title: A comparative analysis of FISH, RT-PCR, and cytogenetics for the diagnosis of bcr-abl-positive leukemias.
abstract: Philadelphia (Ph) chromosome-positive leukemias, with the bcr-abl gene translocation, have a dismal prognosis. The identification of Ph-positive patients is vitally important because only aggressive therapeutic approaches, such as allogeneic bone marrow transplantation, may result in long-term disease-free survival. Routine diagnostic methods, such as Southern blot analysis and cytogenetics, may lead to false-negative results. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis is considered the most sensitive tool for the detection of the bcr-abl translocation, and it is widely used alone or in combination with karyotyping or Southern blot analysis to identify Ph-positive cases. In this study, we used fluorescence in situ hybridization (FISH) with BCR and ABL double-color probes for detecting Ph-positive leukemias. The FISH results were compared with the results of cytogenetic and RT-PCR analyses in 75 patients with leukemia or other myeloproliferative syndromes (chronic myeloid leukemia, 30; acute lymphoblastic leukemia, 24; acute myelogenous leukemia, 6; essential (hemorrhagic) thrombocythemia, 12; chronic myelomonocytic leukemia, 2; and polycythemia vera, 1). FISH analysis proved to be simple, extremely reliable and sensitive; bcr-abl fusion detection was successful in the presence of all types of molecular junctions i.e., (b2a2, b3a2, and e1a2). Furthermore, a Ph-positive case that proved fusion negative by RT-PCR was identified as positive by FISH. The sensitivity of RT-PCR and FISH related to Ph-positive cases were 97% and 100%, respectively. Regarding specificity, in 4 (5%) of 75 patients, RT-PCR provided false-positive results. Cross-contamination was identified because a new specimen was harvested and reanalyzed when FISH, cytogenetics, and RT-PCR results were contradictory. We believe FISH is an optimal diagnostic method to detect bcr-abl translocation that can be used alone or to validate the results of RT-PCR analysis.
authors.affiliation: Department of Hematology, University of Tor Vergata, Ospedale S'Eugenio, Rome, Italy.
authors.forename: M C
authors.initials: MC
authors.lastname: Cox
authors.forename: L
authors.initials: L
authors.lastname: Maffei
authors.forename: S
authors.initials: S
authors.lastname: Buffolino
authors.forename: G
authors.initials: G
authors.lastname: Del Poeta
authors.forename: A
authors.initials: A
authors.lastname: Venditti
authors.forename: M
authors.initials: M
authors.lastname: Cantonetti
authors.forename: G
authors.initials: G
authors.lastname: Aronica
authors.forename: P
authors.initials: P
authors.lastname: Aquilina
authors.forename: M
authors.initials: M
authors.lastname: Masi
authors.forename: S
authors.initials: S
authors.lastname: Amadori
mesh_terms: D001853:Bone Marrow / Q000737:chemistry; D002871:Chromosome Banding; D005456:Fluorescent Dyes / Q000032:analysis; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007621:Karyotyping; D007938:Leukemia / Q000235:genetics*; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics; D015470:Leukemia, Myeloid, Acute / Q000235:genetics; D015477:Leukemia, Myelomonocytic, Chronic / Q000235:genetics; D007962:Leukocytes / Q000737:chemistry; D009196:Myeloproliferative Disorders / Q000235:genetics*; D015513:Oncogene Proteins / Q000235:genetics*; D011087:Polycythemia Vera / Q000235:genetics; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D016315:Proto-Oncogene Proteins c-abl / Q000235:genetics*; D051562:Proto-Oncogene Proteins c-bcr; D012334:RNA, Neoplasm / Q000032:analysis; D013920:Thrombocythemia, Essential / Q000235:genetics; D014178:Translocation, Genetic
chemical_list: D005456:Fluorescent Dyes; D015513:Oncogene Proteins; D011518:Proto-Oncogene Proteins; D012334:RNA, Neoplasm; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl; D016315:Proto-Oncogene Proteins c-abl; C496613:BCR protein, human; D051562:Proto-Oncogene Proteins c-bcr