Quantitative real-time reverse-transcription polymerase chain reaction for diagnosis of BCR-ABL positive leukemias and molecular monitoring following allogeneic stem cell transplantation.
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.
Neumann
Frank
F
Department of Hematology, Oncology and Clinical Immunology, University of Duesseldorf, Moorenstrasse 5, 40225 Duesseldorf, Germany.
Herold
Caroline
C
Hildebrandt
Barbara
B
Kobbe
Guido
G
Aivado
Manuel
M
Rong
Astrid
A
Free
Maren
M
Rössig
Renate
R
Fenk
Roland
R
Schneider
Peter
P
Gattermann
Norbert
N
Royer-Pokora
Brigitte
B
Haas
Rainer
R
Kronenwett
Ralf
R
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
D000970:Antineoplastic Agents; D017931:DNA Primers; D012334:RNA, Neoplasm; D016044:Fusion Proteins, bcr-abl