Myeloid Leukemia

Quantitative PCR for Monitoring CML Patients 69
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Diagnosis and Monitoring of Chronic Myeloid Leukemia
by Qualitative and Quantitative RT-PCR
Susan Branford and Timothy Hughes
Summary
Real-time quantitative reverse-transcription polymerase chain reaction (RQ-PCR) methods
for the quantitation of BCR-ABL mRNA in the blood of patients with chronic myeloid leukemia
(CML) has become the predominant molecular monitoring technique. The BCR-ABL fusion
gene is expressed in over 95% of patients with CML, and RQ-PCR provides a reliable, highthroughput
method to accurately assess the level of treatment response and provides an early
indication of emerging drug resistance. The ABI Prism 7700 Sequence Detection System uses
TaqMan® fluorogenic probes to quantitate specific nucleic acid sequences using RQ-PCR. The
analyzer monitors an increase in fluorescence during the PCR cycle, which is proportional to
the amount of accumulated product. The starting copy number is calculated relative to a series
of standards. The copy number is normalized to a control gene that compensates for variations
in the efficiency of the RT step and for the degree of RNA degradation. In our experience,
reliable and consistent RQ-PCR requires thorough validation of all aspects of the procedure,
including the selection of an appropriate control gene, careful assay design to avoid polymorphisms
in primer or probe binding sites and to exclude the amplification of contaminating
DNA, and monitoring the performance of the RQ-PCR by the use of quality control samples.
Key Words: Chronic myeloid leukemia; BCR-ABL; real-time quantitative PCR; TaqMan;
imatinib mesylate; quality control; atypical; fluorescent probes.
1. Introduction
Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of
the hemopoietic stem cell, which is characterized by the Philadelphia chromosome
translocation (t(9;22)) in approx 95% of patients. The translocation produces
the BCR-ABL gene by juxtaposing part of the ABL proto-oncogene to the
5� portion of the BCR gene. The Bcr-Abl protein is an activated tyrosine kinase
that is causally associated with CML (1,2). The level of BCR-ABL transcript is
a sensitive indicator of the leukemic cell mass. The only chance of cure for
From: Methods in Molecular Medicine, Vol. 125: Myeloid Leukemia: Methods and Protocols
Edited by: H. Iland, M. Hertzberg, and P. Marlton © Humana Press Inc., Totowa, NJ
69