Manal Fouda

Antiplatelet antibodies contribute to thrombocytopenia associated with chronic hepatitis C virus infection

Abstract Thrombocytopenia is one of the most frequent hematological manifestations of hepatitis Cvirus (HCV) infection; which typically worsens with progression of the liver disease and can become a major clinical complication. Several mechanisms have been postulated to explain thrombocytopenia in HCV hepatic patients, including immune mechanisms. The aim of the present work is to investigate the role of immune mechanisms as a causative agent of thrombocytopenia in HCV hepatic patients. The study included 50 hepatic patients with HCV infection (30 with thrombocytopenia and 20 with normal platelets counts). Platelets associated glycoprotein specific antibodies were evaluated by flow cytometry and confirmed by quantitative monoclonal immobilization of platelet antibodies (MAIPA). The frequency of platelet associated immunoglobulin (PAIg) in thrombocytopenic HCV positive hepatic patients by FCM was 86·7, 83·3, 46·7 and 33·3% for total PAIg, PAIgG, PAIgM and PAIgA respectively. MAIPA found platelet specific antibodies in 26/30 (86·7%) of patients. The most likely target antigen for platelets antibodies were glycoprotein (GP) IIb/IIIa (30%), followed by GP IIIa (20·5), GP IIb (13·3%), GPIb (13·3%), then GPIa (10%). The platelets count was inversely correlated to the levels of platelets GP specific antibodies (r=−0·42, p=0·024), and significantly parallel to spleen size (p=0·024). Platelet associated glycoprotein specific antibodies represent a common mechanism inducing thrombocytopenia in patients with chronic HCV infections.

Once weekly recombinant human erythropoietin treatment for cancer-induced anemia in children with acute lymphoblastic leukemia receiving maintenance chemotherapy: A randomized case-controlled study

Abstract Background: Patients receiving chemotherapy for cancer often develop anemia, which can contribute to increased morbidity and reduced quality of life (QOL). Chemotherapy-induced anemia can be successfully treated using recombinant human erythropoietin (rHuEPO). Aim of the study: To demonstrate the effectiveness of once-weekly (QW) rHuEPO dosing to effect improved hemoglobin levels, decreased transfusion use, and improved functional outcomes and QOL in pediatric leukemic patients (ALL) receiving maintenance chemotherapy. Patient and methods: This was a prospective randomized, single-center, open-label, 12-week case-control study of epoetin alfa in pediatric patients with acute lymphoblastic leukemia (ALL) in remission receiving maintenance chemotherapy. Sixty patients were randomly assigned to receive either epoetin alfa (rHuEPO group = 30 cases, 17 males and 13 females, age; 6.8 ± 2.33 years), or no epoetin alfa (control group = 30 cases, 16 males and 14 females, age; 6.76 ± 2.28 years). Both groups were matched as regard age, sex, baseline Hb concentration, remission state, chemotherapy regimen, numbers and amount of blood transfusion, and leukemia state (both were low and standard risk). Epoetin alfa was administered at a dose of 450 IU/kg, once weekly, subcutaneously (s.c.) for 12 consecutive weeks. Endpoints were changes in hematologic and QOL parameters. Results: Among the 30 patients evaluable for hematologic response, the mean increase in Hb from baseline to time of final evaluation was 3.08 ± 1.48 g/dl (p < 0.001). An increase in Hb of ≧ 2 g/dl, in the absence of blood transfusion, occurred in 70% of patients (21 of 30 patients) who were on the study for ≧ 30 days. The overall response rate (Hb increase ≧ 2 g/dl or Hb ≧ 12 g/dl in the absence of blood transfusion) was 90% (27 of 30 patients). In 30 patients who were evaluable for QOL assessment, epoetin-α therapy was found to significantly (p < 0.001) improve mean cancer linear analog scale (CLAS) scores for energy level, ability to perform daily activity, and overall QOL from baseline to the time of final evaluation. QW epoetin-α was found to be well tolerated. Conclusion: Treatment with QW epoetin-α was found to increase Hb levels, decrease transfusion requirement, and improve functional status and QOL in anemic patients with ALL in maintenance receiving chemotherapy. The once-weekly schedule is convenient, safe, and may reduce the burden on patients, parents, and their caregivers by reducing the number of visits to the clinic.

c-Myc oncogene and Cdc25A cell activating phosphatase expression in non-Hodgkin's lymphoma.

