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.

Minimal residual disease diagnostics in myeloid malignancies in the post transplant period

“…In CML, quantitative measurement of the BCR-ABL-expression level was found to be more suitable for the planning of therapeutic interventions than chimerism. 60,124 In patients relapsing with AML after SCT it was shown that the NPM1 mutations precede the decrease of molecular chimerism and the increase of the FLT3-LM in doublemutated cases by intervals of 2-3 weeks, 28 but this has to be confirmed by additional studies and such comparisons are missing for other markers in myeloid malignancies. With respect to chimerism, the different levels of sensitivity as provided by different methods must be taken into account in the interpretation of studies.…”

“…Quantitative real-time PCR was found reliable to monitor the response to these interventions in several studies. 16,17,124,125 Both real-time PCR and nested PCR allow comparable sensitivities of 10 À4 -10 À6 , 8,126 to evaluate the response to imatinib 127 or donor lymphocytes 11 in relapse after SCT. However, the kinetics of disease can only be monitored by quantitative real-time PCR whereas nested PCR is able to confirm the achievement of complete molecular remission.…”

BCR-ABL-transduced human cord blood cells produce abnormal populations in immunodeficient mice

“…The level and degree of phosphorylation exhibited by the p210 BCR-ABL expressed in these cells was equivalent to that seen in K562 cells, a cell line developed from a CML patient with blast phase disease that contains multiple copies of the BCR-ABL oncogene 21,22 and also displays evidence of erythroid differentiation. 23 Thus, some of the features obtained in this in vivo xenograft model are more reminiscent of blast phase CML where higher levels of BCR-ABL expression are typically seen 24,25 and perturbations of differentiation are usually more exaggerated. The use of a weaker promoter to reduce the level of expression of the introduced BCR-ABL transgene may thus be needed to create a model that more closely approximates the chronic phase of human CML.…”

Distinct molecular phenotype of malignant CD34+ hematopoietic stem and progenitor cells in chronic myelogenous leukemia

“…CD34 þ cells was performed with the RNeasy Mini Kit (Qiagen AG, Hilden, Germany) according to the manufacturer's Age, PB counts (leukocytes, thrombocytes, hemoglobin), BCR-ABL transcript levels (BCR-ABL/G6PDH ratio) and purity of enriched CD34+ cells is given for each patient included in this study. BCR-ABL/G6PDH ratio was assessed as previously described (Neumann et al, 2003). For clonogenic assays, no CD34+ cell selection was performed (n.p.…”