Salah Aref

Urokinase Plasminogen Activator Receptor and Soluble Matrix Metalloproteinase-9 in Acute Myeloid Leukemia Patients: A Possible Relation to Disease Invasion

Abstract Extracellular proteolytic enzymes of the urokinase-type plasminogen activator (uPA) and metalloproteinase (MMP) family play a crucial role in the matrix degradation and tissue remodeling process characteristic of malignant disorders. The receptor for urokinase plasminogen activator (uPAR) serves to localize and intensify the action of UPA and is expressed on the surface of malignant cells. Although the biological significance of MMP-9 and soluble urokinase receptor in growth and progression of lymphoid neoplasm is understood, its clinical significance in acute myeloid leukemia (AML) has not been fully elucidated. In this study, we determined the levels of soluble urokinase-type plasminogen activator receptor (suPAR), cellular uPAR and sMMP-9 in 43 newly diagnosed AML patients at diagnosis, before chemotherapy, and also studied 10 normal subjects served as a control group. After chemotherapy suPAR and MMP-9 were determined at remission and relapse. The levels of suPAR, cellular PAR were significantly higher (P=0.001, 0.001) and MMP-9 was significantly lower (P=0.001) in AML patients at diagnosis as compared to controls. suPAR and MMP-9 levels were significantly lower in AML patients who achieved complete remission (CR) as compared to those who did not (P=0.001 for both). Levels of suPAR and MMP-9 were significantly correlated to peripheral blood blast cells (r=0.88, P=0.001; r=0.65, P=0.001, respectively) and blast cell distribution ratio (BCDR, r=0.84, P=0.001; r=65, P=0.001, respectively). suPAR, cellular PAR and MMP-9 were significantly higher in patients with extramedullary infiltration as compared with those without (P=0.001, 0.001, <0.05). The suPAR, cellular uPAR, and MMP-9 levels were uneven in AML FAB subtypes being highest in M5(P<0.05 for all). MMP-9 and suPAR levels were correlated with the disease status. In AML survivors, MMP-9, cellular uPAR and suPAR were significantly lower as compared to non-survivors (P=0.001 for all). In conclusion, MMP-9 and suPAR levels might be used as a marker for disease activity and may contribute to blast cell dissemination. MMP-9 and suPAR may be target molecules in the strategy of treatment of AML.

Angiogenesis factor pattern differs in acute lymphoblastic leukemia and chronic lymphocytic leukemia.

Abstract Angiogenesis is an important event in the survival and progression of solid tumors. The angiogenic status and the exact role of the angiogenic cytokines in lymphoid leukemia has not been fully elucidated. We have investigated the profile of the systemic components of angiogenic regulation in B-lineage acute lymphoblastic leukemia (B-ALL) and B-chronic lymphocytic leukemia (B-CLL), namely vascular endothelial growth factor (VEGF), tumor necrosis factor-alpha (TNF-α), endostatin and matrix metalloproteinase-9 (MMP-9) using enzyme-linked immunosorbent assay (ELISA). In B-ALL patients, sVEGF, and MMP-9 were significantly lower than control levels at diagnosis (p < 0.001) and increased to near control levels in remission (p > 0.05). Both serum TNF-α and endostatin levels showed no significant difference at diagnosis (p > 0.05) and in remission (p > 0.05) compared to control levels. VEGF, TNF-α, MMP-9 and endostatin levels were not significantly correlated with peripheral white cell count or bone marrow blast cell count, but were positively correlated with platelet count. In B-CLL patients, serum VEGF, MMP-9 and TNF-α were significantly higher (p < 0.001 = 0.009, 0.007, respectively) and decreased to near control levels in remission (p > 0.05 for all). Serum endostatin levels showed no significant difference at diagnosis and in remission compared to control levels (p > 0.05). A significant positive correlation between VEGF, TNF-α, MMP-9 and peripheral white cell counts, bone marrow lymphocytic count and platelets count were found. In conclusion, our data suggest that the driving forces of angiogenic factors (VEGF, TNF-α and MMP-9) in adult B-ALL appears different from that in B-CLL patients.

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.