Abstract The product of proto-oncogene c-Myc is a potent activator of cell proliferation. The prognostic importance of the over expression of c-Myc and its transcriptional target Cdc25A in non-Hodgkin lymphoma (NHL) patients remains to be elucidated. To determine the role and the prognostic relevance of c-Myc and Cdc25A over expression in this group, we analyzed the expression of c-Myc oncoprotein by immunohistochemistry and Cdc25A mRNA by reverse-transcription polymerase chain reaction (RT-PCR) in the biopsied lymph nodes of 59 NHL patients. Over expression of c-Myc oncoprotein (P62) was observed in 32 out of 59 samples (54.2%) and Cdc25A in 36 out of 59 (60.1%).The percentage of c-Myc oncoprotein and Cdc25A mRNA over expression was significantly increased from low grade (4/12=25%, 4/16=25%) through intermediate grade (9/20=45%, 10/20=50%) to high grade lymphoma (19/23=82.6%, 22/23=95.6%) respectively (P=0.001 for both). The proportion of patients with positive c-Myc and Cdc25A over expression was significantly higher among patients with elevated serum lactic dehydrogenase (sLDH), and serum beta 2 microglobulin compared to those with normal levels (P<0.05, <0.01, respectively). Moreover, 80 and 90% of NHL patients with bone marrow infiltration at diagnosis had c-Myc and Cdc25A over expression, respectively. On the other hand, positive c-Myc, and Cdc25A over expression were not significantly related to the grade of international prognostic index, or the presence of B symptoms or to histopathological type. The expression of c-Myc and Cdc25A was significantly elevated in those who died when compared to survivors (P<0.001 for both). Moreover, positive c-Myc and Cdc25A over expression was associated with shortened overall survival. In conclusion: over expression of c-Myc and Cdc25A may be poor prognostic factor in NHL and associated with poor outcome. Assessments of c-Myc and Cdc25A expression in NHL at diagnosis are likely to be helpful in predicting patient outcome and selecting optimal therapeutic regimen.UNLABELLED The product of proto-oncogene c-Myc is a potent activator of cell proliferation. The prognostic importance of the over expression of c-Myc and its transcriptional target Cdc25A in non-Hodgkin lymphoma (NHL) patients remains to be elucidated. To determine the role and the prognostic relevance of c-Myc and Cdc25A over expression in this group, we analyzed the expression of c-Myc oncoprotein by immunohistochemistry and Cdc25A mRNA by reverse-transcription polymerase chain reaction (RT-PCR) in the biopsied lymph nodes of 59 NHL patients. Over expression of c-Myc oncoprotein (P62) was observed in 32 out of 59 samples (54.2%) and Cdc25A in 36 out of 59 (60.1%). The percentage of c-Myc oncoprotein and Cdc25A mRNA over expression was significantly increased from low grade (4/12=25%, 4/16=25%) through intermediate grade (9/20=45%, 10/20=50%) to high grade lymphoma (19/23=82.6%, 22/23=95.6%) respectively (P=0.001 for both). The proportion of patients with positive c-Myc and Cdc25A over expression was significantly higher among patients with elevated serum lactic dehydrogenase (sLDH), and serum beta 2 microglobulin compared to those with normal levels (P<0.05, <0.01, respectively). Moreover, 80 and 90% of NHL patients with bone marrow infiltration at diagnosis had c-Myc and Cdc25A over expression, respectively. On the other hand, positive c-Myc, and Cdc25A over expression were not significantly related to the grade of international prognostic index, or the presence of B symptoms or to histopathological type. The expression of c-Myc and Cdc25A was significantly elevated in those who died when compared to survivors (P<0.001 for both). Moreover, positive c-Myc and Cdc25A over expression was associated with shortened overall survival. IN CONCLUSION over expression of c-Myc and Cdc25A may be poor prognostic factor in NHL and associated with poor outcome. Assessments of c-Myc and Cdc25A expression in NHL at diagnosis are likely to be helpful in predicting patient outcome and selecting optimal therapeutic regimen.

