Amplification Of Fusion Gene Research Articles

The Use of ABL and GUS as Control Genes for Normalizing Quantification of Fusion Transcripts by Real Time PCR: Our Experience of More Than 1000 Samples.

Abstract 4727 IntroductionReal-time quantitative PCR (RT-PCR) is a sensitive and accurate tool for monitoring leukemic patients by amplification of fusion gene (FG) transcripts.In order to correct variations in RNA quality and quantity and to calculate the sensitivity of each measurement, a control gene (CG) transcript should be amplified in parallel to the FG transcript.In our laboratory two CG are used: Abelson (ABL) and beta-glucuronidase (GUS).Clinical application for monitoring the treatment of chronic myelogenous leukemia (CML) patients with tyrosine kinase inhibitor makes the quantification of which CG to use very important into the clinical practice today. ObjectiveThe aim of this study was to assess the degree of agreement between the GC, compare the expression of GC (type and time of sampling and pathology), try to redefine the limits between the 4 groups of quality (very good, good, poor and very poor quality samples) and to focus on CML patients. Materials and methodsA total of 1087 samples (peripheral blood and bone marrow) were obtained from 475 patients from January 2005 to December 2007. This study includes patients with acute myeloid leukaemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukaemia (CML) and Hypereosinophilic syndrome (HES).We collected 366 samples of 41 CML patients between 2002 and March 2009 including 32 CML at diagnosis.RT-PCR analysis was performed using the EAC protocols. ResultsComparison between CML, AML, ALL and HES patients showed that ABL and GUS expression did not differ significantly (Bland and Altman method).ABL and GUS gene expressions did not differ significantly between CML patients at diagnosis and the others. ConclusionBased on the literature and our local experience of more than 1000 samples, we propose to use only ABL as CG in routine practice in onco-hematology particularly for CML patients at diagnosis. Disclosures:No relevant conflicts of interest to declare.

Preliminary Studies on the Role of a Small GTPase, RhoA and Its Regulation in Imatinib Resistance

Imatinib has impressive response rates and good tolerability quickly led to its adoption as frontline therapy for all patients with chronic-phase CML (chronic myeloid leukemia), but the therapeutic effect of imatinib is poor in the blast crisis, and imatinib resistance has become a major problem in CML. The possible mechanisms of imatinib resistance include the amplification of BCR-ABL fusion gene and its expression increase, the point mutant of BCR-ABL kinase domain and the effects of other tyrosine kinases such as Src, Hck and Lyn and so on. However, the second-generation tyrosine kinase inhibitors (such as nilotinib and dasatinib), which were developed to overcome imatinib resistance resulting from the point mutant or the activation of other tyrosine kinases, even can not prevent all patients with CML progression to drug resistance. So there would be the other potential factor in imatinib resistance. Our previous studies generated a new imatinib-resistant BCR/ABL-positive cell line, K562-R. The 50% inhibitory concentration of imatinib was 15-fold higher in K562-R than in the wild-type K562. The expression of RhoA gene is up-regulated in K562-R by microarray analyses. RhoA, a small GTPase (24KD), has been found overexpression in breast, colon, head and neck squamous cell carcinoma, bladderand testicular cancer, lung and gastric cancer. It plays an important role in the initiation as well as the progression of human cancer, but the potential role of RhoA related to imatinib resistance has yet been unknown. In this study, we firstly detect the biologic characteristic of K562-R cells with RhoA down-expression by RNA interference. When K562-R cells were transfected with 150nM siRNA-RhoA for 48 hours, the percentage of apoptotic K562-R cells is respectively 12.82% by AnnexinV-PI assay and 9.0% by Hoechst 33258 staining and both have significant increase, cell cycle analysis found significant G0/G1 arrest, the expression of CD29 increase and that of CD71 and GPA have no difference. Secondly, The K562-R cells were treated with three selective inhibitors, including PD98059 (Ras/MAPK inhibitor), LY294002 (PI3K/AKT inhibitor) and AG490(JAK/STAT inhibitor) for 2,4 and 8 hours and the expression of RhoA were analyzed by Western-Blotting. The expression of RhoA is arrested in the K562-R cells treated with PD98059 and AG490 and no different with LY294002. These results indicate that RhoA would be an important target in the down-stream of multi-signal pathways related to imatinib resistance and the potential function of RhoA in imatinib resistance involve in increasing of cell proliferation, resistance to cell apoptosis and changes of cell adhesion.

Fusion genes resulting from alternative chromosomal translocations are overexpressed by gene-specific mechanisms in alveolar rhabdomyosarcoma.

Chromosomal translocations identified in hematopoietic and solid tumors result in deregulated expression of protooncogenes or creation of chimeric proteins with tumorigenic potential. In the pediatric solid tumor alveolar rhabdomyosarcoma, a consistent t(2;13)(q35;q14) or variant t(1;13)(p36;q14) translocation generates PAX3-FKHR or PAX7-FKHR fusion proteins, respectively. In this report, we demonstrate that in addition to functional alterations these translocations are associated with fusion product overexpression. Furthermore, PAX3-FKHR and PAX7-FKHR overexpression occurs by distinct mechanisms. Transcription of PAX3-FKHR is increased relative to wild-type PAX3 by a copy number-independent process. In contrast, PAX7-FKHR overexpression results from fusion gene amplification. Thus, gene-specific mechanisms were selected to overexpress PAX3-FKHR and PAX7-FKHR in alveolar rhabdomyosarcoma, presumably due to differences in regulation between the wild-type loci. We postulate that these overexpression mechanisms ensure a critical level of gene product for the oncogenic effects of these fusions.