Myeloid Leukemia

Detection of BCR-ABL Mutations 95
Fig. 1. Linear representation of the ABL kinase domain, showing the relative location
of the point mutations identified in imatinib-treated patients to date. A number of
nucleotide substitutions resulted in the replacement of the same amino acid. For
example, mutation at nucleotides 756 and 757 both resulted in the Q252H mutation
(nucleotides numbered according to GenBank accession number M14752). In our series
of 279 imatinib-treated patients who are monitored for mutations at least every 6
mo, we have detected mutations in 66 patients (24%). These patients represent all
phases of the disease, and virtually all mutations lead to loss of imatinib response (16).
Multiple mutations have been detected in 23% of the patients with mutations, and the
most frequently detected mutations in our patients are M351T, E255K, F359V, and
T315I.
It is therefore important that emerging mutations be detected early to allow
timely treatment intervention before overt relapse.
A number of procedures have been described for the detection of kinase
domain mutations that involve polymerase chain reaction (PCR) amplification
of either the BCR-ABL kinase domain or the ABL kinase domain (reviewed in
ref. 27). Amplification of the ABL kinase domain does not distinguish the BCR-
ABL allele and is therefore less sensitive because the BCR-ABL target sequence
is diluted by the normal ABL sequence. Various primer combinations have been
used that either amplify the entire kinase domain (28) or restrict the amplification
to exclude the region of the kinase domain beyond the activation loop
(14,17–19). Mutation detection usually involves either direct sequencing of
the amplified product or subcloning of the PCR product and sequence analysis
of multiple clones. Subcloning is a time-consuming technique and is thus less
suitable for widespread testing compared to direct sequencing.
Our method involves RT-PCR amplification of the entire kinase domain of
the BCR-ABL allele using a 5� BCR primer and a 3� ABL primer. A secondstep
PCR using ABL primers may be necessary for samples with low BCR-ABL
transcript levels. A number of mutations have been described in the region
beyond the activation loop, and it is therefore essential to amplify the entire