Chlorambucil Plus Theophylline vs Chlorambucil Alone as a Front Line Therapy for B-Cell Chronic Lymphatic Leukemia

B-cell chronic lymphatic leukemia (B-CLL) has emerged as a prototype of malignancies characterized by a defective apoptosis that leads to a progressive accumulation of monoclonal B cells in the bone marrow, lymphoid tissues and peripheral blood. Chlorambucil, an aromatic derivative of nitrogen mustards, is the most common treatment for chronic lymphatic leukemia (CLL). The response rate with its use is 40 to 60%, with 3 to 10% only of patients achieving a complete response (CR). To improve response rates, chlorambucil has been combined with steroids or other agents. Theophylline, a methylxanthine commonly used as a treatment for asthma, has been shown to induce apoptosis in CLL cells both in vitro and in vivo. Chlorambucil induces apoptosis in CLL cells as well and synergy has been shown between the two drugs without affecting the normal B lymphocytes. The aim of this work was to evaluate the potential utility of this combination as a therapeutic modality for B-CLL. A total of 210 B-CLL patients were recruited and randomized to receive either chlorambucil in an oral dose of 0.1 mg/kg/day indefinitely (109 patients) or chlorambucil 0.1 mg/kg/day plus theophylline 200 mg bid, orally (101 patients). The main endpoints were overall survival from the time of randomization, disease status after 9 months and time to disease progression. After 9 months of treatment, clinical and hematological remission was achieved in 14 patients (12.8%) in the chlorambucil group, compared to 26 (25.7%) in the chlorambucil plus theophylline group (P value 0.01). Partial remission was observed for 38 patients (34.9%) in the chlorambucil group and 36 patients (35.7%) in the chlorambucil plus theophylline group. In patients treated with chlorambucil alone, the median progression-free survival (PFS) was 30 months and in patients treated with chlorambucil plus theophylline it was 44 months. Probabilities of PFS at 24 months for the chlorambucil-treated patients were 59% and 85% for the chlorambucil plus theophylline-treated patients. The difference was statistically significant (P = 0.006). The 3-year and 5-year overall survival rates were, respectively 75% and 38% in the chlorambucil group as opposed to 76% and 46% in the chlorambucil plus theophylline group. The median survival time was 55 months in the chlorambucil group and 56 months in the chlorambucil plus theophylline group. Forty-nine patients died in the chlorambucil group compared to 44 patients in the chlorambucil plus theophylline group (P = 0.371). The trial has demonstrated that adding theophylline to the standard treatment of B-CLL significantly increases effectiveness of treatment in terms of tumor response and time to disease progression. It could not improve the overall survival. Treatment with theophylline does not compromise quality of life or add significant toxicity. As newer drugs have recently become available for treating patients with CLL like fludarabine, further trials are needed to evaluate the effect of combining theophylline with these drugs.

Prognostic cytogenetic markers in childhood acute lymphoblastic leukemia: Cases from Mansoura, Egypt

Abstract The objective of the work was to evaluate children with acute lymphoblastic leukemia (ALL) showing resistance to immediate induction chemotherapy in relation to conventional and advanced cytogenetic analysis. The study was conducted on 63 ALL children (40 males and 23 females) with age range 4.5 months–16 years (mean = 7.76 years). They included 37 cases who attained a true remission and 26 complicated by failure of remission, early relapse or death. They were subjected to history, clinical examination and investigations including CBC, BM examination, karyotyping, FISH for translocations and flowcytometry for immunophenotyping and minimal residual disease diagnosis. Cases aged <5 years; male sex with organomegaly had better remission although statistically insignificant. Initially low HB <8 gm/dl, high WBCs and platelet counts >50.000/mm3 also showed better but non-significant remission rates. Most of our cases were L2 with better remission compared to other immunophenotypes. About 40 informative karyotypes were subdivided into 15 hypodiploid, 10 pseudodiploid, 8 normal diploid and 7 hyperdiploid cases; the best remission rates were noticed among the most frequent ploidy patterns. Chromosomes 9, 11 and 22 were the most frequently involved by structural aberrations followed by chromosomes 5, 12 and 17. Resistance was noted with aberrations not encountered among remission group; deletions involving chromosomes 2p, 3q, 10p and 12q; translocations involving chromosome 5; trisomies of chromosomes 16 and 21; monosomies of 5 and X and inversions of 5 and 11. Our conclusions were that cytogenetic and molecular characterizations of childhood ALL could add prognostic criteria for proper therapy allocation.

