Nahla Farahat

Ser-249 TP53 and CTNNB1 mutations in circulating free DNA of Egyptian patients with hepatocellular carcinoma versus chronic liver diseases

Circulating free DNA (CFDNA) has been shown to be a good source of liver tissue-derived DNA in African and Asian patients with chronic liver disease or HCC. In Egypt, HCC is a frequent carcinoma and mostly occur in the context of chronic infection by HCV, a widespread infection in the Egyptian population. Here we have examined the presence of mutations in TP53 at codon 249 (Ser-249, considered as a hallmark of mutagenesis by aflatoxin) and in CTNNB1 (gene encoding beta-catenin) in CFDNA of patients with HCC or chronic liver disease, from Alexandria, Egypt. The DNA concentrations were significantly higher in HCC patients compared to HBV and HCV carriers without cancer, and to sero-negative individuals. Ser-249 TP53 mutations were determined using PCR-restriction digestion (RFLP) in CFDNA of 255 subjects, and confirmed by sequencing. Ser-249 was found in CFDNA of 12 subjects (4.8%), with the highest prevalence in subjects with chronic liver disease and infection by HBV (6/36; 16.7%) Mutations in CTNNB1 were examined using PCR combined to DHPLC and followed by sequencing. No mutations were found in CTNNB1 neither in CFDNA or in tumour tissue. In parallel, studies on DNA extracted from 20 HCC biopsies showed the presence of ser-249 mutation in two cases (10%). These results indicate that mutagenesis by aflatoxin may play a role in hepatocarcinogenesis in Egypt, and CFDNA may serve as a convenient source of material in monitoring the effects of aflatoxin exposure and viral infections in chronic liver disease and cancer.

A novel gene, FGA7, is fused to RUNX1/AML1 in a t(4;21)(q28;q22) in a patient with T-cell acute lymphoblastic leukemia.

AML1 is among the most frequent targets of chromosomal rearrangements in human leukemias. We report here the molecular analysis of a t(4;21)(q28;q22) that has disrupted AML1 in a patient with de novo T‐cell acute lymphoblastic leukemia. By using 3′‐RACE analysis, we show that this rearrangement results in the fusion of a novel gene immediately downstream of exon 5 or exon 6 of AML1, indicating that the AML1 breakpoint lies in intron 6 and that alternative fusion splice variants are generated. The sequence of the novel gene, located at 4q28, does not have any significant homology with any of the known genes in the human GenBank DNA database. However, the first 118 bases are identical to a part of a human ovarian EST. Also, its high homology with mouse and rat sequences suggests that this sequence most probably represents a part of a novel gene, which we named FGA7 (Fused Gene 7 to AML1). Following the AML1 open reading frame, the FGA7 sequence encodes an unknown protein of 27 amino acids. We isolated three bacterial artificial chromosome (BAC) clones that contain the FGA7 sequence and confirmed the breakpoint of the gene on the patients metaphase spreads by fluorescence in situ hybridization using these BACs as probes. RT‐PCR and Northern blot analyses revealed that FGA7 is expressed in ovarian and skeletal muscle tissues. The predicted AML1‐FGA7 chimeric proteins contained a limited number of residues fused to AML1 in a situation similar to that reported for the AML1‐EAP fusion that is a product of t(3;21). It is possible that the expression of a constitutively shortened AML1 could compete with full‐length AML1 and act as a dominant negative inhibitor of the promoters that the core binding factor activates.

TP53 mutations in circulating free DNA from Egyptian patients with non-Hodgkin’s lymphoma

