FLUORESCENT IN SITU HYBRIDIZATION (FISH) ON PERIPHERAL BLOOD AND BONE MARROW SMARS: 120 MINUTES FOR DETECTION OF PML/RARa FUSION GENE

Acute promyelocytic leukemia (APL) is a clonal hematopoietic stem cell disorder characterized by a chromosomal translocation involving the retinoic acid receptor-a gene on chromosome 17 (RARA). In 95% of cases of APL, RARA gene is involved in a balanced reciprocal translocation with the promyelocytic leukemia gene (PML) on chromosome 15, a translocation denoted as t(15;17)(q22;q12). The long-term outlook is now favorable for the majority of APL patients, when treatment is instituted promptly, due to the availability of therapies such as all-trans retinoic acid (ATRA) with anthracycline-based chemotherapy (idarubicin) and arsenic trioxide (ATO). Therefore, rapid diagnosis of APL contributes to a highly effective therapy. The conventional cytogenetic analysis is an excellent method for detecting the t(15;17)(q22;q12) in APL but it is subject to limitations. Since only dividing cells can be analyzed, sometimes there is no useful results for some patients, because of poor chromosome morphology and/or an insufficient amounts of assessable metaphases; furthermore the conventional chromosome analysis is labour-intensive and time consuming. Fluorescence in situ hybridization (FISH) overcomes some of these limitations and enables the detection of chromosomal rearrangements even on interphase cells, avoiding the requirement of metaphase obtention. Typically, the technique involves multiple step of sample preparation and hybridization of the sample and the probe so taking about 24 hours to analyze the data. Given the need for a rapid diagnosis in patients with APL, we investigated the usefulness and the accuracy of FISH, performed with a commercial probe, to identify the PML-RAR fusion gene, using a quick method applied on peripheral blood and bone marrow smears. Using this 120 minutes lasting procedure, we obtained bright, distinct, compact and easily evaluable hybridization signals with low background and we were able to clearly and unambiguously detect the fusion gene PML/ RARa in 90% of the cases, due to the variable quality of the material available. This study suggests that FISH performed on peripheral blood and bone marrow smears is a reliable method for the rapid detection of PML/RARa rearrangement.