L and E Selectins in Acute Myeloid Leukemia: Expression, Clinical Relevance and Relation to Patient Outcome

Abstract The aim of this study was to assess circulating soluble L (sL) and soluble E (sE) selectins adhesion molecules in acute myeloid leukemia (AML) blasts in order to evaluate their clinical significance. Fifty patients with AML (4 M0, 8 M1, 16 M2, 5 M3, 7 M4, 6 M5, 4 M6) were included in this study. sL and sE selectins were evaluated at diagnosis, remission and in relapsed patients; whole membrane expression of L and E selectins by AML blast was investigated only at diagnosis. In addition, 15 normal persons were studied as a control group. sL and sE selectins were significantly higher in AML patients at diagnosis when compared to controls (P<0.01), but less at remission (P<0.01). Furthermore, elevated sL and sE selectin levels were detected in patients with AML at relapse. sE and sL selectins were significantly higher in AML patients with extramedullary infiltration as compared to patients without extramedullary disease (P<0.001). Membrane expression of L selectin was positive in 20% of AML patients. However, none of the patients showed significant E selectin expression. Patients with higher sE and sL selectins at diagnosis have high probability of relapse compared to those with normal levels (P<0.01 and <0.001, respectively). The overall relapse predictability of sE and sL selectins was 84%. Moreover, patients with higher sE and sL selectins levels had shorter event free survival than patients with lower levels (P<0.001 for both). The overall mortality prediction using sE, sL, and cellular L selectin was 96%. In conclusion: (1) AML blast cell express and release sL selectins but not sE selectin, (2) sE and sL selectins and cellular L selectin may be useful prognostic markers in evaluating AML patients at diagnosis.

Serum Level of Intercellular Adhesion Molecule-1 in Children with Malignant Lymphoma

Objective: Toestimate the serum levels of soluble intercellular adhesion molecule-1 (s-ICAM-1) in children newly diagnosed with lymphoma and to correlate levels of s-ICAM-1 in lymphoma patients with clinical stage, pathological types, clinical and laboratory data and patient outcome. Subjects and Methods: Thirty-five children with newly-diagnosed malignant lymphoma (Non-Hodgkin’s lymphoma, NHL: 23), Hodgkin’s disease (HD: 12), and 8 apparently healthy subjects of matched age and sex taken as a control group were studied. For the patients and control group, the following tests were performed: complete blood count, and the following biochemical investigations: liver function tests, lactate dehydrogenase (LDH), and soluble ICAM-1 estimation using ELISA. In addition, for patients, pathological examination of lymph node biopsy for pathological grading, bone marrow aspiration and biopsy were done. Patients were observed for over 12 months or until death. Results: Serum ICAM-1 increased more in HD and NHL than in the control group (p < 0.000); also s-ICAM-1 increased in advanced stages and high-grade NHL (p < 0.008, 0.04, respectively). LDH levels were higher in patients compared to controls (p < 0.000). There was a positive correlation between high levels of s-ICAM-1 and increased levels of LDH in HD (r = 0.72, p < 0.008) and a positive correlation between high levels of s-ICAM-1 and increased ALT in NHL patients. A positive correlation between s-ICAM-1 levels and the presence of B symptoms in HD and NHL, and a positive correlation between elevated s-ICAM-1 levels and worse outcome in HD and NHL were detected. Conclusions: The data indicate that in children with malignant lymphoma, high serum levels of ICAM-1 correlated with tumor aggressiveness, and quantification of s-ICAM-1 levels may identify a subgroup of children with worse prognosis. Therefore, detection of s-ICAM-1 levels in children with malignant lymphoma might represent an additional disease-associated marker for use in the clinical management of the patients.

Extracellular accumulation of bioactive substances; Interleukin-1β (IL-1β) and plasminogen activator inhibitor-1 (PAI-1) in stored blood units and relation to bacterial contamination

Abstract Bacterial contamination of blood and its cellular components remains an unresolved problem in transfusion medicine and is considered to be the most common microbiological cause of transfusion associated morbidity and mortality. The present work was designed to explore the levels of two bioactive compounds interleukin-1 β (IL-1 β) and plasminogen activator inhibitor-1 (PAI-1) in stored blood units and their relation to bacterial contamination of these units. This study was conducted on 112 blood units obtained from blood bank of Mansoura University Children Hospital. Sequential blood samples were obtained both immediately at donation and after 10 days for measurement of IL-1 β and PAI-1 and for bacterial culture by BACTEC 9050 system. There was statistically significant increase in both IL-1 β and PAI-1 (P = 0.0001) after 10 days of blood units storage. Bacteriological culture revealed no growth in 68% and positive growth in 32% of blood units. The commonest isolated organism was Staphylococcus aureus (15%) followed by Staphylococcus epidermedis (13%) then Yersinia sp. and Enterobacter sp. (2%) for each. From the present study we could conclude that stored blood units contain platelets and WBCs derived bioactive substances PAI-1 and IL-1 β which increase with the duration of blood storage. Furthermore, the extended duration of storage carries the danger of blood contamination by bacteria. Automated blood culture system seems to be helpful in identification of bacterial contamination of blood units. We recommend fresh blood transfusion as early as possible and the practice of Leucofiltration to avoid blood transfusion complications.

