High reliability and sensitivity of the BCR/ABL1 D-FISH test for the detection of BCR/ABL rearrangements

Abstract

The BCR/ABL1 fusion gene is mainly caused by the t(9; 22)(q34; q11.2) translocation, which results in the Philadelphia (Ph) chromosome. The Ph chromosome is the typical hallmark in chronic myeloid leukemia (CML), but can also be present in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). The BCR/ABL1 rearrangement is an important tumor classification marker and a useful prognostic factor allowing an adequate therapy management. Ph chromosome detection by conventional cytogenetics (CC) can be hampered by low quantity and quality of metaphases from tumor cells. Furthermore, BCR/ABL1 rearrangements may be hidden due to cryptic rearrangements or complex aberrations. Therefore, molecular cytogenetic methods turned out to be useful tools for the detection of BCR/ABL1 rearrangements. We performed fluorescent in situ hybridization (FISH) with the recently developed BCR/ABL1 D-FISH probe (QBIOgene, Illkirch, F) on cultured bone marrow and peripheral blood cells of 71 patients with CML, ALL, AML, and myeloproliferative disorder (MPD). FISH results and the results of banding methods were directly compared. Based on the analyses of >200 nuclei per patient, D-FISH correlated closely with CC and allowed an accurate quantification of BCR/ABL1 rearrangements even in a low percentage of aberrant cells. No false-positive or false-negative results were obtained. Furthermore, the D-FISH probe detected three cryptic and one complex BCR/ABL1 rearrangement, which were not visible by CC. We conclude that D-FISH reliably detects standard Ph chromosomes as well as its variant translocations and accurately quantifies BCR/ABL1 rearrangements prior and during cancer treatment as well as in the phase of remission, in daily routine tumor cytogenetic diagnostics.

Evaluation of the Cepheid GeneXpert BCR-ABL Assay

“…5 Sensitivity can theoretically be increased by about 10-fold (to 0.5% leukemia cell content detection) by using fluorescence in situ hybridization and examining 200 white blood cells (WBCs); however, the practical sensitivity of interphase fluorescence in situ hybridization is only about 1%. 6 To date, reverse transcription-polymerase chain reaction (RT-PCR) is considered the most sensitive technique available for detecting low copy numbers of the BCR-ABL gene fusion, and the combination of RT-PCR with real-time fluorescence detection allows for reliable quantitation of the fusion mRNA. The reported sensitivity for the RT-PCR method varies, but it is estimated at one leukemia cell out of 10 5 to 10 6 normal white blood cells.…”

Clinical Applications of BCR-ABL Molecular Testing in Acute Leukemia

“…Cryptic t(9;22) occurs in about 5% of CML cases and also in a small proportion of ALLs, resulting in false negative karyotypic interpretation in metaphase spreads of cells that actually contain the translocation at the molecular level. 4 …”

“…Moreover, FISH detects cryptic and complex BCR-ABL rearrangements such as threeway translocations or breaks outside of the usual major and minor cluster regions. 4 Dual color FISH utilizes two probes, one of which hybridizes to the 5Ј end of the BCR region and the other to the 3Ј end of the ABL region. Fluorescent microscopy is used to visualize these probes which are typically labeled with red and green fluorochromes to produce two red and two green spots representing the two copies of chromosomes 9 and 22 in normal cells.…”

“…In a commercial version of this system (Vysis, Downers Grove, IL) the large ASS-ABL probe mentioned above is combined with a large probe spanning a distance of about 1.5 mb on both sides of the BCR gene, thus providing a second confirmation of the translocation and reducing the rate of false positives to less than 0.5%. 4,[11][12][13] In addition, D-FISH, like S-FISH and ES-FISH, can detect cryptic and complex BCR-ABL translocations that are missed or undecipherable by conventional cytogenetics. 4 …”

“…4,[11][12][13] In addition, D-FISH, like S-FISH and ES-FISH, can detect cryptic and complex BCR-ABL translocations that are missed or undecipherable by conventional cytogenetics. 4 …”