BACKGROUND P53 protein plays important role in the maintenance of genome stability in mammalian cells; it acts in many processes including cell-cycle checkpoint, DNA repair, apoptosis, and angiogenesis. Mutations of P53 have been reported as common mutations in solid tumours, including non-Hodgkin lymphoma, NHL, and have been implicated in drug resistance, aggression and poor prognosis. Chronic infection with hepatitis C, HCV, has been associated in some studies with increased risk of NHL. HCV is a widespread infection in the Egyptian population. Circulating free DNA (CFDNA) has been shown to be a good source of liver tissue-derived DNA in African and Asian patients with chronic liver disease or hepatocellular carcinoma, HCC. Our previous results have shown TP53 mutations in 5% of CFDNA and 10% of tumours of HCC, with underlying HCV. OBJECTIVE Since previous studies have shown p53 mutations in the DNAs extracted from the NH lymphoid tumours, we have assessed the presence of p53 mutations from exons 5 to 9 in CFDNA in patients with NHL, from Alexandria, Egypt, where HCV is highly prevalent, in a first attempt case-control preliminary study. METHODS CFDNA was extracted from sera of 20 cases with NHL and 20 negative control individuals. The retrieved serum DNAs were screened for TP53 mutations from exons 5 to 9 using direct sequencing and a PCR-restriction digestion analysis (RFLP). Concentrations of CFDNA were measured using Fluorometric assay. RESULTS Concentrations of CFDNA were significantly higher among NHL patients compared to the negative control individuals indicating a very high release or turn-over of DNA from the tumour into the blood stream among NHL patients. Mutations of p53 determined in NHL cases (30%) were of Arg-176 (1/20: 5%), Phe-238 (1/20: 5%), Ser-249 (2/20; 10%), Lys-249 (1/20: 5%) and Phe-250 (1/20: 5%). No mutations were detected among controls. However, Arg-213 polymorphism was found in 2 cases of NHL (10%) and in 1 case of controls (5%). CONCLUSION Our findings of higher DNA concentrations with some p53 mutations in CFDNA from patients with NHL that match the previous reported p53 mutations from tumour DNA may hold promises that CFDNA may serve as a convenient source of tumour-derived DNA to serve as a promising tool of a non-invasive, low-cost new strategy for earlier detection, diagnosis and follow up of the disease. A large-scale prospective study utilizing CFDNA and DNA from tumours of NHL patients will be required to validate this first trial of utilizing CFDNA from NHL patients.

Expression of apoptotic markers BCL-2 and bax in chronic hepatitis C virus patients

OBJECTIVES Hepatitis C viral infection(HCV) influence the susceptibility to apoptosis. This could lead to insufficient antiviral immune response and persistent viral infection. DESIGN AND METHODS Group 1: chronic HCV patients with liver cirrhosis and ascites. Group 2: chronic HCV patients without liver cirrhosis and group 3: healthy subjects as control group. Bcl-2 and Bax expression were evaluated by flowcytometry. RESULTS HCV patients (with cirrhosis and ascites) had a statistically significantly low Bcl-2 expression, a significantly high Bax expression and a significantly decreased Bcl-2/Bax ratio compared with controls. While, the results are inverted in the other HCV group. Both groups of HCV, Bcl-2/Bax ratio showed a significant positive correlation with Bcl-2 and a significantly negative correlation with Bax. CONCLUSIONS Chronic HCV exhibit a deregulation of apoptosis with the disease progression. This provides an insight into the pathogenesis of chronic HCV infection, and may contribute to the therapy.

Markers of apoptosis and proliferation related gene products as predictors of treatment outcome in childhood acute lymphoblastic leukemia.

Abstract The aim of the study is to characterize markers of apoptosis in children with acute lymphoblastic leukemia (ALL) in relation to treatment outcome of the disease. The study was performed on 34 children with ALL and 39 healthy children as a control group. Apoptosis was assessed by cell morphology; DNA fragmentation; ELISA and RT-PCR for CD95, CD95L, BcL-2 and nuclear factor-kappa B (NF-κB); and flow cytometry for CD95, CD40, CD49d and CD11a. Apoptosis was significantly lower in patients than controls. Apoptosis detected by CD95 ligand was significantly lower in cases with no remission after treatment than those who achieved remission. Anti-apoptotic factors: CD40, BcL-2, and NF-κB were all found to be higher in cases than controls and in cases with no remission than those achieved remission. CD49d was significantly lower in cases than controls, and significantly lower in cases with who did not achieve remission. CD11a levels were similar in the various groups. Delayed apoptosis of ALL cells is genetically controlled either directly or indirectly by a network of oncogenes and tumor suppressor genes. CD40 appeared to stimulate both T and B lineage and is considered the most potent influencer and predictor of resistance to therapy. Inhibitors for the activity of CD40, Bcl-2 and NF-κB as well as stimulants to CD95 could have a potential therapeutic benefit.

