Expression of Enhancer of Zeste Homolog 2 (EZH2) Gene in Acute Myeloid Leukemia.

BACKGROUND: Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of the chromatin modifying enzyme polycomb repressive complex 2 (PRC2). As a complex, these proteins selectively silence target genes through trimethylation of histone 3 at lysine 27. EZH2 is strongly oncogenic. It has been observed in various malignancies which makes it an interesting therapeutic target. Whether it functions as a tumor suppressor or oncogene in acute leukemia is not settled. Aim of this work: This study aimed at determining the expression levels of EZH2 gene in a cohort of adult Egyptian patients newly diagnosed with acute myeloid leukemia (AML). MATERIALS AND METHODS: The present study included 45 de novo AML patients and 40 healthy subjects of matched age and sex as a control group. All study participants were subjected to complete blood count (CBC), bone marrow examination, immunophenotyping, conventional cytogenetic studies and Detection of EZH2 gene expression levels by real time quantitative polymerase chain reaction (RQ-PCR). RESULTS: EZH2 was significantly downregulated in AML patients compared to controls (p<0.001). There was no significant difference in EZH2 level when considering age, sex, bone marrow blasts count, cytogenetic studies, type or site of infection. Low EZH2 expression was associated with higher mortality (31 patients, 68.9%). CONCLUSIONS: Low EZH2 expression is prevalent in Egyptian AML patients subsequently; it is suggested to function as tumor suppressor gene rather than an oncogene. Moreover, EZH2 downregulation is associated with resistance to chemotherapy and high mortality rate.

MicroRNA 30a Mediated Autophagy and Imatinib Response in Egyptian Chronic Myeloid Leukemia Patients.

Imatinib Mesylate is the drug used for targeted tyrosine kinase inhibition in the beginning of management of all Chronic Myeloid Leukemia (CML) newly diagnosed cases. However, resistance presents a considerable limit to its efficacy. Currently, it is impossible to anticipate IM resistance which makes the recognition of early flags an important treatment goal in CML. In this work we studied the connection between microRNA 30a (miR-30a) and Beclin 1 mediated autophagy and IM resistance in Egyptian CML patients. The study included newly diagnosed (group I, n = 20), imatinib responder (group II, n = 30), imatinib resistant (group III, n = 30) CML patients and a healthy demographically matched control group (group IV, n = 20). miR-30a expression was assayed by quantitative reverse transcription polymerase chain reaction. The variation in expression of miR-30a between CML cases and healthy controls was calculated using relative quantification method (2-ΔΔCT). Beclin 1 was assayed in Peripheral blood mononuclear cells by western blotting. miR-30a was over expressed and Beclin 1 was under expressed in imatinib responders compared to resistant cases median 1.21(0.55-3.02) versus median 0.65 (0.03-1.0) (p = 0.001) and median 950.0 (400.0-2410.0) versus, median 1570.0 (920.0-5430.0) (p < 0.001) respectively. Beclin 1 correlated significantly positively with miR-30a in new cases (p = 0.001) and negatively in imatinib responders (p = 0.021). Receiver Operating Curves demonstrated the performances of miR-30a and Beclin 1 to detect imatinib resistance. They showed sensitivities of 97.14% and 94.29% and specificities of 53.33% and 42.22% at the cut-off values of 1 and 940 respectively. Both miR-30a and Beclin 1 levels showed a relation with imatinib response and can therefore be put forward as valuable markers for detection of resistance and may also have promising future therapeutic implications.

TET2 Single Nucleotide Polymorphism in Myeloid Neoplasms Among Egyptian Patients.

Acute myelogenous leukemia (AML) is a heterogeneous disease characterized by myeloid progenitor cells uncontrolled proliferation gradually replacing normal hematopoiesis. To evaluate Ten Eleven Translocation 2 gene (TET2) single nucleotide polymorphism (SNP) (rs2454206, rs34402524, rs61744960) in AML, and chronic myeloid leukemia (CML) in relation to their disease prognostic criteria. The study included 136 subjects; 52 AML, 54 CML and 30 subjects as control group matched for age and sex. Routine investigations including CBC, bone marrow aspiration, flow cytometry biochemical investigations and cytogenetics and molecular study were performed accordingly. DNA extraction and SNP assay for TET2 gene polymorphism was done using (Thermo-Fisher predesigned SNP, USA) PCR prism 7500. The mean age was 43.4 ± 14.0 years in AML patients, 45.98 ± 15.7 years in CML patients and 39.3 ± 6.587 years in control group (p > 0.05). The frequency of TET2 SNP rs 34402524 ranged from heterozygous to homozygous in both AML (46%, 54%) and CML (48%, 46.2%) groups but was mainly homozygous among the control (80%) group (p = 0.012). TET2 SNP rs 2454206 was mainly wild in CML (65.4%) and control (63.3%) groups compared to AML as wild was only in (46%) and heterozygous in (44%) with only 10% being homozygous (p = 0.046). TET2 SNP rs 61744960 showed a homozygous pattern among all three group (AML CML and control) showing no statistical significance (p = 0.528). Eventhough, higher non responders to treatment were among homozygous and heterozygous groups yet, response to therapy as respect to specific TET2 SNP showed no significant variation (p > 0.05). TET2 SNP in CML cases did not alter the prognostic criteria as no statistical significance was noted (p > 0.05) except for TET2 SNP rs 34402524 where homozygous cases had larger spleen size (p = 0.019). TET2 SNP is common in Egyptian myeloid neoplasm. This is the first study in this field and further studies are recommended to investigate TET2 and relation to other hematological malignancies and leukemogenesis transformation.

