An optimized technology platform for the rapid multiplex molecular analysis of genetic alterations associated with...

Molecular methods play a critical role in the accurate diagnosis of leukemia by complementing morphologic, cytochemical, immunophenotypic, and cytogenetic analyses. We developed a multiplex reverse transcription-polymerase chain reaction (RT-PCR) method combined with liquid bead array cytometry for the rapid detection of genetic alterations associated with leukemia. Fusion transcripts corresponding to the most common recurrent chromosomal translocations were reproducibly detected in as low as 0.1-10 ng of total RNA with an analytical sensitivity of 0.01-1%. Multiday, multilot, multioperator, and multi-instrument precision studies, for a total of 678 independent measures in 46 runs, showed a very high reproducibility with 100% agreement among replicates. Using multiplex panels for four to 20 independent targets, we demonstrate the flexibility of the method to codetect rare splicing isoforms, discriminate among multiple variants generated by unique cytogenetic abnormalities, identify distinct chromosomal partners involved with 11q23 or 17q21 rearrangements, and assess cryptic abnormalities not detectable by standard cytogenetics such as the t(12;21), del(1p32), or NPM1 mutations. Overall, three different internal control transcripts and 34 variants resulting from 18 abnormal chromosomal sites were evaluated. These results underscore the value of the multiplex assay system as a sensitive and reliable technology platform for the characterization of relevant genetic alterations in leukemia.