Sema Sirma

NAD(P)H:quinone oxidoreductase 1 null genotype is not associated with pediatric de novo acute leukemia

NAD(P)H:quinone oxidoreductase1 (NQO1) is a two‐electron reductase that detoxifies quinones derived from the oxidation of phenolic metabolites of benzene. Exposure to benzene metabolites increases the risk of hematotoxicity and leukemia. NQO1 enzyme activity protects the cells against metabolites of benzene. C to T base substitution at nucleotide 609 of NQO1 cDNA (C609T) results in loss of enzyme activity. Low NQO1 activity may play a role in etiology of acute leukemia.

The Novel ETS Factor TEL2 Cooperates with Myc in B Lymphomagenesis

ABSTRACT The human ETS family gene TEL2/ETV7 is highly homologous to TEL1/ETV6, a frequent target of chromosome translocations in human leukemia and specific solid tumors. Here we report that TEL2 augments the proliferation and survival of normal mouse B cells and dramatically accelerates lymphoma development in Eμ-Myc transgenic mice. Nonetheless, inactivation of the p53 pathway was a hallmark of all TEL2/Eμ-Myc lymphomas, indicating that TEL2 expression alone is insufficient to bypass this apoptotic checkpoint. Although TEL2 is infrequently up-regulated in human sporadic Burkitts lymphoma, analysis of pediatric B-cell acute lymphocytic leukemia (B-ALL) samples showed increased coexpression of TEL2 and MYC and/or MYCN in over one-third of B-ALL patients. Therefore, TEL2 and MYC also appear to cooperate in provoking a cadre of human B-cell malignancies.

A genome-wide analysis of lentivector integration sites using targeted sequence capture and next generation sequencing technology

One application of next-generation sequencing (NGS) is the targeted resequencing of interested genes which has not been used in viral integration site analysis of gene therapy applications. Here, we combined targeted sequence capture array and next generation sequencing to address the whole genome profiling of viral integration sites. Human 293T and K562 cells were transduced with a HIV-1 derived vector. A custom made DNA probe sets targeted pLVTHM vector used to capture lentiviral vector/human genome junctions. The captured DNA was sequenced using GS FLX platform. Seven thousand four hundred and eighty four human genome sequences flanking the long terminal repeats (LTR) of pLVTHM fragment sequences matched with an identity of at least 98% and minimum 50 bp criteria in both cells. In total, 203 unique integration sites were identified. The integrations in both cell lines were totally distant from the CpG islands and from the transcription start sites and preferentially located in introns. A comparison between the two cell lines showed that the lentiviral-transduced DNA does not have the same preferred regions in the two different cell lines.

Prognostic significance of the TEL-AML1 fusion gene in pediatric acute lymphoblastic leukemia in Turkey

Purpose The t(12;21) translocation is the most common reciprocal chromosomal rearrangement in pediatric acute lymphoblastic leukemia (ALL). This translocation fuses two genes, TEL and AML1, and results in the production of the TEL-AML1 fusion protein. The authors investigated the incidence and prognostic significance of the TEL-AML1 fusion gene in patients with ALL in Turkey. Methods The authors analyzed 219 children with ALL using the reverse transcription–polymerase chain reaction. Results The TEL-AML1 fusion transcript was detected in 20.1% (44/219) of newly diagnosed children with ALL. TEL-AML1-positive patients had precursor B-cell ALL and were 3 to 10 years old at diagnosis. TEL-AML1-positive patients had a significantly lower rate of relapse compared with TEL-AML1-negative patients. TEL-AML1-positive patients have a higher overall survival rate than TEL-AML1-negative patients. Conclusions These data support that the presence of TEL-AML1 at diagnosis is an independent favorable prognostic indicator in patients with ALL in Turkey.

Real-Time PCR analysis of af4 and dek genes expression in acute promyelocytic leukemia t (15; 17) patients

Among several newly identified oncogenes, dek and af4 are attractive targets for researchers interested with leukemia. In this study quantitative Real-Time RT-PCR technique was used to define alterations in expression of dek and af4 genes associated with acute promyelocytic leukaemia (APL) t (15; 17). RNA samples obtained from bone marrow aspirates of fourteen APL patients, cDNA portions were labelled with Syber Green 1 dye and LightCycler analysis have been performed. Expression changes in patients were found not significant in comparison to healthy donors for af4 (P=0.192) and dek (P= 0.0895). We suggest that af4 gene may have a role in leukomogenesis restricted to lymphoblastic lineage; also further studies must carry on with a larger series of patients in order to understand the relationship between the dek gene and APL. Our study was the first attempt for analysing dek and af4 genes in APL t (15; 17) patients by quantitative Real-Time RT-PCR. This rapid and sensitive method could be used to screen these genes in different types of leukaemia.

