POSTER NO: 267

Quantitative Detection of Leukemic Fusion Gene Transcripts To Assess Minimal Residual Disease

¹Wanli Bi, ¹Kenneth J. Livak, ²James L. Slack, ³Robert E. Gallagher, ⁴Cheryl L. Willman, ⁵Jean Gabert, ⁶Jacques J. Van Dongen, ⁷Niels Pallisgaard, ⁸Gianni Cazzaniga, ⁹Gisela Barbany, ¹⁰Jean-Michel Cayuela, ¹¹Fabrio Pane, ¹²Helene Cave, ¹³David Grimwade, ¹⁴Giuseppe Saglio, ¹⁵S. Viehmann
¹Applied Biosystems, 850 Lincoln Centre Dr, Foster City, CA 94583, USA, ²Department of Medicine, Roswell Park Cancer Institute, Buffalo, New York 14263, USA, ³Department of Oncology, Montefiore Medical Center and Albert Einstein Cancer Center, Bronx, New York 10467, USA, ⁴Department of Pathology, University of New Mexico, Albuquerque, New Mexico, USA, ⁵Department of Biochemistry and Molecular Biology, Hôpital Universitaire Nord/IFR Jean Roche - Université de la Méditerranée, Marseille, France, ⁶Department of Immunology, University Hospital Rotterdam/Erasmus University,, Rotterdam, Netherlands, ⁷Department of Immunohematology, Aarhus University Hospital, Aarhus, Denmark, ⁸Clinica Pediatrica, Universita Milano, Monza, Italy, ⁹Department of Medical Sciences, Uppsala University Hospital, Uppsala, Sweden, ¹⁰Central Laboratory of Hematology and INSERM U462, Saint Louis Hospital, Paris, France, ¹¹Department of Biochemistry and Molecular Biology, University Hospital, Naples, Italy, ¹²Department of Genetic Biochemistry, Robert Debré Hospital, Paris, France, ¹³Department of Hematology, University College London Hospitals, London, UK, ¹⁴Department of Clinical and Biological Science, University of Turin, Turino, Italy, ¹⁵Biogenetics Laboratory, Children's University Hospital, Giessen, Germany

Gene translocation is commonly seen in leukaemia such as childhood and adult AML, ALL, and CML. Quantitative detection of fusion gene transcripts promises to be an effective method for monitoring disease status, predicting relapse, and conducting risk-adapted therapy. This application will require standardized, robust, specific, sensitive, and precise assays. Applied Biosystems has collaborated with 29 European/American research laboratories to develop assays for the targets PML-RARa, E2A-PBX1, MLL-AF4, TEL-AML1, BCR-ABL, SIL-TAL1, CBFb-MYH11, AML1-ETO. The assays are based on the 5' nuclease activity of Taq DNA polymerase. In addition to two primers for PCR amplification, a third fluorogenic oligonucleotide probe is included in the reaction to specifically detect amplified product. In order to adjust for sample variation due to integrity of RNA, presence of RT and/or PCR inhibitors, and differences in RT efficiency, 14 reference genes were evaluated. ABL, GUS and b2MG were shown the best suited for serving as control genes. The concept of detection limit was also introduced to reduce the incidence of false negative results. Inter-laboratory studies demonstrated excellent reproducibility and high concordance between laboratories. Sensitivity for these assays is extremely high (<10 copies). Analyses of PML-RARa mRNA in APL patients showed significant correlation between the RNA level at the end of consolidation and relapse risk.