Myeloid Leukemia

Duplexed QZyme RT-PCR for APL Analysis 129
disease (MRD) could serve as an independent prognostic indicator (5). Detection
of PML-RARα transcripts at the conclusion of consolidation therapy, or
subsequent recurrence of detectable transcripts (molecular relapse), was predictive
of imminent clinical relapse. This observation translated into immediate
clinical benefit, because pre-emptive therapy at the point of molecular
relapse, rather than hematological relapse, improved the long-term survival for
individual patients (6). However, qualitative PCR fails to detect MRD in a
subgroup of patients that ultimately relapse. Such false negatives are likely to
reflect poor specimen RNA quality, which remains difficult to accurately
determine with qualitative methods. Recent studies indicate quantitative realtime
PCR will be a more powerful tool for monitoring APL (7–11). These
studies suggest that quantification of fusion transcripts may further improve
prediction of patient relapse and help tailor therapy to individuals. As a consequence,
molecular monitoring is already being incorporated into protocols for
current multi-center therapeutic trials (12).
1.2. Real-Time Quantitative PCR Using QZyme
QZyme PCR (BD Biosciences Clontech) is a novel method that allows
real-time detection and quantification of genomic DNA, cDNA, or mRNA targets.
The protocol is well suited to assessment of MRD because of its capacity
to accurately measure differences in target concentration over a broad dynamic
range, typically extending over five orders of magnitude (13). Further, the
method can detect low numbers of copies of a target transcript, giving a clinician
the best chance of detecting the earliest stages of molecular relapse.
Finally, QZyme PCR is readily amenable to duplex analysis, which has multiple
advantages for analysis of clinical specimens (14). Duplex analysis allows
the investigator to maximize the amount of information obtained from each
clinical specimen, increasing sample throughput and reducing the cost per data
point. Most importantly, duplex reactions allow the inclusion of an internal as
opposed to a parallel control. The internal control enables a more accurate and
reliable measurement of RNA integrity and its ability to be amplified. The
inclusion of a quantitative internal control helps to minimize false-negative
results resulting from poor-quality specimens, which pose a serious risk in the
management of leukemia patients during remission.
1.3. The QZyme Strategy: Analysis of APL Specific Fusion Transcripts
Two duplex, single-tube QZyme RT-PCR assays were developed to simultaneously
quantify PML-RARα fusion transcripts (either L-type and most Vtype
[see Note 1], or S-type) together with an internal control transcript, BCR.
Our reasons for choosing BCR as the control have been discussed previously
(15). These QZyme assays are suitable for quantifying fusion transcripts in