A 2-COLOR BCR-ABL PROBE THAT GREATLY REDUCES THE FALSE-POSITIVE AND FALSE-NEGATIVE RATES FOR FLUORESCENCE IN-SITU HYBRIDIZATION IN CHRONIC MYELOID-LEUKEMIA

The t(9;22) translocation resulting in the fusion of BCR and ABL genes is pathognomonic in chronic myeloid leukemia (CML) and may be investi gated at the molecular level using fluorescence in situ hybridization (FISH). Two-color BCR-ABL probes visualizing one fusion signal (1F FIS H) have high false positive rates (FPR) and false negative rates (FNR) . The FPR is a result of the random spatial association of probe signa ls within normal interphase cells so that some cells appear to contain the BCR-ABL fusion gene. The FNR of 1F FISH probes depends on the dis tance between the BCR and ABL probes hybridized to the BCR-ABL fusion gene (less than or equal to 368 kb); the ''gap'' between the signals c ausing the cell to be interpreted as normal. To overcome there difficu lties, a two-color probe was used. employing four yeast artificial chr omosome (YAC) sequences that span the breakpoint regions of the BCR an d ABL genes and that visualize the two fusion signals BCR-ABL and ABL- BCR in CML cells (2F FISH). The FNR for the 2F FISH probes was assesse d on clonal Ph+ granulocyte-macrophage-colony-forming cell (CFU-GM) de rived colonies and was reduced to 0.4% (2/450), compared with an FNR o f 13.5% (111/823) with 1F FISH. The FPR in normal mononuclear cells fo r the 2F FISH was 0.19 +/- 0.12% (3/1,700), whereas the FPR using 1F F ISH was 4.5 +/- 2.3% (63/1,294). The 2F FISH can thus be used to evalu ate very small frequencies of BCR-ABL-positive and -negative interphas e cells and may be of use in the clinical monitoring of CML. (C) 1998 Wiley-Liss, Inc.