Acute myeloid leukemia (AML) patients show high relapse rates and some develop conventional chemotherapy resistance. Leukemia Stem Cells (LSCs) are the main player for AML relapses and drug resistance. LSCs might rely on the B-cell-specific Moloney murine leukemia virus integration site-1 (BMI-1) in promoting cellular proliferation and survival. Growth of LSCs in microenvironments that are deprived of nutrients leads to up-regulation of the signaling pathways during the progression of the disease, which may illustrate the sensitivity of LSCs to inhibitors of those signaling pathways as compared to normal cells. We analyzed the expression of LSC markers (CD34, CLL-1, TIM-3 and BMI-1) using quantitative RT-PCR in bone marrow samples of 40 AML patients of different FAB types (M1, M2, M3, M4, M5, and M7). We also studied the expression of these markers in 2 AML cell lines (Kasumi-1 and KG-1a) using flow cytometry and quantitative RT-PCR. The overexpression of TIM-3, CLL-1, and BMI-1 was markedly correlated with poor prognosis in these patients. Our in vitro findings demonstrate that targeting BMI-1, which markedly increased in the leukemic cells, was associated with marked decrease in leukemic burden. This study also presents results for blocking LSCs' surface markers CD44, CLL-1, and TIM-3. These markers may play an important role in elimination of AML. Our study indicates a correlation between the expression of markers TIM-3, CLL-1, and especially of BMI-1 and the aggressiveness of AML and thus the potential impact of prognosis and therapies that target LSCs on improving the cure rates.

The lymph node (LN) is the site of chronic lymphocytic leukemia (CLL) cell activation and proliferation. Aberrant microRNA (miRNA) expression has been shown to have a role in CLL pathogenesis; however, a comparison of miRNA expression between CLL cells in the LN and the peripheral blood (PB) has previously not been reported. On the basis of the analysis of 17 paired LN and PB samples from CLL patients, we identify a panel of miRNAs that are increased in LN CLL cells correlating with an activation phenotype. When evaluated in CLL cells from 38 patients pre and post treatment with ibrutinib, a subset of these miRNAs (miR-22, miR-34a, miR-146b and miR-181b) was significantly decreased in response to ibrutinib. A concomitant increase in putative miRNA target transcripts (ARID1B, ARID2, ATM, CYLD, FOXP1, HDAC1, IBTK, PTEN and SMAD4) was also observed. Functional studies confirmed targets of ibrutinib-responsive miRNAs to include messenger RNA transcripts of multiple tumor suppressors. Knockdown of endogenous miR-34a and miR146b resulted in increased transcription of tumor suppressors and inhibition of cell proliferation. These findings demonstrate that ibrutinib downregulates the expression of a subset of miRNAs related to B-cell activation leading to increased expression of miRNA targets including tumor suppressors and a reduction in cell proliferation.

Several methods were tried for Giardia detection in stool. This study aimed to compare between the results of ordinary microscopy, direct immunofluorescence assay (DIF), and flow cytometry (FC) for the detection of Giardia cyst in human stool samples. The study included 84 children recruited from outpatient clinics of Mansoura University Children Hospital. Fecal samples were processed and examined for Giardia cysts using conventional microscopy, DIF, and FC. Among 84 fecal samples, 40 (47.6%) were diagnosed as Giardia-positive by saline wet mount, while DIF and FC detected 52 (61.9%), and 38 (45%) Giardia-positive cases, respectively. When compared with DIF as a gold standard method, ordinary microscopy had 76.9% sensitivity and 100% specificity while the FC had a sensitivity of 73.1% and 100% specificity, with statistically significant differences between DIF and the other two methods (P < 0.05). DIF was able to detect as few as 500 cysts/g of concentrated stool, yielding a threshold higher than ordinary microscopy (1,800 cyst/g) even after concentration. It is concluded that direct microscopic examination is reliable in Giardia diagnosis as a first choice test. DIF is an excellent technique in clinically suspected cases after negative microscopy. FC was found to be less sensitive to obtain accurate organisms' count but it could be an effective alternative method for the detection of Giardia cysts, especially for large-scale epidemiological studies or extensive surveillance programs as it has the beneficial attribute of speed and do not depend on an experienced microscope viewer. However, DIF remains the gold standard while FC still requires significant technical improvements before it can compete with DIF for Giardia diagnosis. V C 2012 International Clinical Cytometry Society

It has been known that the insertion/deletion mutation of CALR gene is the second deriver mutation in myeloproliferative neoplasm (MPN) of essential thrompocythemia (ET) and primary myelofibrosis (PMF). As the molecular workup has been incorporated for the prospective screening and diagnosis of MPN in our Oncology Center. An Egyptian 87 cohort of patients with non-mutated JAK2 (58 ET and 29 MF) were investigated using polymerase chain reaction (PCR) as a pilot study. We found that 37 out of 87 patients (42%) were carrying CALR mutations (30 out of ET (52%) and 7 out of MF (24%)). Sanger sequencing was used to determine the type of CALR mutations in all positive patients and we found that 13 out of 37 (35%) had type 1/type 1-like and 36 out of 37 (97%) with type 2/type 2-like. This CALR mutation profile in Egyptian patients appear different from the western status as type2/type 2-like is the highest in our patients (97%) versus 35-45% and type1/type 1-like was 35% versus 55-65% compared to western results. Meanwhile, the clinical course and phenotype of our cohort of patients is not similar to that in western as there is no significant difference of overall survival between type1/type1-like and type2/type2-like. This finding might be due to the different environmental and genetic backgrounds of Egyptian population. A part of it might be related to the HCV infection as 12 out of 37 (32%) had HCV infection. Further study is in progress on a large number of patients to correlate that with the clinicopathological status, response to therapy and the mechanistic pathway of oncogenic transformation. Disclosures No relevant conflicts of interest to declare.

