Comparison of sensitivity of nested PCR and quantitative PCR in Bcr-Abl p210 transcript detection in chronic myelogenous leukemia

Background: For minimal residual disease detection in chronic myelogenous leukemia (CML) patients who have achieved complete clinical remission and complete cytogenetic response, nested PCR (nPCR) and quantitative real-time PCR (qPCR) can be used. Achieving of molecular remission is the goal of therapy, so it is of critical importance and clinical utility to obtain high sensitivity of molecular testing. We compared the level of sensitivity of nPCR and qPCR in the detection of BCR-ABL p210 transcripts in a Bcr/Abl-positive cell dilution model. Materials and Methods: For determination of sensitivity level, serial dilutions of K562 cell line (Bcr-Abl-positive) in NB4 cell line (Bcr-Abl-negative) were made (range of dilution of positive cells: 10-3-10-7). Isolated RNA samples were transcribed into cDNA and tested for p210 transcript by nPCR and qPCR. Bone marrow and peripheral blood samples of two CIVIL patients on imatinib mesylate therapy were also tested by both methods. Results: In the cell dilution test, nPCR showed sensitivity for detecting Bcr-Abl positive cell of 10-5, and qPCR showed a sensitivity of 10-6. Both methods detected p210 transcripts in bone marrow samples of CML patients. However, qPCR also detected transcripts in peripheral blood samples, with a lower transcript level in comparison to bone marrow samples. Conclusion: Although frequently quoted as nPCR being by approximately I log more sensitive than qPCR, in our marker-positive cell line K562 dilution experiment we documented higher sensitivity of the standardized Europe Against Cancer Program developed qPCR method.