High-resolution analysis of acquired genomic imbalances in bone marrow samples from chronic myeloid leukemia patients by use of multiple short DNA probes.

Chronic myeloid leukemia (CML) is a biphasic hematopoietic malignancy associated with a single cytogenetic aberration, the Philadelphia translocation t(9;22)(q34;q11), resulting in the BCR-ABLI fusion oncogene. Molecular heterogeneity was recently demonstrated in the form of extensive deletion of chromosomes 9 and 22 material from the der(9)t(9;22) in 15% of CML patients. The deletions were associated with a worse disease prognosis. Further genetic heterogeneity is seen during the terminal blast crisis stage of CML, in the form of additional non-random chromosome abnormalities. These include most frequently an extra copy of the Ph chromosome, trisomy 8, and isochromosome 17q. We used the genetic heterogeneity of CML as a framework to explore a new technique for high-throughput assessment of locus copy number in malignancy. Multiplex amplifiable probe hybridization (MAPH) relies on the ability of numerous short (100-300 bp) DNA probes to be recovered quantitatively by use of a common primer pair after hybridization to genomic DNA. Derivative chromosome 9 deletions were successfully mapped in a CML cell line (MC3) and nine patient bone marrow samples by simultaneous hybridization of 10 MAPH probes. All results were confirmed by fluorescence in situ hybridization. MAPH was found to be informative in the presence of up to 50% of normal cells, thus establishing the sensitivity of the technique in clonal tumor cell populations. MAPH was performed effectively on DNA samples extracted from fresh or methanol/acetic acid-fixed clonal cell populations. Amplifications of BCR-ABLI were also detected and quantified in four CML cell lines by use of MAPH probes specific for ABL1 exon 11 and BCR exon 1. Our results demonstrate that MAPH is a reproducible high-throughput method suitable for the assessment of genomic imbalances of multiple loci in tumor DNA samples with heterogeneous cell populations at a resolution of 100-300 bp. (C) 2003 Wiley-Liss, Inc.