The foetal like tyrosine kinase 3 mutation (Flt3) gene encodes a tyrosine kinase receptor that regulates proliferation and differentiation of haematopoietic stem cells. In children with acute myelogenous leukaemia (AML), internal tandem duplication of the Flt3 gene (Flt3/ITD) was previously reported and correlated to poor prognosis. Limited data are available about childhood acute lymphoblastic leukaemia (ALL). We analysed bone marrow specimens from 55 newly diagnosed acute leukaemia cases including 30 AML and 25 ALL by genomic PCR for the presence of Flt3/ITD and correlated its presence with clinical outcome. Tandem duplication was found in 6/30(20%) AML cases: 2/8 M1, 1/8 M2, 2/6 M3, 1/6 M4 with loss of heterozygosity (LOH) in two cases. Four of the cases with duplication were males and the other two cases were females. Complete remission was achieved in 16.6% of cases with duplication vs. 45.8% in cases without duplication. Failure to achieve induction remission was noted in 50% of cases with duplication vs. 29.1% in cases without duplication. FLt3/ITD was not found in ALL cases. Lineage restriction analysis revealed that Flt3/ITD was not present in lymphocytes, suggesting a lack of stem cell involvement for this mutation. Flt3/ITD was the most significant prognostic factor as declared by multivariate analysis. Patients with Flt3/ITD appear to be refractory to primary induction therapy, and for those who achieve remission, there is a high rate of relapse and death so there may be an association between this type of mutation and patient outcome.

Immune thrombocytopenic purpura is an acquired disorder, in which accelerated platelet consumption is due to platelet autoantibodies. The aim of this study was to investigate the clinical value of platelet autoantibodies assay in children with ITP and to evaluate fl ow cytometry in the detection of platelet autoantibodies in comparison with monoclonal antibody specifi c immobilization of platelet antigen (MAIPA) assay. We measured platelet autoantibodies by fl ow cytometry and MAIPA in 18 children with ITP (6 acute, 7 chronic and 5 in remission), in addition to 5 healthy children with matched age and sex as a control group. Signifi cant elevation of platelet-associated immunoglobulin G (PAIgG), PAIgM and PAIgA was demonstrated in children with acute ITP compared to controls and children with chronic ITP (P < 0.05). There was signifi cant elevation of PAIgG and PAIgM in children with acute ITP compared to children with ITP in remission (P < 0.05). There was signifi cant negative correlation between platelet count and PAIgG levels in ITP children (r = -0.717; P = 0.001). Flow cytometry found PAIgG in 94.4% of ITP children. MAIPA has detected platelet specifi c IgG autoantibodies in 83.3% of ITP children. ROC analysis revealed sensitivity of 94%, specifi city of 57% with overall accuracy of 83% for detection of PAIgG by fl ow cytometry compared to MAIPA.Conclusions Platelet autoantibodies testing in ITP can discriminate acute from chronic forms of the disease and is helpful in follow up of patients. Determination of PAIgM in combination of PAIgG could be of interest in the investigation of ITP. Flow cytometry is a sensitive method of detection of platelet autoantibodies that could be used in screening of suspected ITP and should be followed by MAIPA assay in positive cases to establish the diagnosis.

BackgroundThe prevalence of Hepatitis C virus in Egypt reaches 15%, which is considered the highest in the world. Genotype 4 represents 93 % of Egyptian HCV infections. Non-Hodgkin lymphoma (NHL) is the 5th most common cancer in Egypt. The association between HCV infection and occurrence of B-cell NHL is well known while data are scarce in Eastern countries.ObjectivesWe aimed to evaluate the prevalence of HCV infection among patients with B-cell NHL and the clinical characteristics of HCV associated B-cell NHL in the Delta region (Mansoura-Egypt).MethodsBetween March 2012 and March 2013, 110 adult patients newly diagnosed with B-cell NHL were enrolled in the current study. This study was carried out at Oncology Center, Mansoura University. Study subjects provided serum for HCV testing.ResultsThe prevalence of HCV infection among these patients was 61% (67/110 patients). Among them, 80% (32/40 tested patients) presented with viremia. In contrast with the histological distribution previously described in Northern regions, the majority of HCV associated lymphomas were DLBCLs (72%) followed by SLL/CLL (13%), follicular lymphomas (7.5%) and marginal zone lymphomas (7.5%).ConclusionsB-cell lymphomas are highly associated with HCV infection in Egypt. Further developments are needed to give access to antiviral treatment for these patients.

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