randomization constituted comparable groups, including international Prognostic Index variables. Compared to C and CC, COP induced lower overall response rates (75%, 85%, 51%, χ2 test p<.005), including lower complete remission rates (43%, 62.5%, 5.5%, χ2 test p<.001). With a median follow-up of 10 months, median FFP was superior in patients receiving cladribine containing regimens (8 versus 11 versus 6 months, respectively, χ2= 15.1, log-rank test p<.001). No difference in median OS was detected (9 versus 12 versus 7 months, respectively, χ2= 1.15, log-rank test p=.56). Incidences of infections (7% versus 15% versus 11%) and non-hematological side effects (7% versus 7.5% versus 16%) were similar in the randomize groups, whereas CC but not C induced more frequent cytopenias compared to COP (30% versus 11%, χ2 test p<.05). This resulted in more frequent intervals’ prolongation between CC versus COP cycles (respectively 32.5% and 11%, χ2 test p<.05) but dose reductions because of hematological or other toxicity were comparable in C, CC, and COP groups (11% versus 17.5% versus 5.5%). In pts with low grade B-cell NHL, first line C and CC regimens provided similar response rates, FFP, and OS, which were superior to those obtained with COP. The first interim analysis has resulted in discontinuation of accrual in the COP arm. An observed trend toward a better tolerance of C over CC, which may influence the choice between these regimens as front-line treatments, warrants larger number of pts and longer follow-up.
Effects of a New Pathogen-Reduction Technology (Mirasol PRT) on Platelet Function.
Authors: Diaz-Ricart Maribel , Perez-Pujol Silvia , Tonda Raul , Lozano Miguel , Fuste Berta , Lopez-Vilchez Irene , Galan Ana M. , Li Junzhi , Goodrich Raymond , Escolar Gines
Published: October 2019

Several strategies for pathogen reduction are being developed to reduce the risk of pathogen transmission associated with platelet transfusion. There is concern that these techniques could have a deleterious effect on platelet integrity and functions. We evaluated the impact of a new technology for pathogen reduction based on riboflavin + illumination at 6.2 and 12.3 J/ml (Mirasol PRT) on functional and biochemical characteristics of platelets. Platelet concentrates (PCs) obtained by apheresis (n=8) were treated with Mirasol PRT and then stored at 220 C. Modifications in major platelet glycoproteins (GPIb, GPIV, and GP IIbIIIa), adhesive ligands (vWF, Fg, and Fn), activation dependent antigens (P-selectin and LIMP), and apoptotic markers (Annexin V binding and factor Va) were analyzed by flow cytometry. Adhesive and cohesive functions of platelets in the concentrates were evaluated using well-established models with circulating human blood. Studies were performed the preparation day (day 0) and along storage (days 3 and 5) of PCs. Aliquots were taken for aerobic and anaerobic microbiologic cultures. No bacterial growth was observed. Exposure to PRT treatment results in very mild modifications in platelet counts. Levels of GPIb, GPIIb-IIIa and GPIV remained stable during storage in PCs treated with PRT 6.2J/ml and in the same range as those observed in non treated PCs. However, when PRT 12.3 J/ml was applied, levels of GPIb moderately decreased along days of storage 3 and 5. VWF, Fg, and factor Va did not modify their expression, neither by treatment nor by storage period. Fn appeared more elevated in all PRT samples when compared with non-treated samples. A progressive increase in P-selectin and LIMP expression and in Annexin V binding was observed during storage of PRT-treated PCs. Functional studies in perfusion models indicated that Mirasol PRT 6.2 J/ml treated platelets preserved adhesive and cohesive function to levels compatible with those observed in the respective control PCs. In conclusion, platelet function was well preserved in PCs treated with Mirasol PRT 6.2 J/ml and stored for 5 days.
ADAMTS13 Activity in Sickle Cell Disease: A Role in the Development of Clinical Vaso-Occlusion?.
Authors: Schnog John-John B. , Kremer-Hovinga Johanna , Akin Sakir , Ozveren Ernan , Krieg Soraya , Lammle Bernhard , Brandjes Dees , Mac Gillavry Melvin R. , Muskiet Fred D. , Duits Ashley J.
Published: October 2019

