Myeloid Leukemia

Detection of BCR-ABL Mutations 103
Fig. 3. Acquisition of mutations is associated with resistance to imatinib, as shown
by rising levels of BCR-ABL transcripts. The graph represents the log reduction of
BCR-ABL transcripts from the baseline level in a newly diagnosed chronic-phase patient
treated with imatinib. The patient achieved a complete cytogenetic response by 3
mo of imatinib therapy, after which there was a steady increase in the BCR-ABL level
and loss of a major cytogenetic response (defined as Philadelphia chromosome present
in 0–35% of metaphases). Two kinase-domain mutations became detectable at the
time of the BCR-ABL increase, which corresponded with the loss of imatinib response.
samples. The volume of product may be increased successfully by up to 8 µL for
samples producing very faint bands on an agarose gel. The volume of water added
to the reaction is adjusted accordingly.
5. Analysis of the sequence is performed using software that aligns the forward and
reverse sequences against a reference sequence of the ABL kinase domain. Any
variation from the reference sequence is highlighted. The sequence we use is
GenBank accession number M14752. However, we have found that this sequence
has a number of nucleotides within the kinase domain that differ from other ABL
sequences in GenBank, including mRNA sequence X16416 and the DNA
sequence U07563 as well as the sequence found in patients. The discrepant nucleotides
are 894 A>G, 1062 A>G, 1334 G>T, 1335 T>G, 1375 A>G (the first nucleotides
are those published in M14752). It is therefore important that a correct
reference sequence be used to determine true nucleotide substitutions. The entire
chromatogram is carefully inspected to ensure that the sequencing is of adequate
quality to allow true mutations to be detected. Poor quality sequencing may have
high background levels that interfere with analysis. In this situation, the sequencing
reaction should be repeated. When mutations are detected in patients for the
first time, the whole procedure, including RT-PCR and quantitative PCR, is repeated
using a second RNA sample from the same time-point if available, or