Detection of chimeric BCR-ABL genes on bone marrow samples and blood smears in chronic myeloid and acute lymphoblastic leukemia by in situ hybridization

Abstract

The presence of BCR-ABL fusion genes has important diagnostic and prognostic implications in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). The CML-specific chimeric BCR-ABL gene with a break involving the major breakpoint cluster region (M-bcr) of the BCR-gene has been detected by means of fluorescence in situ hybridization (FISH). In this study, we present a FISH protocol that allows the detection of breaks in both the major and the minor breakpoint cluster region (m-bcr). Three hybridization signals of D107F9, a yeast artificial chromosome (YAC)-derived probe spanning the breakpoint regions of the BCR gene, were indicative of the translocation events. To increase the specificity further, this probe was combined with cos-abl 8, a cosmid probe flanking the breakpoint within the ABL gene for dual-color hybridization. Samples of 21 patients with CML, the ALL-derived cell line SUP-B15, and of seven patients with Philadelphia chromosome (Ph1)-positive ALL (three of them with breakpoints within m-bcr) were examined. BCR-ABL fusion was detected in all cases with high specificity (false-positive nuclei: mean, 0.1%). On cytogenetic preparations, the percentages of BCR-ABL- positive interphase cells ranged from 53% to 91%. Comparable efficiencies were achieved on blood smears. In conclusion, hybridization with D107F9 and cos-abl 8 allows unambiguous diagnosis of BCR-ABL genes and is likely to become an important tool for the monitoring of therapies in patients with CML and ALL.

Interphase cytogenetics and competitive RT‐PCR for residual disease monitoring in patients with chronic myeloid leukaemia during interferon‐α therapy

“…The technical limitation of the interphase analysis for BCR-ABL fusion is that it cannot identify small numbers of leukaemic cells due to the presence of false-positives in normal cell populations. The frequency of nuclei with artefactual co-localization of fluorescent signals in normal marrow is usually of about 1-2% and depends on the DNA probes used (Amiel et al, 1994;Bentz et al, 1994). In our study the highest frequency of false-positive readings for an individual normal control was 2·8%.…”

Diagnosis and monitoring of chromosome aberrations in hematological malignancies by fluorescence in situ hybridization

“…C) Detection of a reciprocal translocation, e.g., the t(9;22). In this example, two genomic clones are used: i) a YAC clone spanning both the minor and the major breakpoint cluster region of the BCR gene; breaks in either region result in splitting of one of the BCR YAC signals (illustrated by the green signals); ii) to increase specificity and sensitivity of detection, cohybridization is performed with a cosmid clone recognizing DNA sequences from the ABL gene (illustrated by the red signals) that are distal to the breakpoint on chromosome 9, and thus are translocated to the Philadelphia chromosome (Ph') [21].…”

Rapid detection of BCR/ABL and PML/RARA using fluorescence in situ hybridization in cytospin preparations

“…Published studies have shown that FISH analysis is particularly sensitive for the detection of the chimeric genes arising from t(8;21), t(15;17), inv(16) in AML (Mancini et al 1995;Fischer et al 1996) and the Philadelphia chromosome in CML and acute lymphoblastic leukaemia (ALL) (Bentz, Cabot & Moos 1994). The molecular biology of the chimeric BCR-ABL gene and the PML-RARa fusion has been well documented (Melo 1996;Minden, Imrie & Keat-ing1996) The present paper describes a pilot study for the rapid detection of genetic abnormalities from cytospin preparations of bone marrow and peripheral blood specimens.…”