Endothelial cell activation occurs continuously in sickle cell disease (SCD). As VWF and especially the ultra large VWF multimers, released from activated endothelial cells, can serve as bridging proteins between the vessel wall and sickle red blood cells, we determined ADAMTS13 (the vWF-cleaving protease) activity as well as VWF:Rcof and VWF:Ag levels in SCD. Fourteen sickle cell patients (13 HbSS, 1 HbSC; 39 samples collected) and 9 HbAA controls were included. VWF:Rcof and VWF:Ag were assessed by commercially available assays, ADAMTS13 activity was determined by quantitative immunoblotting of proteolysed VWF-substrate. VWF:Rcof and VWF:Ag levels were higher in patients, as compared to controls. ADAMTS13 activity ranged from 45–128% in asymptomatic patients, from 38–205 in patients during acute vaso-occlusive events (VOE) and from 43-75% in controls. The ratios of ADAMTS13 to vWF:Ag levels and activity were lower in patients (SCD asymptomatic: 0.44 [interquartile range 0.34–0.55], SCD acute VOE: 0.29 [0.21–0.43], HbAA: median 0.61 [0.33–1.23], Kruskar Wallis test p=0.003). The ADAMTS13 - VWF:Rcof and the ADATS13 - VWF:Ag ratios dropped during acute vaso-occlusive events in 3 of 6 patients. In patients from whom multiple samples were collected (n=4), ADAMTS13 activity and VWF:Ag levels (and to a lesser extent VWF:Rcof) were inversely related. One patient showed no change in ADAMTS13 activity despite strong increments in VWF:Ag and VWF:Rcof during vaso-occlusive pain crises prior to his death (pulmonary embolism). VWF:Ag levels at initial presentation of acute VOE were related to the duration of pain (rs=0.53, p=0.02), whereas the ratios of ADAMTS13 to VWF:Ag levels and ADAMTS13 to VWF:Rcof were inversely related hereto (rs=−0.67, p=0.002, rs=−0.53, p=0.02 respectively). These data demonstrate that ADAMTS13 activity is related to VWF:Ag levels and VWF:Rcof in SCD and that the role of ADAMTS13 and VWF in the pathophysiology of SCD deserves further evaluation.
A Report of Transplantation of 224 Patients with Beta Thalassemia Major in Tehran, 1991– 2004.
Authors: Ghavamzadeh Ardeshir , Jahani Mohamad , Alimoghaddam Kamran , Iravani Masoud , Bahar Babak , Mousavi Asadolah , Gholibeikian Simin , Pejhan Shervin , Orouji Zahra , Babhadiashar Nima , Ashouri Asieh
Published: October 2019

Introduction: Beta Thalassemia Major is one of the most common single gene disorders and the most common type of hemoglobinopathies in Iran, with more than 20,000 patients. North and South parts of Iran have the highest prevalence, although it exists all over the country. In Iran, the first thalassemic patient was transplanted in our center, in 1991.

Methods and patients: Till now (August 2004) 224 Beta Thalassemia Major Patients have been transplanted in this center. Age range =2–17 years, M/F=123/101, Class I=99, class II=81 and class III= 44. 124 patients (55.4%) received Bone Marrow, 94 (42%) Peripheral Blood stem cell and 6 (2.7%) Cord Blood for transplantation. Their conditioning regimen in class I and II included Busulfan 3.5mg/kg for 4days, Cyclophosphamide (CY) 50mg/kg for 4 days and their GVHD prophylaxis regimen was Cyclosporine (CYX) 3mg/kg (IV from day −2 till day +5) and 12.5mg/kg (PO after then) and in class III, included Busulfan 4mg/kg for 4days and CY 40mg/kg for 4days and their GVHD prophylaxis regimen was CYX plus MTX 10mg/m2 on day +1 and 6mg/m2 on days +3 and +6.

Results:188 (84%) patients are alive and 36(16.1%) deceased.188 (84%) patients have passed 100 days after transplantation.168 (75%) patients had developed clinical acute GVHD after transplantation (grade II–IV= 57.1%). 67(35.6%) patients developed chronic GVHD (44 limited, 23 extensive). Three to ten year overall survival was 81% and disease free survival was 70 %.

Conclusion: Blood and marrow transplantation (based on other documented studies and our experience) is the treatment of choice for class I& II and is indicated in class III thalassemic patients.
Smoking as a Cause for Mild Chronic Neutrophilia.
Authors: Stemmelin German R. , Doti Carlos A. , Shanley Claudia M. , Ceresetto Jose M. , Rabinovich Oscar M. , Vicente Reparaz Maria A. , Vukovic Matias Gonzalez , Bullorsky Eduardo O.
Published: October 2019

Mild chronic neutrofilia is a frequent reason for hematologic consultation and in 70% of the cases there is no identifiable cause.

Objective: to determine if smoking habit could be the etiology for leucocytosis with neutrophilia in smokers with no other pulmonary associated disease.

Materials and Methods: We questioned 300 consecutive blood donors from our institution, elaborating a complete record of smoking habit. Immediately before blood donation an automated complete blood count was performed. Leucocytosis was defined as a white cell count above 11 x 109/L, and neutrophilia as a neutrophil count over 7.7 x 109/L. All data is stated as mean value ± 1 SD.

Results: we studied 195 (65%) men and 105 (35%) women (n=300). Mean age was 36.5 y.o. (18–69). Fifty-five percent (165/300) smoke or were previous smokers; 8.4% (14/165) of these, did not smoke at the time of this investigation, and only two have quit smoking a year prior to questioning. Mean time of duration of smoking habit was 16.4 years ± 10.6 and the average amount of cigarettes smoked through life (estimated from data referred along different phases of each subject’s history) was 1.26 x 105 ± 1.21 x 105. In the following table results from smokers (previous or actual) vs. non-smokers are compared.

Leucocytosis was present in 37/165 (22.4%) of smokers and in 3/135 (2.2%) of non-smokers (p<0.001) and neutrophilia was noted in 19/165 (11.5%) of smokers and in 2/135 (1.5%) of non-smokers (p<0.001). None of the volunteers had a WBC count over 20 x 109/L. A direct association was established for the number of cigarettes smoked and the WBC count and neutrophil count.

Discussion: smoking habit affects ciliar movement, inhibits alveolar macrophage function and produces hyperplasia on mucous glands within bronquial walls. These alterations result in entrapment of mucous secretions that will ultimately lead to bacterial colonization. The neutrophilia observed in smokers would be the result of a normal physiologic response to a potential infectious focus. In conclusion, is our understanding that the smoking habit should be considered as a common etiology for mild leucocytosis and neutrophilia.