Analysis of MYH Tyr165Cys and Gly382Asp variants in childhood leukemias

DNA-repair gene mutations have been suspected as being a predisposing factor in the development of leukemia (Horwitz 1997). Our group identified one of the first examples of a DNA-repair gene mutation to be causally linked to childhood hematological malignancies and neurofibromatosis type I, which involves a homozygous germ-line mutation in the mismatch repair (MMR) gene MLH1 (Ricciardone et al. 1999; Wang et al. 1999). Subsequently, homozygous inactivation of MSH2, another MMR gene, was also found to be associated with early onset leukemia (Whiteside et al. 2002). It is well known that heterozygous germ-line mutations in the MMR pathway genes MLH1, MSH2, PMS2, PMS1, and MSH6 lead to hereditary nonpolyposis colorectal cancer (HNPCC) (Peltomaki 2001). Thus, tumorigenesis through an ‘‘MMR deficiency pathway’’ appears to be associated with two different disease phenotypes which are dependent on the status of the germ-line mutation: (a) HNPCC when the mutation is present on only one allele (heterozygous), and (b) hematological malignancies when the mutation(s) is present on both alleles (homozygous or compound heterozygous). Base excision repair (BER) is another important DNA-repair pathway and plays a significant role in the repair of mutations generated by reactive oxygen species during aerobic metabolism. BER was not linked with any human genetic disorder until recently, when a British family in which three siblings affected by multiple colorectal adenomas and carcinoma was shown to be compound heterozygous for MYH missense variants Tyr165Cys (Y165C) andGly382Asp (G382D) (Al-Tassan et al. 2002). MYH is a homologue of E. coli mutY, and the mutations mentioned above affect residues that are conserved (Tyr82 and Gly253). Tyrosine 82 is predicted to function in mismatch specificity and is located in the pseudo-helix-hairpin-helix (Guan et al. 1998). Adenine glycosylase activity assays of the Tyr82Cys and Gly253Asp mutant proteins with 8-oxoG:A and G:A substrates show that their rate for adenine removal at 37 C is reduced by approximately 98% (Tyr82Cys) and 86% (Gly253Asp) (Al-Tassan et al. 2002). Furthermore, bi-allelic germ-line mutations in MYH were identified in seven unrelated patients with colorectal adenomas (six with colorectal cancer) (Jones et al. 2002). Interestingly, the missense variations Tyr165Cys and Gly382Asp, which significantly reduce the adenine glycosylase activity of MYH protein, were each identified once in a normal control group of 100 British individuals with no history of colorectal adenoma or carcinoma (Al-Tassan et al. 2002). Since a connection between DNA-repair gene mutations and the path to hematological malignancy is now well established, and individuals who carry heterozygous MYH missense mutations Tyr165Cys and Gly382Asp have been documented in a control group (Al-Tassan et al. 2002), we investigated the association between these two MYH missense mutations and childhood leukemia risk.

Real-time PCR analysis of the apoptosis related genes in ATRA treated APL t(15;17) patients

All-trans retinoic acid (ATRA) treatment of the acute promyelocytic leukemia (APL) have subsequently resulted in cell apoptosis, but the molecular mechanism of this effect remains elusive. In order to understand a possible involvement of genes regulating apoptotic signal pathways, expression levels of bcl2, bax, dapk1, myc, bad, wt1, and mcl genes were analyzed during ATRA treatment in five APL patients with t (15;17) using Real- time PCR (LightCycler). Two samples from each patient were compared to each other: primary diagnostic sample and a sample taken at remission. Effect of the ATRA treatment was demonstrated by the concomitant induction of cd14 and il1β genes in four patients. Also other apoptosis related genes were found down-regulated in general but especially the down regulated levels of wt1 and bax attract attention. Result suggested that ATRA dependent apoptosis of APL was under the control of both internal and external pathways without relationships to the amount of the blast populations. Ratio of bcl2 to bax may be more important for this regulation than the ratio of bcl2 to bad. Either bcl2 family or less known apoptosis related genes as wt1 will still be required to further studies in this setting.

Comparison of the cytogenetic and molecular analyses in the assessment of imatinib response in chronic myelocytic leukemia.

We aimed to compare the cytogenetic and molecular analyses in the assessment of imatinib mesylate response in patients suffering the chronic phase of chronic myelocytic leukemia who were refractory to alpha-interferon treatment. A total of 117 patients in the chronic phase of chronic myelocytic leukemia were included. The patients were treated with 400 mg/day imatinib mesylate. Bone marrow samples were obtained for the cytogenetic and molecular analyses. Patients without the Ph chromosome were defined as complete cytogenetic responders. Partial cytogenetic response was determined when the Ph chromosome was detected in 1-35% of the cells. Molecular response was determined by quantitative real-time reverse transcriptase polymerase chain reaction (QR-PCR) and defined as no detection of BCR-ABL mRNA. The frequencies of complete and partial cytogenetic response were 29% (n = 34) and 15% (n = 18), respectively. No cytogenetic response was achieved in 56% (n = 65) of the patients. Molecular response was achieved in 62% (n = 21) and 33% (n = 6) of the complete and partial cytogenetic responders, respectively. All of the 65 patients with no cytogenetic response were also molecular nonresponders. We conclude that there is reasonable agreement between the cytogenetic and molecular analyses. Both methods are complementary in the assessment of response to therapy.