Thrombophilic Risk of Factor V Leiden, Prothrombin G20210A, MTHFR, and Calreticulin Mutations in Essential Thrombocythemia Egyptian Patients.

OBJECTIVES: Essential thrombocythemia (ET) is one of the myeloproliferative neoplasms characterized by a sustained elevation of platelet numbers with a tendency for thrombosis and hemorrhage. The aim of this work is to establish the relation between calreticulin, factor V Leiden, prothrombin G20210A, and MTHFR mutations in ET patients and the thrombotic risk of these patients. METHODS: This study was carried out on 120 ET patients and 40 apparently healthy individuals as a control group. RESULTS: There were increases in WBCs, PLT counts, PT, fibrinogen concentration factor V Leiden, and MTHFR mutation in ET patients as compared to the control group (P < 0.05). Also, there were increases in WBCs, PLT counts, and hematocrit value in thrombosed ET patients as compared to the nonthrombosed ones (P < 0.05). On the contrary, there was no significantly statistical difference in ET patients with JAK2 V617F positive mutation versus the JAK2 negative group (P > 0.05) and in patients with cardiovascular risk factors versus patients with noncardiovascular risk factors (P > 0.05). ET patients with factor V Leiden, prothrombin gene, and CALR mutations were more prone to thrombosis (odds ratio 5.6, 5.7 and 4.7, respectively). On the contrary, JAk2V 617F and MTHFR mutations have no effect on the thrombotic state of those patients. CONCLUSION: There is a significant increase risk of thrombosis in ET patients with CALR mutation, thrombophilic mutations, as well as factor V Leiden and prothrombin gene mutation with a risk of developing leukemic transformation.

Assessment of annexin A5 and annexin A2 levels as biomarkers for pre-eclampsia: A pilot study.

Deficient anticoagulant activity of annexin A5 and deficient profibrinolytic activity of annexin A2 have been linked to increased risk of thrombotic events. Placental dysfunction due to fibrin deposition/microthrombi has been implicated in the pathogenesis of pre-eclampsia (PE). In this study, we aimed to assess serum levels of annexin A5 and annexin A2 in a cohort of PE patients and investigate their role as biomarkers for the development of the disease. We examined 80 women in total; 40 healthy pregnant women and 40 pregnant women with PE after 20weeks of pregnancy. Women were subjected to full clinical assessment, ultrasonography, and laboratory testing including complete blood picture, liver and kidney function tests and assessment of serum and urine proteins. Annexin A5 and annexin A2 were analyzed using enzyme-linked immunosorbent assay. The study showed serum annexin A2 but not annexin A5 was significantly reduced (P=0.029) in women with PE (total and severe cases) compared to those with normal pregnancy. The ROC analysis of annexin A2 level for the prediction of development of PE showed an area under the curve of 0.64 (P=0.029), and the best cut-off value was 0.89ng/ml with a sensitivity of 70.0% and a specificity of 70.0%. Univariate analysis showed annexin A2 of <0.89ng/ml, proteinuria, lower platelet count and higher BP were associated with significantly higher risk to develop PE. Based on this pilot study, serum annexin A2 levels may be a useful biomarker for pre-eclampsia. However, a larger study is required before a final conclusion is made.

Low cost biological lung volume reduction therapy for advanced emphysema.

BACKGROUND: Bronchoscopic lung volume reduction (BLVR), using biological agents, is one of the new alternatives to lung volume reduction surgery. OBJECTIVES: To evaluate efficacy and safety of biological BLVR using low cost agents including autologous blood and fibrin glue. METHODS: Enrolled patients were divided into two groups: group A (seven patients) in which autologous blood was used and group B (eight patients) in which fibrin glue was used. The agents were injected through a triple lumen balloon catheter via fiberoptic bronchoscope. Changes in high resolution computerized tomography (HRCT) volumetry, pulmonary function tests, symptoms, and exercise capacity were evaluated at 12 weeks postprocedure as well as for complications. RESULTS: In group A, at 12 weeks postprocedure, there was significant improvement in the mean value of HRCT volumetry and residual volume/total lung capacity (% predicted) (P-value: <0.001 and 0.038, respectively). In group B, there was significant improvement in the mean value of HRCT volumetry and (residual volume/total lung capacity % predicted) (P-value: 0.005 and 0.004, respectively). All patients tolerated the procedure with no mortality. CONCLUSION: BLVR using autologous blood and locally prepared fibrin glue is a promising method for therapy of advanced emphysema in term of efficacy, safety as well as cost effectiveness.

Association of Hemostatic Gene Polymorphisms With Early-Onset Ischemic Heart Disease in Egyptian Patients.

The association between hereditary thrombophilia and venous thrombosis is well established but controversial data exist with respect to arterial thrombosis. We performed a pilot study on 31 patients with acute myocardial infarction (AMI), 21 patients with unstable angina (UA), and 20 healthy volunteers to investigate the role of various hemostatic gene polymorphisms in young Egyptian patients, who survived their first ischemic heart disease (IHD). Thrombophilic gene polymorphisms were tested using multiplex polymerase chain reaction and reverse-hybridization technique. We showed an increased risk of AMI with factor V (FV) Leiden and prothrombin G20210A heterozygosity. The increased risks of UA was associated with GA and A allele of fibrinogen ß-455G→A polymorphism. Conversely, factor XIII (FXIII) Val34Leu GT and T allele were protective in the UA group. Nevertheless, the prevalence of FV H1299R, plasminogen activator inhibitor 1 4G/5G, glycoprotein IIIa C1565T, 5,10-methylenetetrahydrofolate reductase C677T, and A1298C mutations did not differ between patients with IHD and controls. The data have clinical implications regarding screening and thromboprophylaxis in high-risk individuals younger than 40 years.