Smokers (n:165) Non-smokers (n:135) p (t-test) WBC x 109/L 9.6 ± 2.1 6.8 ± 1.3 < 0.001 Neutrophils x 109/L 5.7 ± 1.6 3.9 ± 1.0 < 0.001 Hct % 45.9 ± 3.7 45.2 ± 3.6 NS Platelets 207 ± 48.7 206.4 ± 45.8 NS
Cytokine or Cytokine Receptor Treatment Using Hematopoietic Secretory Lysosomes as Vehicles.
Authors: Hansson Markus , Calafat Jero , Gao Ying , Persson Ann-Maj , Tapper Hans , Olsson Inge
Published: October 2019

Immunological responses are tightly controlled through a balance between stimulatory and inhibitory cytokines. This is used therapeutically in chronic inflammatory diseases such as rheumatoid arthritis or Crohn’s disease, where anti-cytokine therapy has proven to be useful. Furthermore, stimulatory cytokines are used in treatment of some malignant diseases. However, both anti-cytokine therapy and cytokine therapy are systemic treatments with severe side-effects: thus local delivery of the treatment may improve the therapeutic efficacy and reduce the systemic side-effects.

We therefore propose the concept of local delivery using secretory lysosomes of hematopoietic cells as vehicles. A secretory lysosome is a compartment with a dual function of storage and degradation found in secretory cells, e.g. in neutrophils and lymphocytes. The secretory lysosomes are released by degranulation at sites of inflammation or infection. Therapeutically-active protein could be induced for expression and storage in secretory lysosomes and thereby be locally released by degranulation resulting in local delivery of therapeutically active protein.

In support of this concept, we have shown that: I) gene transfer and granule loading can be achieved using the soluble TNF-a receptor (sTNFR1) after cDNA expression in hematopoietic cell lines and hematopoietic progenitor cells; II) endoplasmic reticulum export can be facilitated by the addition of a transmembrane domain; III) constitutive secretion can be prevented by incorporating a cytosol-sorting signal resulting in secretory lysosome targeting; IV) the sTNFR1 is released from the transmembrane domain into the secretory lysosome by proteolytic cleavage; V) regulated sTNFR1-secretion can be triggered by activation or calcium signal.

In vivo investigations are currently determining the feasibility of local protein delivery at sites of inflammation or malignancy.
Intermittent Low Dose Interleukin 11 Can Lead to Transfusion Independence in Patients with Chronic Myelo-Monocytic Leukemia and Thrombocytopenia.
Authors: Shah Chirag , Gentile Teresa C.
Published: October 2019

Recombinant Interleukin- 11 (IL-11) is a thrombopoietic cytokine that stimulates megakaryocytopoiesis in vitro and platelet production in vivo. It attenuates post chemotherapy thrombocytopenia at a dose of 50 mcg/ kg/ day subcutaneously (SC). Unfortunately, prolonged administration is associated with significant toxicity including peripheral and pulmonary edema at this dose. Administration of low dose IL-11 at 10 mcg/kg/day SC has shown efficacy in bone marrow failure states without significant toxicity. We report two cases of chronic myelo-monocytic leukemia (CMML) with transfusion dependent thrombocytopenia who received intermittent low dose IL-11 without significant toxicity.

Case Reports- Patient 1 is a 79-year-old male with history of CMML with pancytopenia of three years duration. Recently he required transfusion of platelets with his platelet counts falling to less than 15 x 109/L. His cytogenetic study showed normal karyotype, 46 XY. He required platelet transfusion at every 14 days. He initially was started on IL-11 at 10mcg/kg/day, 5 days per week.. His platelet count increased to above 30 x 109/L and he became transfusion independent within two weeks. Unfortunately on this schedule he developed edema and mild CHF. IL-11 was stopped for two weeks and upon resolution of toxicity, restarted at 10 mcg/kg/day on Monday, Wednesday and Friday. He has remained transfusion independent without recurrence of edema at 5 months on this schedule.

Patient 2 was a 63-year-old male with previous history of chronic lymphocytic leukemia and diffuse large B cell lymphoma who developed CMML with severe pancytopenia. His karyotype was 46, XY, −7, +21. His platelet count was consistently less than 10 x 109/L. He required platelet transfusion twice a week. He was started on IL-11 at 10mcg/kg/day for 5 days per week, two weeks on and two weeks off. His platelet count increased to as high as 64 x 109/L after 2nd cycle. His platelet transfusion requirement decreased from every 3rd day to every 10th-14th day. He experienced no peripheral or pulmonary edema.

Conclusion: Administration of low dose IL-11 in other bone marrow failure states has been reported but its use has not been described in CMML. Our observation in these 2 patients suggests that IL-11 has efficacy in CMML and is very well tolerated at low doses on an intermittent administration schedule. IL-11 may decrease the transfusion requirement in transfusion dependent patients. Further studies are needed to evaluate overall impact on larger number of patients who require regular platelet transfusion.
A Simple Technique to Determine Thrombopoiesis in Thrombocytopenic Patients Using Immature Platelet Fraction (IPF).
Authors: Wada Hideo , Sakakura Miho , Matsumoto Takeshi , Kaneko Toshihiro , Nobori Tutomu , Shiku Hiroshi
Published: October 2019

Introduction

Reticulated platelets (RP) were suggested that those cells might contain increased amounts of cytoplasmic RNA, reflecting the rate of thrombopoiesis in bone marrow. The flow cytometric analysis has been used for RP assay, however it was difficult applying to routinely testing due to complicated operations. We developed a new automated RP testing method named as immature platelet fraction (IPF) using XE-2100 automated hematology analyzer equipped with modified software and a polymethine dye. The aim of the study is to evaluate the IPF as indicator to discriminate level of thrombopoiesis in thrombocytopenic patients.