Thrombopoietin (TPO) Levels in Hepatic Patients with Thrombocytopenia

Abstract Thrombocytopenia is a common problem complicating the course of liver disease. One of the postulated mechanisms in chronic liver disease is impaired production of the hormone, thrombopoietin (TPO). The aim of present study was to evaluate the role of TPO on the occurrence of thrombocytopenia. Serum TPO levels was determined by ELISA in 40 patients with liver disease (11 seropositive with hepatitis C; 10 with mixed liver cirrhosis; 19 with bilharzial hepatic fibrosis), plus 14 normal healthy subjects as a control group. The sTPO levels were unevenly distributed among the liver disease subgroups being the highest in the group with HCV (median 1232.0, range 154.7-2042.0 pg/ml) followed by the mixed cirrhosis group (556.5; 342.0-1497.0 pg/ml) and lowest among the bilharzial hepatic fibrosis group (130.0; 22.0-204.0 pg/ml) ( P <0.01). While sTPO levels in HCV and cirrhotic group were significantly higher when compared to the control group (97.0; 19.0-377.0 pg/ml), those in the Bilharzial hepatic fibrosis group were not significantly elevated ( P > 0.05). There is significant negative correlation between sTPO levels and spleen size ( R =−0.3, P =0.043); but there was no correlation with platelet count ( R =0.09, P >0.05). In addition, sTPO levels were significantly higher in patients with platelet counts ≥60×10 9 /l as compared to those with platelet counts <60×10 9 /l ( P =0.04). Using the receiver operating curve (ROC) at sTPO cut off value ≥ vs. < 368 ng/ml, most of HCV and cirrhotic patients had higher sTPO levels (81.8 and 80.0%, respectively), while all Bilharzial hepatic fibrosis group (100%) had lower sTPO levels. In conclusion, sTPO levels had no role in the occurrence of thrombocytopenia in liver disease patients and other factors appear to be more important. It also appears that the mechanism controlling sTPO levels might be different in cirrhotic patients compared to Bilharzial hepatic fibrosis patients.

Soluble Hepatocyte Growth Factor (sHGF) and Vascular Endothelial Growth Factor (sVEGF) in Adult Acute Myeloid Leukemia: Relationship to Disease Characteristics

Abstract There is little understanding of the factors controlling the mobilization of blast cells from bone marrow to peripheral blood and tissues. The aim of this study was to evaluate the soluble hepatocyte growth factor (sHGF) and vascular endothelial growth factor (sVEGF) levels in newly diagnosed patients with acute myeloid leukemia (AML) and to correlate these levels with the clinico-pathological features. Sixty-three patients with AML and 15 normal controls were included in this study. The levels of sHGF and sVEGF were determined by enzyme linked immunosorbent assay at diagnosis and after remission induction chemotherapy. Our results revealed significantly increased plasma levels of sHGF and sVEGF at diagnosis when compared to both control and remission levels (P=0.000 for both). The sHGF and sVEGF levels differed between AML FAB subtypes (P=0.000). The highest concentrations were found in M5 followed by M4. SHGF and sVEGF were directly correlated with peripheral white cell counts (WBC) (r=0.836, P=0.000, r=0.718; P=0.000, respectively), but inversely correlated with blast cell distribution ratio (BCDR) (r=−0.785, P=0.000, r=−0.664, P=0.000, respectively). Moreover, both sHGF and sVEGF levels were significantly elevated in AML patients with extra-medullary infiltration as compared to those without (P=0.000, 0.006, respectively). The sHGF but not sVEGF levels were significantly elevated in patients who died compared to those who relapsed and to patients in complete remission (P=0.02, 0.08, respectively). Logistic regression analysis revealed that the sHGF level at diagnoses is a powerful predictor of the patient outcome, compared to sVEGF. In conclusion: our data support the hypothesis that angiogenic factors play a functional role in blast cell movement from the bone marrow to peripheral tissues. Assessment of sHGF at AML diagnosis is likely to be helpful in predicting patient outcome and selecting optimal therapeutic regimen.

