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

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.