Methods

We have analyzed IPF in 129 healthy volunteers (40 females and 89 males; range, 21–60 years), and in 127 consecutive, unselected patients in Mie University Hospital from June 1, 2002 to March 31 2003. IPF was measured in the patients categorized to three groups which are different level of thrombopoiesis phase as ‘suppressed’ (Chemotherapy-induced hypoplasia (n=80)), aplastic anemia (AA) (n=24)), ‘increased’(idiopathic thrombocytopenic purpura (ITP) (N=51), Thrombotic thrombocytopenic purpura (TTP) (n=9)), the peak value of IPF preceding with platelet recovery (peripheral blood stemcell transplant (N=5)), bone marrow transplant (N=1), chemotherapy (N=101)), liver transplant (N=2))), high of FDP (>10μL/mL) group (N=19)) and ‘normal’(healthy volunteers (N=129)). Peripheral blood was collected in EDTA anti-coagulant. Total platelet counts and IPF were determined by the XE-2100 (Sysmex). Analysis of IPF was performed by special designed software. A receiver operating characteristic (ROC) curve was plotted for choosing optimized cut off value of IPF to discriminate different level of thrombopoiesis.

Results

Mean IPF value in healthy volunteer was 3.3±1.7 (%). IPF in ‘increased’ group was significantly higher than ‘suppressed’ and ‘normal’ group (p<0.01). In ‘increase’ group, IPF was significantly elevated in ITP (especially active phase; 16.5±9.2 %), TTP (13.4±3.1 %) and the peak value of IPF prior platelet recovery (11.4±5.4 %). The best cutoff point of IPF was chosen to discriminate ‘increased’ group from ‘suppressed + normal’ group was 6.15 (%). The sensitivity and specificity of this cutoff value are 80.0% and 90.1% for distinguish ‘suppressed + normal’ group, 80.0% and 83.7 % for distinguish ‘suppressed’ group. The positive predictive value was 89.4%, and negative predictive value was 70.7% to detect the ‘increased’ group from ‘suppressed’ group.

Summary

Our results suggest that the IPF reflects the level of thrombopoiesis in thrombocytopenic disorders. This simple technique of IPF measurement is useful for the differential diagnosis and analysis of platelet kinetics in thrombocytopenic patients.
Identification of Gene Expression Profiles in Acute Lymphoblastic Leukemia Cells That Discriminate Intracellular Thioguanine Nucleotide Accumulation in ALL Cells after In Vivo Treatment with Mercaptopurine.
Authors: Zaza Gianluigi , Cheok Meyling , Yang Wenjian , Deqing Pei , Cheng Cheng , Pui Ching-Hon , Relling Mary V. , Evans William E.
Published: October 2019

Thioguanine nucleotides (TGN) are considered the principal active metabolites exerting the antileukemic effects of mercaptopurine (MP). Numerous clinical studies have reported substantial inter-patient variability in intracellular TGN concentrations during continuation therapy of acute lymphoblastic leukemia (ALL). To identify genes whose expression is related to the intracellular accumulation of TGN in leukemia cells after in vivo treatment with MP alone (MP) or in combination with MTX (MP+MTX), we used oligonucleotide microarrays (Affymetrixâ HG-U95Av2) to analyze the expression of approximately 9,670 genes in bone marrow leukemic blasts obtained at diagnosis from 82 children with ALL. TGN levels were determined in bone marrow aspirates of these patients 20 hours after mercaptopurine infusion (1 g/m2 I.V). Because, as previously reported, patients treated with MP alone achieved higher levels of intracellular TGN compared to those treated with the combination, we used Spearman’s rank correlation to identify genes associated with TGN levels separately for the 33 patients treated with MP alone and the 49 with the combination (MP: median TGN: 2.46 pmol/5x106 cells, range: 0.01–19.98; and MTX+MP: median TGN: 0.55 pmol/5x106 cells, range: 0.005–3.31). Hierarchical clustering using these selected probe sets clearly separated the 33 patients treated with MP alone into two major groups according to TGN concentration (< 2.46 and > 2.46 pmol/5x106 cells; n=60 genes) and two major branches were also found for patients treated with the combination (< 0.55 and > 0.55 pmol/5x106 cells; n=75 genes). Interestingly, there was no overlap between the two sets of genes, indicating that different genes influence the accumulation of TGN when this drug is given alone or in combination with MTX. The association between gene expression profiles and TGN levels determined by leave-one-out cross-validation using support vector machine (SVM) based on Spearman correlation, was rho=0.60 (p<0.001) for MP alone and rho=0.65 (p<0.001) for MTX+MP, with false discovery rate (FDR) computed using Storey’s q-value (MP: 50% true positive, MTX+MP: 70% true positive respectively). Genes highly associated with the post-treatment TGN level in ALL patients treated with MP alone encode transporters, enzymes involved in the MP metabolic pathway and cell proliferation. Genes associated with post-treatment levels of TGN after combined therapy have been implicated in protein and ATP biosynthesis. Together, these in vivo data provide new insights into the basis of inter-patient differences in TGN accumulation in ALL cells, revealing significant differences between treatment with MP alone or in combination with MTX.
Progressively Intensified Chemotherapy: A Phase II Trial in Patients with Multiple Myeloma.
Authors: Farjami Sassan , Akhtar Adnan , Kancherla Rama Mohana , Liu Delong , Seiter Karen , Ahmed Tauseef
Published: October 2019

Background: A number of randomized and non-randomized studies have demonstrated the efficacy of the high-dose therapy with stem cell rescue. The efficacies of these treatment modalities were mainly related to the degree of myeloablation and destruction of the bone marrow microenvironment. We decided to evaluate the efficacy and toxicities of a progressively intensified chemotherapy followed by stem cell rescue in patients with multiple myeloma.