Increased VWF antigen levels and decreased ADAMTS13 activity in preeclampsia

Abstract This study aimed to assess the von Willebrand factor (VWF) antigen levels and its proteolytic enzyme ADAMTS13 activity in preeclampsia. The study includes 10 non-pregnant women, 50 normal pregnancy, and 110 preeclamptic (PE) women at the same period of pregnancy. For all studied groups plasma ADAMTS13 activities were determined with the FRETs-VWF 73 assay, while VWF antigen levels with an immunoturbometric assay. The plasma ADAMTS13 activity was significantly reduced in PE as compared with normal pregnancy and non-pregnant women (P < 0.01 for both). In contrast, plasma VWF antigen and VWF RCO were significantly elevated in PE, as compared with normal pregnancy group as well as non-pregnant women (P < 0.01 for both). In conclusion, reduction of plasma ADAMTS13 and elevation in VWF might have a role in the pathogenesis of PE.

Cyclin D1 expression in acute leukemia

Abstract Background: Disorders of the cell cycle regulatory machinery play a key role in the pathogenesis of cancer. Over expression of cyclin D1 protein has been reported in several solid tumors and certain lymphoid malignancies, but little is known about the involvement of cyclin D1 in acute leukemia. Patients and methods: In this study, we analyzed the expression of cyclin D1 at protein level in, 40 newly diagnosed patients with acute myeloid leukemia (AML), 10 patients with acute lymphoblastic leukemia (ALL), and 11 normal controls using flow cytometry. Results: The expression of cyclin D1 was not significantly different in AML group as compared to normal controls. On the other hand, over expression of cyclin D1 was evident in ALL group (4/10) as compared to that in healthy control. The ALL cases with cyclin D1 over expression were significantly correlated to blast cell counts in the peripheral blood and bone marrow (BM) but not with hemoglobin level, WBC, and platelets count. The ALL group with lymphadenopathy and organomegaly express significantly higher cyclin D1 over expression as compared to those without. Conclusion: The biological value of cyclin D1 over expression might be different in AML and ALL.

Assessment of Bcl-2 Expression as Modulator of Fas Mediated Apoptosis in Acute Leukemia

Abstract Apoptosis is the primary mechanism through which most chemotherapeutic agents induce tumor cell death. The balance in the expression of pro (Fas/CD95) and anti-apoptotic protein (Bcl-2) may control the response of leukemic cells to chemotherapy and subsequently affect the patients prognosis. The aim of this study was to determine the levels of Bcl-2 and Fas expression on blast cells from patients with acute leukemia and to correlate the degree of expression to the clinical and laboratory prognostic factors and the patients outcome. Forty newly diagnosed patients with acute leukemia (16 ALL, 24 AML) were included in the study. Ten normal subjects of matched age and sex were studied as a reference control group. The degree of Bcl-2 and Fas expression on acute leukemia blast cells were assessed before the start of therapy and on mononuclear cells after 1 year of follow up, using flow cytometry. The degree of Bcl-2 and Fas expression were significantly higher in AML (P<0.01,<0.05, respectively) and ALL (P<0.01, <0.05, respectively) as compared to controls. The expression of Fas and Bcl-2 was related to FAB type with the highest Bcl-2 and lowest Fas expression in M5 and T-ALL (P<0.01, for all). In ALL, patients responding to induction chemotherapy revealed lower Bcl-2 and higher Fas expression when compared to non-responders (P<0.05). In contrast, in AML the difference between responders and non-responders to induction chemotherapy regarding Bcl-2 and Fas expressions was not statistically significant (P>0.05). Bcl-2 and Fas expression were significantly elevated in the relapsed acute leukemia group (in both AML and ALL) when compared to those in remission (P<0.01, <0.05, respectively). Bcl-2 and Fas expression at diagnosis was not significantly different when those surviving were compared to the group who had died, either in the ALL or AML groups (P>0.05). Bcl-2 expression was significantly correlated to bone marrow blast cell counts (R=0.6, P<0.01), blast cell distribution ratio (R=0.4, P<0.05) and lymphadenopathy (R=0.33, P<0.05). Whereas Fas expression was significantly correlated to bone marrow blast cell counts (R=0.52, P<0.01). In conclusion, assessment of Bcl-2 and Fas expression at diagnosis in acute leukemia (1) could predict responsiveness to induction chemotherapy in ALL but not in AML group but (2) could not predict patients out come both in ALL and AML groups.

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.