title: Spectrophotometric detection of RT-PCR-amplified BCR/ABL fusion transcripts. A survey performed on archival hematologic slides.
abstract: We describe a method of spectrophotometric detection of BCR/ABL chimeric sequences amplified by multiplex reverse transcriptase-polymerase chain reaction (RT-PCR), enabling the use of archival hematologic slides as RNA sources. Multiplex PCR amplified b3a2, b2a2, and e1a2 break points of the BCR/ABL translocation and the normal ABL gene product. Assessment of sensitivity, performed on K562 cells, showed that the threshold approximated radioactive methods of detection (i.e., 1 positive cell in 1 x 10(6) negative cells for single round PCR and lower than 1 positive cell in 1 x 10(7) negative cells for nested PCR). Then, we assayed 38 different archival hematologic slides from 18 patients, including 11 cases of chronic myelogenous leukemia or chronic myelogenous leukemia-like disease, such as a case of myelofibrosis and a case of chronic neutrophilic leukemia, 6 cases of acute lymphoblastic leukemia, and 1 case of acute myelogenous leukemia. Amplification and spectrophotometric detection of BCR/ABL fusion messenger RNAs gave an unambiguous positive result in 24 (89%) of 27 expected positive slides, among which 17 (63%) were positive after a single PCR round. Concordant unambiguous results were obtained from 35 (92%) of 38 slides, as verified through parallel analyses of corresponding cryopreserved cells. Retrospective analysis on archival hematologic slides yielded identification of the presence or absence of the t(9;22) translocation and break point in 14 previously uncharacterized cases. The application of this method can help define the diagnosis of cases lacking other appropriate material and assist in the retrospective analysis of large patient series for which only smears are available.
authors.affiliation: Division of Experimental Oncology 2, Centro di Riferimento Oncologico IRCCS, Aviano, Pordenone, Italy.
authors.forename: V
authors.initials: V
authors.lastname: Attadia
authors.forename: M
authors.initials: M
authors.lastname: Alosi
authors.forename: S
authors.initials: S
authors.lastname: Improta
authors.forename: V
authors.initials: V
authors.lastname: Zagonel
authors.forename: G
authors.initials: G
authors.lastname: Martinazzo
authors.forename: A
authors.initials: A
authors.lastname: Carbone
authors.forename: A
authors.initials: A
authors.lastname: Pinto
mesh_terms: D002460:Cell Line; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000175:diagnosis / Q000235:genetics; D015470:Leukemia, Myeloid, Acute / Q000175:diagnosis / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000175:diagnosis / Q000235:genetics; D012189:Retrospective Studies; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods*; D014178:Translocation, Genetic; D014407:Tumor Cells, Cultured
chemical_list: D016044:Fusion Proteins, bcr-abl