DETECTION OF PHILADELPHIA-CHROMOSOME USING PCR AND EUROPIUM-LABELED DNA PROBES

More than 95% of the patients with chronic myelogenous leukemia (CML) carry translocations between protooncogene abl of chromosome 9 and bcr gene of chromosome 22, resulting in the Philadelphia chromosome (Ph(1 )). After allogeneic bone marrow transplantation (BMT) it is important to detect possible residual malignant cells in CML patients. A new se nsitive hybridization method combined with polymerase chain reaction ( PCR), based on the detection of the europium (Eu3+) label by time-reso lved fluorescence, was applied for the detection of Ph(1) chromosome. Total RNA from 10(6) peripheral blood leukocytes was isolated by the a cid guanidinium thiocyanate-phenol-chloroform extraction. After cDNA s ynthesis by reverse transcriptase, the PCR amplification (30 cycles) w as carried out. In the detection phase two oligonucleotide probes were used in the hybridization reaction, one biotinylated (bcr gene, exon 2) and one (abl gene) labeled with Eu3+. The hybrids were collected in a streptavidin-coated microtitration well and the bound Eu3+ was meas ured in a time-resolved fluorometer. To assess the sensitivity of the method, different numbers of CML cell line K562 cells were mixed with 10(5) apparently normal human leukocytes. Five K562 cells/10(5) leukoc ytes could be detected. Six patients with CML confirmed by clinical an d cytogenetic criteria were studied. Three of the patients underwent a n allogeneic BTM 6-18 months before the investigation and all of them were Ph(1)-negative. The other three patients who were nontransplanted were positive as expected.