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Long-Template DNA Polymerase Chain Reaction for the Detection of the bcr/abl Translocation in Patients with Chronic Myelogenous Leukemia¹

Department of Internal Medicine I, Hematology/Oncology, Albert Ludwigs University Freiburg Medical Center, D-79106 Freiburg, Germany

In most patients with chronic myelogenous leukemia (CML) primitive hematopoietic progenitors carry the acquired reciprocal bcr/abl gene rearrangement t(9;22)(q34.1;q11.21). However, not all of the progenitor cells express the bcr/abl hybrid mRNA or the p210 fusion protein. These cells, therefore, might escape detection by techniques that are based on expression of the fusion gene. To circumvent this problem, we established a new detection method for the rearrangement at the DNA level. Because breakpoints might occur in a very large genomic region (>200 kb), we developed a long-template DNA-PCR (LT-DNA-PCR). In 22 of 59 CML patients, fragments of up to 19 kb could be amplified. Furthermore, 6 of 7 leukapheresis products of three bcr/abl-positive patients which were collected after mobilization chemotherapy and had been shown to be negative for the bcr/abl rearrangement by FISH and by RT-PCR were clearly positive by LT-DNA-PCR. Using a specific pair of primers, it is possible to detect the presence of, and to characterize, the individual gene rearrangement. This approach could serve for diagnostic purposes as well as detection of minimal residual disease under cytotoxic therapy or after purging regimens, being independent of expression of the bcr/abl hybrid mRNA or the fusion protein.

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