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The acute phase of chronic myeloid leukemia (CML) is accompanied by secondary chromosomal changes. The additional changes have a non-random pattern; however, highly abnormal (marker) chromosomes are reported in some 20% of abnormal karyotypes. These marker chromosomes have proved to be beyond the resolution of conventional G-banding analysis. We used molecular cytogenetic techniques to determine the structure of complex chromosome markers in 10 CML-derived cell lines after our investigations of CML patients in blast crisis. Multicolor fluorescence in situ hybridization identified a multitude of structural chromosome aberrations. In addition, genomic gains identified by comparative genomic hybridization (CGH) were mapped to highly complex marker chromosomes in more than one cell line. The most common genomic loss detected by CGH affected chromosome 9, whereas the most common genomic gains affected, in order of frequency, the sequences of 8q,6, and 13q. The smallest discrete amplification on 8q was identified in cell line MEG-01. This amplicon contains sequences represented by the marker D8S263/RMC08P029 but did not contain the proximal MYC gene or a more distal marker, D8S256/RMC08P025. We determined the size of the amplicon to be less than the chromosome segment 8q24.12-q24.13. The use of region- and locus-specific probes to analyze the organization of highly complex marker structures aided the identification of preferentially amplified genomic regions. The resultant amplifications could harbor gene(s) driving disease progression. (C) 2003 Wiley-Liss, Inc.

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-ABL1 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-ABL1 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.

A range of fluorescent in situ hybridization techniques have been used to reveal hidden variant Philadelphia translocations in two cases of Ph-positive chronic-phase chronic myeloid leukaemia. In one patient, a highly complex variant Ph translocation affecting four chromosomes had resulted in the formation of structures with the appearance of i(17q) and +8. Misinterpretation of these karyotypes has direct clinical relevance. Our findings illustrate that even established cytogenetic abnormalities may contain cryptic abnormalities beyond the resolution of conventional cytogenetic methods.

Structural rearrangements involving the short arm of chromosome 12 are common in acute lymphoblastic leukemia (ALL) and often involve the TEL locus at 12p13. The balanced t(12;17)(p13;q12) is a rare but recurrent aberration in ALL. Whereas the TEL gene has been postulated as a likely candidate for involvement in the t(12;17), the precise molecular consequences of this translocation have not yet been elucidated. We identified a t(12;17) in 2 of 398 childhood ALL patients karyotyped at presentation in our institute. Both cases had a precursor-B immunophenotype and were CD10 negative and CD33 positive. Fluorescence in situ hybridization excluded involvement of the TEL locus in the t(12;17) and provided no evidence for concomitant cryptic deletion of the 12p commonly deleted region. Comparison of these and previously published cases demonstrates that the translocation predominately occurs in children and young adults with precursor B-ALL and is typically characterized by low CD10 expression and high CD33 expression. Our data support the involvement of a new locus telomeric to TEL in the pathogenesis of t(12;17)-positive ALL.

Chromosome band specific FISH probes allow improved detection of terminal translocations in leukaemic metaphases