Methods: Between 1997 and 2000 twenty-eight patients (median age, 51years; range 38–70) consecutively enrolled in this study. Patients received VAD combination chemotherapy as induction regimen. Blood progenitor cells were collected following two cycles of cyclophosphamide, etoposide and granulocyte-colony stimulating factor (G-CSF). The chemotherapy intensified by giving melphalan at a dose of 140 mg/m2and mitoxantrone at a dose of 60 mg/m2 on day -7of 1st myeloablation followed by stem cell rescue on day 0. Between 56–94 days following 1st transplant patients received 2nd intensification by Melphalan at a dose of 200 mg/m2 on day -3 of 2nd myeloablation followed by stem cell rescue on day 0. After recovery from 2nd intensification patients received Interferon-alfa2b and prednison as maintenance therapy for 18 months.

Results: After induction there was 7%(2) complete response (CR), 72%(20) partial response and in 21%(6) their disease progressed (PD). Following escalation with cyclophosphamide and etoposide the CR rate was 21%(6), with 86%(24) overall response. After dose intensification with melphalan, mitoxantrone and 1st stem cell rescue the CR increased to 61%(17) with 89%(25) overall response. Following 2nd dose intensification with melphalan and stem cell rescue the CR increased to 68%(18) and overall response reached to 90%(24) Most non-hematologic toxicities were in the range of grade 0–2. Mucositis and diarrhea were the most severe toxicities associated with 1st and 2nd intensification. After median duration of follow up of 66.67 months the survival probability was 56.47% with 95 percent confidence interval of 42.65% and 70.29%.

Conclusion: Our study suggests that combination of cyclophosphamide with etoposide could induce remission in significant number of patients prior to aphresis and by decreasing tumor burden could reduce stem-cell contamination (in vivo purging). Also mitoxantrone in addition to melphalan as a standard preparative regimen could result in high rates of complete remission and overall response, which could be translated in to better overall survival.
Conditioning Regimens in Stem Cell Transplantations Facilitate Homing by Increasing Bone Marrow SDF-1 through Induction of SDF-1 Gene Transcription.
Authors: Zweegman Sonja , Janssen Jeroen J.W.M. , Kessler Floortje L. , Mus Adriana M.C. , van den Born Jacob , Huijgens Peter C. , Drager Angelika M.
Published: October 2019

Stromal Derived Factor-1 (SDF-1) is a key regulator of engraftment. The clinical relevance of SDF-1 is suggested by the relation between in vitro migratory capacity of CD34-positive cells towards SDF-1 and engraftment after stem cell transplantation (SCT). For better insight in engraftment after SCT clarifying the determinants of SDF-1 is therefore important. We investigated SDF-1 responses after chemotherapy and irradiation of the murine stromal M210B4 cell line and in allogeneic SCT patients. SDF-1 protein levels were measured in conditioned medium (CM) of the M210B4 cell line, in peripheral blood plasma and bone marrow plasma by ELISA. The migration supportive capacity of CM was tested by allowing CD34-positive cells to migrate over fibronectin coated transwells for 4 hours. Furthermore, sdf-1 gene expression was investigated by means of a quantitative PCR using mRNA from M210B4 cells or from buffy coats of peripheral blood and bone marrow cells.

Both irradiation and cytarabine treatment of M210B4 cells resulted in a significant ~2-fold increase in SDF-1 levels and a significant ~1.5-fold increase in migration of CD34-positive cells. Changes in protein levels correlated with those of mRNA: both treatments resulted in a significant ~2-fold increase in SDF mRNA copies.

In agreement with in vitro results, a significant ~3-fold increase in SDF-1 protein levels was induced by in vivo myeloablative and non-myeloablative conditioning regimens. The increase in SDF-1 was higher in BM plasma as compared to PB plasma, thereby significantly increasing BM-PB gradients (BM minus PB SDF-1 levels: 11.0±3.5 after versus 4.0±2.8 ng before conditioning). Corresponding with protein levels, sdf-1 mRNA increased in bone marrow cells of 4/5 patients (~40-fold increase, from 0.3±0.3% to 9.7±15.2% relative to GAPDH expression, n=4 of whom 2 received myeloablative and 2 non-myeloablative regimens). Wide ranges of increase in SDF-1 message (ranging from 0.26 to 104-fold increase) and protein levels (1.26 to 5.16-fold increase) were observed, which may be accounted for by differences in conditioning regimens.