Relationship between HLA-DRB1*0101, DRB1*0301 alleles and interleukin-12 in haemophilic patients and hepatitis C virus positive hepatocellular carcinoma patients.

The immune system can effectively eliminate hepatitis C virus (HCV) in 15 % of acute hepatitis cases. It is assumed that certain HLA-DR alleles present HCV epitopes more effectively to CD4 helper T cells than do others resulting in vigorous proliferative response to these epitopes and probably HCV recovery. So, we aimed at investigating the frequency of HLA-DRB1*0101 and DRB1*0301 alleles in child and adult haemophilics and in HCV positive hepatocellular carcinoma (HCC) patients in a trial to predict patients who require early therapeutic intervention. We also evaluated interleukin (IL)-12 levels in these patients since IL-12 induces interferon (IFN)-gamma production. This study was conducted on 50 antiHIV negative male patients subdivided into: 25 HCV negative haemophilics (group I), 10 HCV positive haemophilics (group II) and 15 HCV positive HCC (group III). Fifteen healthy persons of matched age and free of HCV and HIV infections were chosen as controls (group IV). All patients and controls were subjected to thorough history taking and clinical examination, routine and diagnostic investigations, viral markers, DRB1*0101 and DRB1*0301 amplification by polymerase chain reaction and plasma IL-12 quantitation by enzyme linked immunosorbent assay (ELISA). The frequencies of DRB1*0101 and DRB1*0301 were 20% and 30% respectively in HCV positive haemophilics and 13.3% and 40%, respectively in HCC. IL-12 levels were significantly lower in HCC cases than in HCV positive haemophilics. Among the haemophilics, IL-12 levels were non-significantly higher in children than in adults and were associated with the given number of blood product bags. DRB1*0101 and DRB1*0301 may have a role in HCV clearance and persistence in Egyptian patients with haemophilia and HCC. Low IL-12 levels encountered in HCV positive haemophilics suggest its relation to immunopathogenesis and outcome of HCV infection.

Leptin in acute leukaemias: relationship to interleukin-6 and vascular endothelial growth factor.

Leptin alone and in combination with other cytokines has a stimulatory effect on proliferation of leukaemic cells. This effect may be due to prevention of apoptosis of progenitor cells or upregulation of specific receptors on leukaemic precursors that make them more responsive to stimuli. This work investigates the relationship between serum leptin level, serum interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) in acute leukaemic patients. The relationship to blood cell counts, haemoglobin and response to chemotherapy was also investigated. The study included 25 acute leukaemic male patients [15 acute myeloid leukaemia (AML) and 10 acute lymphoblastic leukaemia (ALL)] and 15 age and sex matched healthy controls. All were subjected to thorough history taking, clinical examination, complete blood picture, hepatic and renal function tests and determination of serum leptin, IL-6 and VEGF levels. In addition, patients were subjected to bone marrow aspiration, cytochemistry and immunophenotyping study and serum leptin assay after chemotherapy. Serum leptin level showed statistically significant elevation only in AML group (p<0.01). This elevation was unrelated to the presence of extramedullary infiltration or response to chemotherapy and correlated only with body mass index (p<0.05). In ALL, the mean serum leptin level was insignificantly different from the controls. In both AML and ALL, there was no significant difference in serum leptin level before and after treatment. Statistically significant elevation of IL-6 and VEGF, uncorrelated with serum leptin level was detected in AML patients when compared with the controls. No correlation was found between serum leptin level and any of the studied haematological parameters. It is concluded that the release of leptin, IL-6 and VEGF may be regulated by different mechanisms leading to diversity in clinical features of the disease.