Erythrocyte Phosphatidylserine Exposure in β-Thalassemia

Introduction: Phospholipid asymmetry is well maintained in erythrocyte (RBC) membranes with phosphatidylserine (PS) exclusively present in the inner leaflet. Eryptosis, the suicidal death of erythrocytes, is characterized by cell shrinkage, membrane blebbing and cell membrane phospholipids scrambling with PS exposure at the cell surface. Erythrocytes exposing PS are recognized, bound, engulfed, and degraded by macrophages. Eryptosis thus fosters clearance of affected erythrocytes from circulating blood which may aggravate anemia in pathological conditions. Thalassemia patients are more sensitive to the eryptosis depletion and osmotic shock which may affect RBC membrane Phospholipid asymmetry. Aim: We aimed in this work to determine the erythrocyte PS exposure in splenectomized and non splenectomized β-Thalassemia (β-TM) patients and correlate it with the clinical presentation and laboratory data. Methods: RBCs were stained for annexin V (AV) to detect phosphatidylserine (PS) exposure in 46 β-TM patients (27 splenectomized and 19 non splenectomized) compared to 17 healthy subjects as a control group. Results: We reported significant increase in erythrocyte PS exposure in β-TM patients compared to control group (p=0.0001). Erythrocyte PS exposure was significantly higher in splenectomized β-TM patients as compared with non splenectomized β-TM patients (p=0.001). No correlation was found between erythrocyte PS exposure and clinical or hematological data of β-TM patients but there was positive correlation between erythrocyte PS exposure and ferritin level in β-TM patients. Conclusion: These findings suggest that β-TM patients have higher level of erythrocyte PS exposure and splenectomy was shown to aggravate erythrocyte PS exposure without aggravation of anemia.

P086 Once weekly recombinant human erythropoietin treatment for cancer-induced anemia in children with acute lymphoblastic leukemia receiving maintenance chemotherapy: A randomized case-controlled study

Background: Patients receiving chemotherapy for cancer often develop anemia, which can contribute to increased morbidity and reduced quality of life (QOL). Chemotherapy-induced anemia can be successfully treated using recombinant human erythropoietin (rHuEPO). Aim of the study: To demonstrate the effectiveness of once-weekly (QW) rHuEPO dosing to effect improved hemoglobin levels, decreased transfusion use, and improved functional outcomes and QOL in pediatric leukemic patients (ALL) receiving maintenance chemotherapy. Patient and methods: This was a prospective randomized, single-center, open-label, 12-week case-control study of epoetin alfa in pediatric patients with acute lymphoblastic leukemia (ALL) in remission receiving maintenance chemotherapy. Sixty patients were randomly assigned to receive either epoetin alfa (rHuEPO group 1⁄4 30 cases, 17 males and 13 females, age; 6.8 ^ 2.33 years), or no epoetin alfa (control group 1⁄4 30 cases, 16 males and 14 females, age; 6.76 ^ 2.28 years). Both groups were matched as regard age, sex, baseline Hb concentration, remission state, chemotherapy regimen, numbers and amount of blood transfusion, and leukemia state (both were low and standard risk). Epoetin alfa was administered at a dose of 450 IU/kg, once weekly, subcutaneously (s.c.) for 12 consecutive weeks. Endpoints were changes in hematologic and QOL parameters. Results: Among the 30 patients evaluable for hematologic response, the mean increase in Hb from baseline to time of final evaluation was 3.08 ^ 1.48 g/dl ( p , 0.001). An increase in Hb of ^ 2 g/dl, in the absence of blood transfusion, occurred in 70% of patients (21 of 30 patients) who were on the study for ^ 30 days. The overall response rate (Hb increase ^ 2 g/dl or Hb ^ 12 g/dl in the absence of blood transfusion) was 90% (27 of 30 patients). In 30 patients who were evaluable for QOL assessment, epoetin-a therapy was found to significantly (p , 0.001) improve mean cancer linear analog scale (CLAS) scores for energy level, ability to perform daily activity, and overall QOL from baseline to the time of final evaluation. QW epoetin-a was found to be well tolerated. Conclusion: Treatment with QW epoetin-a was found to increase Hb levels, decrease transfusion requirement, and improve functional status and QOL in anemic patients with ALL in maintenance receiving chemotherapy. The once-weekly schedule is convenient, safe, and may reduce the burden on patients, parents, and their caregivers by reducing the number of visits to the clinic.