We conclude that generation of an SDF-1 gradient over the blood-bone marrow barrier by conditioning regimens plays a role in engraftment. The introduction of reduced intensity stem cell transplantations challenged the idea that sublethal conditioning is a prerequisite for engraftment by preparing vacant niches. Our observation that both non-myeloablative and myeloablative regimens result in an increase in SDF-1 gradient, can be proposed as an explanation. Interestingly, the variation in increase of SDF-1 after different conditioning regimens indicate that homing efficiency may be improved by reconsidering the optimal moment of stem cell infusion and the agents used in conditioning regimens.
Immunological Recovery after Nonmyeloablative Transplant: A Prospective Study of 24 Patients Treated in a Single Center.
Authors: Bron Dominique , Hyunh Van , Dewilde Virginie , Bennani Jalil , Lewalle Philippe , Huynh Phuong , Leroy Rita , Crombez Patrick , Debusscher Louisette , Feremans Walter , Bernier Michel , Martiat Philippe
Published: October 2019

Background: Nonmyeloablative conditioning regimens are more and more widely used in the setting of stem cell transplant due to a reduced transplant related mortality. However, these conditioning regimens include more immunosuppressive agents and little data in terms of immunological recovery are found in the literature.

Population: 24 patients (pts) admitted for nonmyeloablative transplant from HLA identical donors were prospectively followed during a period of 1 year. All pts received a conditioning regimen consisting of rabbit ATG (fresenius), Fludarabine phosphate, Cyclophosphamide ( ARA-C for myeloid malignancies) cyclosporin A and mycophenolate mofetil (MMF). Lymphocytes markers expression as well as lymphocytes absolute numbers were studied to evaluate the cellular immunologic recovery. IgG, IgA and IgM levels were also monitored during one year. Pts were stratified according to the presence of GVHD(acute and chronic) or not.

Results: 24 pts with a median age of 52 (20–64) years old transplanted with an attenuated conditioning regimen, were evaluated. Median delay to recover 500 ANC and 20000 plt/ul was 8 days. Without GVHD, the pts recover a normal level of CD3, CD8, CD19,CD56 and CD45RO positive lymphocytes within 6 months. CD4+ and CD45RA + lymphocytes did not reach normal values within 1 year; Therefore the CD4/CD8 ratio remains low for more than 12 months. This observation explains the high incidence of oppotunistic infections in these pts even 6 months after transplantion. In our small seies,with or without GVHD neither IgA, nor IgM recovered within 1 year. IgG recovery is not evaluable because of human IVIG administration monthly after transplant.

Conclusion: our small series confirms the rapid hematologic recovery after nonmyeloablative transplant.Cellular immunological recovery without GVHD is achieved within 6 months for most of the lymphocytes subsets excepted the CD4+ lymphocytes who need more than 12 months to recover. IgM and IgA recovery is also very slow.
Haploidentical Allogeneic Versus Gene Modified Autologous Stem Cell Transplantation for Severe Combined Immunodeficiencies (S.C.I.D). The Risk/Benefit Balance Is in Favor of Gene Therapy.
Authors: Cavazzana-Calvo Marina , Hacein-Bey-Abina Salima , Saint-Basile Genevieve , Le Deist Françoise , Wulfraat Nicolas , Alexander Ian , Landais Paul , Blanche Stephane , Fischer Alain
Published: October 2019

For the last 35 years or so, allogeneic hematopoietic stem cell transplantation (HSCT) has been the only curative approach for patients affected by severe combined immunodeficiencies (SCID), a medical emergency. According to the most recent European survey haploidentical transplants allow a survival rate at 3 years of 75% (taking only into account patients transplanted since 1995). The mortality rate is heavily influenced by age at transplantation (being very low within young infants), in correlation with the infection burden and also the incidence of acute graft-versus-host reaction (GVHD). Between 1998 and 2003, 6 patients with NK(−) B(+) SCIDs (either gc or JAK3 deficiency) received an haploidentical HSCT at Necker Hospital, 3 of them died from an infection, one in the context of a severe GVHD. In addition, despite the improvement in the survival rate, over the time a number of long-term concerns have been detected including a frequent persistence of a B cell deficiency, and a decline in T-cell functions related to the absence of donor stem cell engraftment and may be also to a premature decline in the thymus function.

Therefore, several patients have received a second transplant with a low efficiency in an haploidentical setting.

These significant limitations set the rationale for the development of an alternative strategy such as gene therapy. From March 1999 up to May 2002, ten children with gc deficiency under the age of one year were enrolled.

All of them are alive today. The gc gene transfer into patients’ CD34(+) cells led in 9 out of 10 patients to the emergence of T and NK lymphocytes. It took 10 to 12 weeks to detect mature T cells in the periphery, a delay which is significantly faster than the one observed after haploidentical HSCT. In all but 2 patients, T cell counts normalized up to 5.3 years after gene therapy. The occurrence of two severe adverse events led to put transiently the trial on hold in order to understand the pathophysiology of these events and assess its overall risks. Following a thorough analysis of the retrospective data and a prospective analysis, it was concluded that the benefit/risk balance in favor of the gene therapy approach at least for patients older than 3 months, hence our gene therapy protocol has been reopened. An update of the clinical data will provided at the meeting.
t(3;14)(P14.1;Q32) Involving IGH and FOXP1 Is a Recurrent Chromosomal Aberration in MALT Lymphoma.
Authors: Streubel Berthold , Vinatzer Ursula , Lamprecht Andrea , Raderer Markus , Chott Andreas
Published: October 2019

