Simultaneous Detection of Major Fusion Transcripts in CML, ALL and AML Leukemias in a Multiplex Bead-Array Assay.

INTRODUCTION: Accurate sub-classification of leukemia-associated chromosome translocations is essential for precise risk-stratified diagnosis and improvement of treatment outcomes. We have developed a rapid assay system to detect the nine most common chromosomal translocations covering three different leukemia disease groups: CML, ALL and AML. The purpose of our study was to confirm clinical detection of twelve translocation markers.

MATERIALS AND METHODS: The liquid bead array assay combined multiplex RT-PCR with signal readout in a 96-well plate format. RNA fusion transcripts of leukemia-associated chromosome translocations were reverse transcribed into cDNA and amplified by multiplex PCR using biotin-modified primers. An endogeneous control gene, GAPDH, was co-amplified in each sample and concurrently analyzed. A single-tube bead array contained 11 marker specific oligonucleotide probes with sequences complimentary to the fusion transcript splice junction for the following markers: b2a2, b3a2 for CML; e1a2, E2A-PBX1, TEL/AML1 and MLL/AF4 (e10/e4, e9/e5) for ALL; PML/RARa (long and short forms), AML1-ETO and inv(16) (A and D types) for AML. Biotinylated amplified products were hybridized to the array without intervening purification steps. After incubation with streptavidin-conjugated fluorophore, the reaction mixtures were analyzed by the Luminex platform.

RESULTS: RNA samples from translocation-positive cell lines and IVT products were detected with less than 10% CVs in approximately five hours. Long and short forms of PML-RARa and A and D types of inv(16) were specifically resolved. The assay can detect two MLL/AF4 subtypes: MLL/AF4 (e10/e4) or MLL/AF4 (e9/e5). Eighty-seven leukemia patient samples (RNAs extracted from peripheral blood and bone marrow) were screened and all translocations were correctly detected. A software system for selective readout based on disease class was implemented.

CONCLUSIONS: This multiplex array-based assay can be an effective and reliable tool in the clinical screening of leukemia patients for the presence of specific fusion transcripts with important diagnostic and prognostic implications.