Use of Direct Sequence PCR for ABl Kinase Mutations in Patients with CML Blast Crisis, Treated Prior to the Availability of Imatinib Therapy.

We identified all patients who developed blast crisis before the advent of imatinib therapy and in whom stored marrow samples were available from a database of CML patients in a major teaching hospital. mRNA was extracted and cDNA synthesized successfully in 20 of 22 cases. The cDNA was subjected to real time PCR for quantification of BCR-ABL transcripts. The ABL allele not involved in the t(9;22) translocation was excluded from mutational analysis by subjecting the cDNA to nested PCR. This was achieved by amplifying from exons 13 and 10 of the BCR and ABL moieties of the BCR-ABL fusion gene using primers B2A and JAMR, respectively. The resulting amplicon was then subjected to nested PCR using primers, NTPB+ and NTPE-, sited within exons 4 and 10 of the ABL gene. The nested PCR yielded an 863 bp fragment in length containing the entire BCR-ABL kinase domain. To test for successful amplification an aliquot of the PCR products were electrophoresed through 2.0% agarose gel. In all cases, a single amplicon was observed and the PCR reaction subjected to magnetic purification. The purified amplicon were then sequenced by Sanger’s dideoxy chain termination reaction using Big-Dye ABI 310 sequencer (Applied Biosystems, Foster City, USA). In each case, the sequence obtained was compared with the published ABL type 1a sequence, Genbank M14752, using Blast 2 software. No mutations were found in any patient. This suggests the absence of a mutated kinase in a proportion of greater than approximately 30% of the total BCR-ABL kinase, due to the limit of detection of the technique. Our results suggest that the predominant form of BCR-ABL in the setting of blast crisis (in the absence of imatinib) is wild type. This goes against theories that kinase mutations confer an intrinsic ‘gain of function’ or a proliferative advantage to a CML clone. Instead, it is likely that mutant forms of BCR-ABL which are relatively resistant to imatinib binding gain prominence through positive selection with the presence of imatinib within the cell milieu.