• 2018-07
  • 2019-04
  • 2019-05
  • Real time quantitative polymerase chain reaction RQ PCR was


    Real-time quantitative polymerase chain reaction (RQ-PCR) was performed to quantify BCR/ABL1 transcript as described previously [1]. The quantitative results were expressed as percent ratios relative to an ABL1 internal control using the following formula (p210 BCR–ABL/ABL1×100×CF) [2]. The level of BCR/ABL1 expression was 31% at diagnosis (IS). The patient was first treated with hydroxyurea at 3g/day for 7 days, after which imatinib (Gleevec, Novartis, Basel, Switzerland) was administered at a dosage of 600mg/day. In January 2011, cytogenetic analysis revealed an unexpected t(11;22)(p15;q11) with aldose reductase 9 apparently not being involved (Fig. 1 lower). D-FISH signal pattern proved to be 2F1G1O, with one fusion signal on the der(22), a second fusion signal on 11p15, and one orange and one green signal on normal chromosomes 9 and 22, respectively (Fig.2B). The karyotype was then interpreted as t(9;11;22)(q34;p15;q11) [3]. BCR/ABL1 expression had increased to 39.5% (IS). The patient was treated with the ICE chemotherapy protocol consisting of idarubicin 8mg/m2/day, cytarabine (ARA-C) 800mg/m2 and etoposide 150mg/m2/day. The patient was treated again with imatinib 800mg/day without hematological response.
    Results At diagnosis the patient was in accelerated phase CML with standard translocation. After three months of therapy with imatinib, cytogenetic analysis revealed the persistence of Ph in all examined metaphases but with a variant translocation involving a third chromosome, t(9;11;22) which had not been observed in the previous cytogenetic analysis (Fig.1). ICE-chemotherapy, which proved to be clinically unsatisfactory, apparently reduced the variant translocation clone but led to the expansion of the Ph+ clone with standard translocation. The following cycle of imatinib 800mg/day before BMT was also ineffective. The variant translocation reappeared with additional chromosomal abnormalities including Ph duplication. Quinacrine-bandend (Q-banded) and FISH techniques revealed the presence of the two Ph translocations (standard and variant) alternatively with ABL1/BCR construct location on 9q34 or 11p15 and without adjacent deletions in the junctions of the BCR and/or ABL1 genes (Fig. 2). Considering the alternation of the type of translocation, which would appear to be related to the kind of therapy, we observed that cells carrying the standard translocation were sensitive to imatinib, while the cells bearing the variant translocation were sensitive to ICE chemotherapy. The BM samples that we had stored at diagnosis and after three months of imatinib therapy were therefore screened for kinase domain (KD) mutations in the BCR–ABL1 gene using direct sequencing as described previously [4]. The BM sample taken at diagnosis showing the standard translocation had no detectable BCR–ABL1 KD mutation (Table 1, Fig. 3A), while two different KD mutations were identified in the sample carrying the variant translocation: the first mutation has already been reported and was at codon position 255 (E255V), while the second mutation, which has never previously been described, was at position 258 (E258V) (Table 1, Fig. 3B–D) [5]. The two mutations led to an A to T transition that resulted in a glutamic acid to valine substitution in the ABL1 protein and both mutations should be present at higher than 20%. These point-mutations in the tyrosine kinase domain of BCR–ABL1 may cause progressive clinical resistance to imatinib and are associated with a greater likelihood of progression to blast crisis and shorter survival [6].
    Discussion Our case presents two points of interest: Of the mechanisms of resistance to imatinib, point mutations in the ABL1 KD are among the most frequently investigated. Several mutations are known to confer differing levels of resistance to the available tyrosine kinase inhibitors [5]. The modalities of occurrence and/or selection of mutations in the ABL1 KD sequence are under investigation, but some authors believe that the mutations can antedate treatment with imatinib [9]. This theory is in agreement with the hypothesis that the KD mutations in our patient were present in a number of proliferating leukemic cells, and that imatinib rapidly selected the clone bearing the mutations. Establishing the presence of leukemic clones with KD mutations as early on as possible could therefore lead to the timely use of different tyrosine kinase inhibitors [10].