Myeloid Leukemia

70 Branford and Hughes
selected patients is an allogeneic transplant; however, the recent introduction
of the highly successful Bcr-Abl kinase inhibitor imatinib mesylate (Glivec,
Novartis Pharmaceuticals, Basel, Switzerland) has substantially improved
treatment response. In patients with a complete cytogenetic remission, residual
leukemic cells expressing BCR-ABL mRNA are usually detectable by the polymerase
chain reaction (PCR) technique. Qualitative PCR provides limited
information of treatment response, whereas serial monitoring of BCR-ABL levels
by real-time quantitative reverse transcription (RT) PCR (RQ-PCR) can
identify patients in need of therapeutic intervention before the onset of overt
relapse.
The reliability of RQ-PCR techniques is very dependent on careful experimental
design and appropriate validation of all aspects of the procedure
(reviewed in refs. 3,4). The technique is based on the generation of a fluorescent
signal coupled with instrumentation for the amplification, detection, and
quantification of gene expression. There are now a number of probe systems
and instruments for RQ-PCR (reviewed in ref. 5). The method described here
uses TaqMan® fluorescence hybridization probes on the 7700 Sequence
Detection System (Applied Biosystems, Foster City, CA). The probe consists
of an oligonucleotide with both a fluorescent reporter and a quencher dye
attached. When the probe is intact, the proximity of the reporter dye to the
quencher dye results in suppression of the reporter fluorescence, primarily by
fluorescence resonance energy transfer (FRET). The probe is cleaved by the 5�
to 3� nuclease activity of AmpliTaq DNA polymerase during the extension
phase of the PCR cycle, which results in an increase of reporter fluorescence
signal. The increase in fluorescence is proportional to the amount of accumulated
product, and the SDS software is used to determine the starting copy
number relative to a series of standards (Fig. 1). A control gene is quantitated
to control for variation in the efficiency of the RT step and for variation in the
degree of degradation of the RNA. The quantitative results of the target gene
are reported relative to the control gene, which is the normal BCR gene in our
assay. The assay performance is monitored by quality control samples that are
performed with every RQ-PCR assay.
RQ-PCR for monitoring the level of BCR-ABL transcripts is sensitive and
reliable, and enables a high-throughput analysis (6–9). Rising levels of BCR-
ABL are strongly predictive of cytogenetic and hematological relapse after an
allogeneic transplant (10–12). Monitoring imatinib-treated CML patients by
quantitative RT-PCR has proven effective for defining patient response. Early
reduction of BCR-ABL can predict a subsequent cytogenetic response (13–16).
The level of BCR-ABL predicts for disease-free survival (17,18) and clinical
outcome (15,19), while the proportional reduction in BCR-ABL is closely correlated
with the level of cytogenetic response (19).