Translocations t(11;18)(q21;q21), t(14;18)(q32;q21), and t(1;14)(p22;q32) are known to occur in MALT lymphoma. In a patient with MALT lymphoma of the thyroid a novel t(3;14)(p14.1;q32) was observed by cytogenetic analysis. Subsequent fluorescence in situ hybridization studies showed that the immunoglobulin heavy chain locus (IGH) was rearranged on chromosome 14. Long-distance inverse polymerase chain reaction identified FOXP1 as the partner gene on chromosome 3. Two FISH-assays were established to screen 90 MALT lymphomas, 8 splenic and 6 nodal marginal zone lymphomas for this aberration. Overall, 8 MALT lymphomas (9%) harbored t(3;14)(p14.1;q32); the aberration was detected in MALT lymphomas of the ocular adnexa (4 of 20), thyroid (2 of 5), and skin (2 of 20), but not in the stomach (n=20), salivary gland (n=20), and lung (n=5). Splenic and nodal marginal zone lymphomas were t(3;14)(p14.1;q32) negative. Further 38 MALT lymphomas with known t(11;18)(q21;q21), t(14;18)(q32;q21), or t(1;14)(p22;q32) did not reveal a t(3;14)(p14.1;q32). Most t(3;14)+ MALT lymphomas (7 of 8) harbored additional genetic abnormalities, such as +3. Real-time quantitative RT-PCR showed up-regulation of FOXP1 in cases with t(3;14)(p14.1;q32) or trisomy 3. This study identifies FOXP1 as a new translocation partner of IGH in MALT lymphomas.
Global DNA Methylation Profiling Demonstrates That Idiopathic Myelofibrosis Is Characterized by a Distinct Epigenetic Signature with Aberrant Methylation Changes in Genes Involved in Inflammation and Hematopoiesis
Authors: Opalinska J. , Sohal D. , Thompson R. , Zhou L. , Li Y. , Pahanish P. , Kotla V. , Kambhampati S. , Parmar S. , Friedman E. , Figueroa M. , Ye K. , Wickrema A. , Melnick A. , Greally J. , Pardanani A. , Verma A.
Published: October 2019

Chronic idiopathic myelofibrosis (MF) is a clonal hematopoietic disorder that leads to progressive marrow fibrosis and peripheral cytopenias. Very little is known about the role of aberrant DNA methylation in the pathobiology of this disease. Whole genome methylation was analyzed by a recently described novel method, the HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific restriction digestion by HpaII and MspI followed by amplification, two color labeling and cohybridization to quantitatively determine individual promoter methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. Peripheral blood leucocytes from 9 patients with IMF were compared to 9 age-matched normal and anemic controls. Gene expression analysis was performed using 37K oligo maskless arrays using cDNA from the same samples. Methylation analysis showed that myelofibrosis samples clustered separately from normal and anemic controls when grouped by unsupervised clustering based on Pearson’s correlation coefficient. On the other hand, global gene expression demonstrated no clear cut differences between myelofibrosis and control samples suggesting that methylation profiling has greater discriminatory power when compared to conventional gene expression profiling. A high correlation (r=0.88–0.96) was observed between whole genome methylation profiles of matched sets of bone marrow and peripheral blood leucocyte samples from selected patients demonstrating that peripheral blood leucocytes can act as a valid surrogates for epigenomic analysis. Further analysis showed that genes aberrantly methylated in all myelofibrosis samples included v-myc, histone 2A, TNF, TNF Receptor1, FGF14 and others. Functional pathway analysis by Ingenuity showed that pathways involved in Inflammation and Cell signaling were the most affected by epigenetic silencing. Most interestingly, a large proportion of gene promoters were also aberrantly hypomethylated. These included genes for chemokine CXCL13, APC, IL-3, STAT2 and others. The pathways most activated by hypomethylation were involved in hematopoiesis and cell growth and proliferation demonstrating the biological validity of our analysis. Thus, our data demonstrates that myelofibrosis is characterized by distinct epigenetic aberrations that are preserved in peripheral blood leucocytes. These can be the basis of future studies on pathogenesis and diagnosis for this disease and lead to translational studies with agents targeting DNA methylation.
Dynamic Imaging of Fibrin Network Formation Correlated with Other Measures of Polymerization.
Authors: Weisel John W. , Chernysh Irina N.
Published: October 2019

The clotting time or gel point is commonly used in a variety of clinical assays, but little has been known about what it represents structurally because of the limitations of imaging technologies. The necessity of fixation and dehydration for electron microscopy means that only final clot structures, often distorted by preparation artifacts, can be observed, and there are severe limitations of image quality for conventional light microscopy. For similar reasons, we know almost nothing about the branching and lateral aggregation of fibrin that are essential for clot or thrombus stability. Turbidity, light scattering, or clot stiffness are useful to follow the time course of polymerization but reflect only overall clot properties. We visualized in real time fibrin network formation in the hydrated state, using deconvolution microscopy, which allows optical sectioning without the bleaching that accompanies confocal microscopy. Thus, the events during polymerization could be followed quantitatively over long periods of time. Videos will demonstrate the major observations. Individual mobile fibers were observed before the gel point. After gelation, an initial fibrin network, or scaffold, was seen, which evolved over time by addition of new fibers and elongation and branching of others. Furthermore, some fibers in the network moved chaotically for some time. A detailed, quantitative morphological analysis of network formation was carried out by superposition of images from different time points colorized to distinguish changes. We quantified network formation by the number of branch points, and longitudinal and lateral growth of fibers as a function of time. The distributions of fibers that reached a maximum of longitudinal growth and branch point formation both had maxima at the gel point but, surprisingly, some longitudinal growth continued and new branch points appeared after the gel point, requiring modification of existing models of fibrin polymerization. The cumulative percentage of fibers reaching their final length and the number of branch points attained maximum values at the time corresponding to that at which the turbidity reached approximately 60% of its maximum. Lateral growth reached a plateau at the same time as turbidity. Measurements of clot mechanical properties revealed that the clots achieved maximum stiffness and minimum plasticity well after branch point, as well as length changes and lateral growth of fibers, were completed. These results provide new information on the time sequence of events during fibrin network formation, which is important to understand both clotting and thrombosis and to allow modulation of clot properties.
Pilot Results: Pre-Treatment with Deferasirox Increases the Chances of Rapid Viral Response in Patients with Chronic Hepatitis C Infection Treated with PEG-Interferon/Ribavirin.
Authors: Goubran Hadi A. , Essmat Gamal , Morcos Hanaa H. , Amin Sherif N.
Published: October 2019