VKORC1 and CYP2C9 genotypes in Egyptian patients with warfarin resistance.

Warfarin is the most commonly prescribed anticoagulant drug; however, a narrow therapeutic range and a high risk of bleeding or stroke complicate its clinical use. Warfarin resistance was defined as prolonged warfarin requirements of more than 15 mg/day to achieve therapeutic anticoagulation or failure to achieve therapeutic anticoagulation with more than 20 mg/day. The resistance is associated with polymorphisms of the vitamin K epoxide reductase-oxidase complex (VKORC1) and cytochrome P450-2C9 (CYP2C9) genes, which affect warfarin pharmacodynamics and pharmacokinetics, respectively. Identification of the VKORC1 -1639 (A/G) and CYP2C9 (*1/*2/*3) allelic variants was performed using the PGX-Thrombo Strip in 41 patients with warfarin resistance compared with 30 patients with normal warfarin response out of 352 diagnosed cases of deep vein thrombosis. In warfarin-resistant patients, the VKORC1-1639 genotype frequencies were GG 0.756, GA 0.244 and AA 0.0, whereas in warfarin responder patients, they were: GG 0.333, GA 0.400 and AA 0.276 with P ≤ 0.001. The CYP2C9 genotype frequencies showed nonsignificant difference in both group of patients (P = 0.31). Our results suggest that the VKORC1-1639 GG and the wild type CYP2C9*1*1genotypes are associated with the high-dose requirement for warfarin therapy, and that VKORC1-1639 GG is responsible for warfarin resistance and failure in Egyptian patients.

Prognostic relevance of Wilms tumor 1 (WT1) gene Exon 7 mutations in-patient with cytogenetically normal acute myeloid leukemia.

This study aimed to assess the prognostic influences of Wilms tumor 1 (WT1) gene mutations in cytogenetically normal acute myeloid leukemia (CN-AML) among Egyptian patients. Exon 7 of WT1 was screened for mutations in samples from 82 CN-AML patients out of 203 newly diagnosed AML patients, using a high-resolution capillary electrophoresis. Seven out of 82 AML patients (8.3 %) harbored WT1 mutations. There was no significant difference between the mutant WT1 and wild type AML patients as regard age, sex, French-American-British subtypes and the prevalence of success of induction remission therapy (P < 0.5). AML patients with mutant WT1 had shorter overall survival as compared to those patients with wild WT1 (HR = 1.38; 95 % CI 4.79-6.86; P = 0.004). In conclusion, CN-AML patients with WT1 gene mutation have poor clinical outcome. We recommend testing the WT1 mutations as part of molecularly based risk assessment and risk-adapted treatment stratification of patients with CN-AML.

The condensed MCMDM-1 VWD bleeding questionnaire as a predictor of bleeding disorders in women with unexplained menorrhagia.

Menorrhagia is a common clinical problem and is unexplained in more than 50% of women. Many studies have suggested that underlying bleeding disorders are prevalent in menorrhagic women. However, the assessment and quantifying of hemorrhagic symptoms are still limited and not widely used. Thirty women aged 11-31 years with a clinical diagnosis of unexplained menorrhagia were investigated to assess the diagnostic utility of the condensed MCMDM-1VWD bleeding questionnaire as a predictive of bleeding disorders in these women. In addition to administration of the questionnaire, comprehensive hemostatic testing was performed to all women. The incidence of inherited bleeding disorders among this group was 66.6% (20/30). Eight patients had von Willebrand disease (VWD) and seven had possible Glanzmann's thrombasthenia. Rare bleeding disorders including hemophilia A carrier, Afibrinogenemia, Factor V deficiency and combined factor V and factor VIII deficiency were also identified. The receiver operator characteristic analysis of the condensed MCMDM-1 VWD bleeding questionnaire in menorrhagic women showed that the cutoff, sensitivity, specificity, positive and negative predictive values were 3.5, 85, 90, 89 and 86%, respectively. Bleeding score was strongly correlated to bleeding time in women with possible Glanzmann's thrombasthenia. In VWD, a significant inverse correlation between the bleeding score and the VW factor levels was detected with a significant increase of bleeding score in type III VWD compared with type I. Bleeding disorders are common in women with unexplained menorrhagia and the condensed MCMDM-1VWD bleeding questionnaire can distinguish between menorrhagic women with and without bleeding disorders and help assess their severity.

Reverse hybridization StripAssay detection of beta-thalassemia mutations in northeast Egypt.

AIM: beta-Thalassemias are widely distributed in Mediterranean and Middle Eastern countries. Reverse hybridization StripAssay method is reported to be rapid, simple, reproducible and less expensive. The aim of this study is to evaluate reverse hybridization StripAssay method for detection of beta-thalassemia mutations in Egyptian children. SUBJECTS AND METHODS: Forty children with beta-thalassemia major with mean age of 10.33+/-4.75 years were recruited consecutively from outpatient Hematology Clinic of Mansoura University Children's Hospital. Mutation analysis was performed by the beta-Globin StripAssay MED. RESULTS: The most frequent mutant alleles detected were; IVS 1.110, IVS 1.1 and IVS 1.6 accounting for 33.75, 27.5 and 18.75% respectively. The detection rate of the used method in our population was 90%. CONCLUSION: beta-globin StripAssay is a fast, easy-to-perform and reliable method for genetic screening of beta-thalassemia patients in Egypt. IVS 1.110, IVS 1.1 and IVS 1.6 are the most frequent mutant alleles with poor phenotype/genotype correlation.

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.