Study of microRNA Profile as a Molecular Biomarker in Egyptian Chronic Lymphocytic Leukemia

MicroRNAs target mRNAs for cleavage or translational repression. They play a critical role in the progression of malignancies and leukemias including chronic lymphocytic leukemia (CLL). However, microRNA expression levels in Egyptian patients with CLL, and their prognostic value remain elusive. Our main aim was to assess the expression pattern of a panel of microRNAs in CLL patients to create an informative microRNA profile. The study subjects were 40 newly diagnosed CLL patients of both sexes and 40 age and sex matched controls. The expression levels of 12 microRNAs were evaluated by qRT-PCR, including miR-15a, 16, 23b, 24, 29a, 29c, 34a, 146a, 155, 181a, 195, and 221. Flow cytometry was used to determine the expression levels of BCL2, CD38, and ZAP-70 in CLL patients. We identified various degrees of upregulated miRNAs (miR-29a, miR-29c, miR-34a, miR-155, miR-146a, and miR-195) and down-regulated ones (miR-15a, miR-16, miR-23b, miR-24, miR-181a, and miR-221) in CLL patients relative to controls. The mean fluorescence intensity ratio (MFI-R) of BCL2 was recorded and was significantly upregulated in CLL patients compared with normal controls. In addition, inverse correlations were observed between microRNAs (miR-15a, miR-16, miR-155, and miR-195) and BCL2 MFI-R while positive correlations were observed between miR-29a and miR-29c, and BCL2 MFI-R. These findings suggest that these miRNAs regulate BCL2 levels. Moreover, we found that miR-15a, miR-16, miR-155, miR-181a, miR-195 and miR-221 were significantly upregulated, while miR-29a and miR-29c were significantly downregulated in ZAP-70 positive CLL patients. Various miRNAs may play an important role in the pathogenesis of CLL and have the potential to be used for the prognosis of patients with CLL.

PTU-110 Circulating Bone Marrow-Derived Stem Cells and Stem Cell Factor Serum Level in Chronic Hepatitis C: Relation to Hepatic Proliferation and Fibrosis

Introduction Bone marrow-derived stem cells (BMSCs) are pluripotent cells that can be mobilised into circulation and recruited to sites of inflammation where they promote local tissue repair. Therefore, the present work was designed to study circulating BM-derived hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) and serum levels of stem cell factor (SCF), a stem cell mobilising factor, in patients with chronic hepatitis C (CHC) in relation to hepatic proliferation and fibrosis. Methods Thirty treatment-naïve patients with CHC and 15 healthy subjects were included in the study. The BM-derived HSCs and MSCs cells in fresh blood samples were identified as CD34+CD45+CD117+ and CD34-CD45-CD106+ cells respectively using flow cytometric assay. Serum SCF levels were measured using an enzyme linked immunosorbant assaykit. Liver biopsies were examined to assess METAVIR histological activity grade and fibrosis stage and steatosis grade. Immunohistochemical staining of liver specimens was done using monoclonal antibodies against cytokeratin (CK)7 for detection of hepatic progenitor cells (HPCs), Ki-67 as proliferation marker and a-smooth muscle actin (a-SMA) for identification of activated hepatic stellate cells (HpSCs). Results Patients with CHC showed significant increases in the percentages of HSCs and MSCs in peripheral blood and serum SCF levels compared with healthy subjects (P < 0.05). Numerous CK7+ HPCs were detected mostly lining primitive bile ducts or as individual positive cells in the portal tracts. Hepatic proliferative activity evidenced as nuclear positivity for Ki-67, was observed in proliferated ductal profiles in portal tracts and within hepatocytes and directly correlated with HPC expansion. Based on the percentages of BMSCs in peripheral blood, patients with CHC were distinguished into two types of patients: “mobilizers” and “non-mobilizers”. BMSC mobilizers showed a significant increase in serum SCF levels and significant decreases in HPC expansion, hepatic proliferative activity, serum levels of aminotransferases, histological activity grade, fibrosis stage and a-SMA expression compared with BMSC non-mobilizers (P < 0.01). Conclusion Chronic HCV infection is associated with mobilisation of BMSCs from the BM into the circulation in parallel with an increased production of SCF, particularly when HPC activation and hepatic proliferative activity are impaired. Although the mobilised BMSCs are not sufficient to bring about hepatic repopulation, they may play an important role in limiting hepatic necroinflammation and fibrosis in HCV-induced liver damage. Disclosure of Interest None Declared

