title: Quantitative real-time reverse-transcription polymerase chain reaction for diagnosis of BCR-ABL positive leukemias and molecular monitoring following allogeneic stem cell transplantation.
abstract: Real-time reverse-transcription polymerase chain reaction (RT-PCR) (qPCR) of the BCR-ABL mRNA is a suitable technique to measure the amount of circulating leukemic cells in chronic myelogenous leukemia (CML). In this study, we evaluated a BCR-ABL-specific qPCR method using the LightCycler technology in 95 patients with Philadelphia chromosome positive acute leukemia (n = 7) or CML in different stages (n = 88). Primers and hybridization probes were chosen to detect the most prevalent variants of BCR-ABL (b2a2, b3a2, b2a3, b3a3, e19a2, e1a2) with a sensitivity of 10-5 for b2a2 and b3a2. With median BCR-ABL/G6PDH ratios of 10.7% in the untreated chronic phase, 43.2% in the newly diagnosed accelerated phase, and 131.4% in newly diagnosed blast crisis the BCR-ABL mRNA levels varied significantly between different stages of CML whereas no difference was found between blast crisis and untreated acute leukemias (136.9%). There was a strong relationship between qPCR results and cytogenetics in patients treated with imatinib, interferon-alpha, or following allografting. Thirteen patients with CML were sequentially examined by qPCR following myeloablative or non-myeloablative allogeneic peripheral blood stem cell transplantation. Five patients received donor lymphocytes and became BCR-ABL negative as confirmed by nested RT-PCR. The gradual disappearance of BCR-ABL positive cells could be monitored by qPCR following non-myeloablative transplantation. Comparison of BCR-ABL levels with the degree of donor chimerism showed that 91% of samples with complete donor chimerism were BCR-ABL negative. In 22% of BCR-ABL negative samples chimerism between 71% and 98% was observed, indicating the persistence of normal recipient's hematopoietic cells. In conclusion, the qPCR protocol used in this study is a reliable and fast method for monitoring molecular response in CML.
authors.affiliation: Department of Hematology, Oncology and Clinical Immunology, University of Duesseldorf, Moorenstrasse 5, 40225 Duesseldorf, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Neumann
authors.forename: Caroline
authors.initials: C
authors.lastname: Herold
authors.forename: Barbara
authors.initials: B
authors.lastname: Hildebrandt
authors.forename: Guido
authors.initials: G
authors.lastname: Kobbe
authors.forename: Manuel
authors.initials: M
authors.lastname: Aivado
authors.forename: Astrid
authors.initials: A
authors.lastname: Rong
authors.forename: Maren
authors.initials: M
authors.lastname: Free
authors.forename: Renate
authors.initials: R
authors.lastname: Rössig
authors.forename: Roland
authors.initials: R
authors.lastname: Fenk
authors.forename: Peter
authors.initials: P
authors.lastname: Schneider
authors.forename: Norbert
authors.initials: N
authors.lastname: Gattermann
authors.forename: Brigitte
authors.initials: B
authors.lastname: Royer-Pokora
authors.forename: Rainer
authors.initials: R
authors.lastname: Haas
authors.forename: Ralf
authors.initials: R
authors.lastname: Kronenwett
mesh_terms: D000970:Antineoplastic Agents / Q000008:administration & dosage; D001752:Blast Crisis / Q000175:diagnosis / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005260:Female; D016044:Fusion Proteins, bcr-abl / Q000235:genetics; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000175:diagnosis* / Q000235:genetics / Q000628:therapy; D008297:Male; D025202:Molecular Diagnostic Techniques; D018365:Neoplasm, Residual / Q000175:diagnosis / Q000235:genetics; D009360:Neoplastic Cells, Circulating; D012334:RNA, Neoplasm / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D018183:Transplantation Chimera / Q000235:genetics; D014184:Transplantation, Homologous
chemical_list: D000970:Antineoplastic Agents; D017931:DNA Primers; D012334:RNA, Neoplasm; D016044:Fusion Proteins, bcr-abl