Myeloid Leukemia

Detection of BCR-ABL Mutations 101
of the RNA is assessed prior to sequencing using real-time quantitative PCR to
determine the levels of BCR-ABL mRNA and a control mRNA. A cutoff level
based on the number of detectable BCR control transcripts in the sample was
established as an indicator of degraded RNA. When samples that did not comply
with these cutoff values are used for mutation analysis, unreliable results may be
obtained, as evidenced by conflicting results between repeat analyses. The cutoff
value varies depending on the level of the BCR-ABL transcripts. For patients with
very low BCR-ABL levels it is particularly important that the sample is of high
quality; otherwise, the sample will not amplify in the two-step PCR. The indicators
used in our analysis are: BCR values of >100,000 transcripts if the BCR-
ABL/BCR ratio is 50,000 transcripts if the BCR-ABL/BCR ratio is 0.1% to 1.0%, and BCR values
>7,000 transcripts if the BCR-ABL/BCR ratio is >1.0%. Individual laboratories
will need to determine an appropriate method to indicate adequate quality
RNA if the real-time technique is not available. To allow amplification of samples
with very low BCR-ABL values in the second-step PCR, the amount of cDNA
added to the first-step PCR is increased. For patients with BCR-ABL/BCR ratios
of less than 1.0%, 3µL of cDNA (150 ng) is added. These patients usually have a
complete cytogenetic response to imatinib and a lower incidence of acquired resistance.
However, we have detected mutations in some patients with this level
of response, and it is therefore important that the mutation technique be optimized
to allow analysis in these patients (16). For patients with higher BCR-ABL
levels, 2µL of cDNA (100 ng) is usually adequate to amplify a suitable product in
the first-step PCR. The amount of master mix and cDNA added for each patient
is therefore tailored to the BCR-ABL level.
2. The accuracy of mutation detection is also dependent on the efficiency of the
PCR reaction. For this reason a quality control sample that has a mid-range BCR-
ABL level (approx 1.0–8.0% BCR-ABL/BCR%) is used to monitor the PCR. This
level of BCR-ABL is within the range of values that is equivalent to a partial
cytogenetic response (Philadelphia chromosome level 1–35%). As an indication
of adequate efficiency of PCR amplification, the positive control should amplify
in the first-step PCR. We have found that failure to amplify correctly in the firststep
PCR may lead to inaccuracy of mutation analysis, and low-abundance mutations
may not be detectable if the second-step PCR proceeds. For this reason, the
first-step PCR is repeated if the quality control sample fails to produce a band of
the correct size on the agarose gel.
3. On occasion, additional bands are visualized on the agarose gel after PCR amplification
that may interfere with the sequencing analysis. In the second-step PCR,
a product of approx 1600 bp is sometimes present in addition to the correct band
of 863 bp. The larger band is the first-round product and will not interfere with
the sequencing reaction. However, bands of a smaller size than 863 bp represent
artifact amplification and will interfere with the sequencing reaction. During
method optimization it was found that alternative transcription occurred in some
samples and produced a PCR product with ABL exon 7 spliced out. When the