Role of bone marrow-derived stem cells, renal progenitor cells and stem cell factor in chronic renal allograft nephropathy

Abstract Introduction Chronic allograft nephropathy (CAN) is a poorly understood clinico-pathological entity associated with chronic allograft loss due to immunologic and non-immunologic causes. It remains the leading cause of late allograft loss. Bone marrow derived stem cells are undifferentiated cells typically characterized by their capacity for self renewal, ability to give rise to multiple differentiated cellular population, including hematopoietic (HSCs) and mesenchymal stem cells (MSCs). Characterization of HSCs includes their multipotency, expression of typical surface markers such as CD34 and CD45, while characterization of MSC includes their multipotency, expression of typical surface markers such as CD90 and CD105, and the absence of hemopoietic lineage markers. Aim & methods The aim of the present work was to study the role of bone marrow-derived HSCs and MSCs, renal progenitor cells and SCF in chronic renal allograft nephropathy in relation to renal hemodynamics and histopathological changes. We studied 30 patients with kidney transplantation for more than 6 months, divided into 15 patients with stable serum creatinine and 15 patients who developed CAN. Detection of HSCs and MSCs in the peripheral blood using flow cytometry via detection of CD34, CD45, CD117 and CD106, as well as immunohistochemical detection of CD34, CD133, VEGF and αSMA in transplanted kidney biopsies of patients with CAN were done. Results There was a significant increase in the levels of SCF, number of peripheral blood HSCs and MSCs in both transplanted patient groups than the controls and they were higher in patients of group Ia than patients of group Ib, (F = 39.73, P < 0.001), (F = 13.28, P < 0.001), (F = 11.94, P < 0.001), respectively and this was accompanied by evident expression of markers of renal repair. Conclusion Stem cells might have a role in renal regeneration in CAN and this may pave the way toward the use of stem cells in correction of CAN.

Clinical significance of sCD86 levels in patients with acute myelogenous leukemia

Abstract Introduction CD86 (B72) molecules are surface glycoproteins and members of Ig superfamily that are expressed only on professional APCs and are important in the early interactions between APCs and T cells during the induction of immune response. It is well established that mCD86 is expressed by AML blasts in a considerable proportion of patients. The release of soluble forms of membrane molecules provides a powerful means by which leukocytes can either inhibit or enhance the biological effects relative of their membrane-bound counterparts, and there is now considerable evidence to support the possibility that the release of a soluble form of CD86 (sCD86) has an immunoregulatory role in vivo. The observation that sCD86 levels are highest in the FAB subtypes with the highest AML blast levels, together with the observation that high levels of sCD86 are associated with poor prognosis, strongly suggests that sCD86 is derived from the malignant cells in these patients. The aim The present study was to assess levels of sCD86 in de novo acute myeloid leukemia patients and to determine any possible correlation with outcome following induction chemotherapy. The study was carried out on 30 patients with de novo acute myeloid leukemia and 20 healthy controls. Method Levels of soluble CD86 (sCD86) in the serum was measured using ELISA technique at presentation and after one cycle of induction chemotherapy. Conclusion We found that sCD86 was detected in both patients and controls. Levels of sCD86 were higher than the cut-off value in 36.6% of patients. There was a significant difference between levels of sCD86 before and after treatment. Patients (54.5%) with high sCD86 levels had monocytic morphology. Patients with high levels of sCD86 had a lower rate of complete remission.