Quantitative real‐time reverse‐transcription polymerase chain reaction for diagnosis of BCR‐ABL positive leukemias and molecular monitoring following allogeneic stem cell transplantation

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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‐α, 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.

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