Myeloid Leukemia

50 Picard, Silvy, and Gabert
quantification or a poor sensitivity of analysis (discussed later). For fusion gene
transcripts, at least four dilutions are recommended (100,000 to 10 copies),
because the FG assay must be sensitive. To avoid contamination, careful attention
is required, notably when using plasmids. Thus, we recommend separate
locations for handling cDNA samples and plasmids. For cell line dilutions, all
dilutions are required.
As for any PCR analysis, negative and positive controls are required for
RQ-PCR assays. Negative controls (like water or Tris-EDTA) allow identification
of contamination of reaction mix. A no-amplification control that contains
sample and no enzyme could be necessary to rule out the presence of
fluorescent contaminants in the sample or in the block of the thermal cycler.
To monitor sample contamination, we recommend performing a negative control
for each sample—i.e., an RT sample in which reverse transcriptase has
been omitted. Positive PCR controls, such as a cell line dilution, can also be
used as a calibrator. Moreover, to control the extraction step, which is probably
the most variable part of a PCR assay, an aliquot of a frozen pool of PBMCs
spiked with a cell line positive for the fusion transcript can be added to each
extraction batch and can be run in each RQ-PCR analysis.
Ideally, all samples, standard curve and negative as well as positive controls,
should be amplified at minimum in duplicate in order to limit experimental
pitfalls in a well. It is noted that one well is generally used in LightCycler
for CG detection.
3.2. Fusion Gene Transcripts and AML
Thirty percent of AML cases express one of three fusion gene (FG) transcripts:
PML-RARA, AML1-ETO, or CBFB-MYH11. In the literature, different
sequences for FG transcript primers and probes are described (Table 2). However,
only one study has led to the development of standardized RQ-PCR
achieving sensitivities of at least 10 –5 , suitable for detection of MRD as well as
diagnostic screening (30). Therefore, in 2004, standardization of RQ-PCR is
possible by two means: the EAC protocol or diagnostic kits.
3.2.1. The EAC Protocol
In 1999, 26 laboratories from 10 European countries designed a joint project
of the health and consumer protection of the European Commission (SANCO)
via the EAC program in order to develop standardization and quality control
for RQ-PCR analysis, based on the ABI 7700 platform (Applied Biosystems).
The major aim was to establish a standardized protocol allowing comparison
of MRD data within multicenter therapeutic trials in order to assess the relative
efficiency of each therapeutic strategy for leukemias bearing an appropriate
marker (30,31). The EAC primers and probes for amplification of PML-RARA,