Chronic hepatitis C infection [CHCV] is a major cause of morbidity and mortality. It is treated with a combination of interferon and ribavirin [INF/RIBV] for 1 year in strain 1 & 4 prevalent in our geographic area 1. Iron overload is an important cofactor of disease outcome, enhancing inflammation, fibrosis and cirrhosis and predisposing to hepatocellular carcinoma. It also affects the response to conventional therapy2–3. We evaluated the impact of pre-treatment iron chelation using deferasirox on the outcome of combination therapy (160μg PEG-Interferon α-2-a (Reiferon-Retard) and ribavirin) in terms of early viral response defined as clearance or 2 log reduction in viral load at 12 weeks and rapid viral response defined as viral clearance by four weeks. The latter may imply a shortened duration of therapy to 6 months rather than a year, improving cost effectiveness and reducing side effects 4,5. The study included 30 patients with CHCV candidates for INF/RIBV therapy with a Metavir score of less than F3 by liver biopsy or fibrotest. The mean serum ferritin of patients prior to therapy was 520+/−98 ng/ml. Ten patients were subjected to iron chelation using deferasirox (15mg/Kg/day p.o.) for 6–8 weeks prior initiation of therapy (group I), bringing their serum ferritin to 309+/−74 ng/ml, whereas 20 patients started therapy without pre-treatment chelation (group II). Patients were followed up periodically both clinically and laboratory. HCV PCR both quantitative and qualitative were conducted at week 4 and 12. In 7 patients (70%) from group I, early viral clearance at 12 weeks, was reported compared to 12 (60%) in group II (p>0.05). Six patients out of the 7 cleared in group I and 6 patients out of the 12 in group II (p<0.01) had a rapid virologic response with complete viral clearance at 4 weeks. Pre-treatment with deferasirox may improve early viral response rates. It seems to favor rapid virologic response in CHCV patients treated with INF/RIBV with a potential shorter duration of therapy, significant cost reduction and less side effects. Larger studies are needed to confirm the results of this pilot work.
A GMCSF/Interleukin-15 Fusokine Leads to the Generation of a Novel Type of CD2/MHCII Immune Regulatory Cell with Potent Immune Suppressive Properties as Demonstrated in the EAE Mouse Model of Multiple Sclerosis.
Authors: Rafei Moutih , Hsieh Jeremy , Li Meng Yang , Zehntner Simone , Forner Kathy , Galipeau Jacques
Published: October 2019

Multiple sclerosis (MS) is an autoimmune disease characterised by the infiltration of autoreactive T-cell causing damages to the central nervous system. So far, interferon-β and glatiramer acetate are the only two immunomodulatory coumpounds that have been approved as non-curative disease managing strategies. Therefore, there is an urgent need for the development of novel efficient therapies that can be both safe and potent in inhibiting MS progression and promote reversal of disease state. We have recently published a report describing a novel synthetic GMCSF and IL15 Fusion Transgene (GIFT15) and have described its paradoxical and potent immune suppressive properties in vivo [Rafei et al., Blood (March 2007)]. Its mechanism of action relies on STAT3 hyperactivation arising from aberrant signalling taking place downstream of the IL15 receptor. We have now further studied the effect of GIFT15 on mouse spleen cells in vitro and here demonstrate that it leads to the conversion of murine T-cells to a novel suppressive regulatory cell type. Indeed, GIFT15-treated splenocytes (hereafter GIFT15 regs) shed their TCR and loose expression of CD3, CD4 and CD8, retain CD2 expression and acquire expression of MHC II. Distinct to classic T-regulatory cells, GIFT15 regs do not express CD25 or FOXP3. GIFT15 regs were able to suppress an in vitro two-way MLR by a contact-dependent mechanism as well as by the contemporaneous production of interleukin (IL)-10. Furthermore, GIFT15 regs were able to block antigen-specific activation of CD4-T-cells in response to autologous macrophage stimulation. As a proof-of-principle in vivo study, GIFT15 regs were injected intravenously in mice with pre-established experimental allergic encephalitis (EAE) and disease score was monitored over time. Interestingly, mice recovered significantly faster than controls following administration GIFT15 regs and a blockade in EAE progression was also noticed over time. In conclusion, our data suggests that GIFT15 can be used as a method to ex vivo generate suppressor cells of a new type which are distinct from classic Tregs or Tr1 cells. We propose that GIFT15 regs derived from autologous lymphocytes may be exploited for the treatment of autoimmune disease such as MS and may also be of use for other autoimmune ailments as well.