HGM2002 Poster Abstracts: 7. Medical Genomics
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
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.
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