In vivo kinetics of kinase domain mutations in CML patients treated with dasatinib after failing imatinib

A significant minority of newly diagnosed patients with chronic myeloid leukemia (CML) in chronic phase (CP) fail to respond adequately or lose their response to imatinib mesylate (IM) after an initial reduction in leukemia cell load. Some of these patients will still respond to one or other of the newer tyrosine kinase inhibitors (TKI). Resistance to IM is due to various possible mechanisms,^1,,–4  but perhaps the best characterized is expansion of a subclone bearing a BCR-ABL kinase domain (KD) mutation that impairs IM binding.^5,,–8  Here we report finding KD mutations in 12 patients judged to have failed imatinib. The kinetics of the mutant subclones evolved very differently after the start of dasatinib.

We studied 46 consecutive CML patients judged to be resistant to or intolerant of imatinib, who started treatment with dasatinib at the Hammersmith Hospital in London between April 2005 and July 2006. Dasatinib was administered at a dose of 70 mg twice daily by mouth, but dosage was reduced if necessary in the presence of myelosuppression or for other toxic effects. The majority of patients were treated with dasatinib in a phase 2 multicenter study sponsored by the manufacturer and approved by the Research Ethics Committee of the Hammersmith Hospital. All patients gave informed consent for their participation in accordance with the Declaration of Helsinki.

cDNA was synthesized from peripheral blood or bone marrow aspirates and subjected to real-time quantitative polymerase chain reaction (RQ-PCR) as previously described.⁹  cDNA from samples from patients with evidence of resistance to TKI were screened for KD mutations by direct sequencing and subjected to quantitative single nucleotide polymorphism (Q-SNP) analysis by pyrosequencing if appropriate, as detailed previously.¹⁰  If a patient's therapy was changed but he/she achieved no lasting response, cDNA was rescreened by direct sequencing and subjected to Q-SNP analysis as necessary.¹⁰ 

When the results of Q-SNP studies suggested that 2 mutations were present in the same allele, an 863-bp amplicon containing the entire KD generated by nested PCR was cloned using TOPO TA Cloning kit (Invitrogen, Glasgow, United Kingdom) in accordance with recommendations from the manufacturer. Following transformation of competent cells followed by 2-mL overnight cultures, the plasmid DNA was extracted using SV Minipreps (Promega, Southampton, United Kingdom) as recommended by the manufacturer. DNA was subsequently subjected to Sanger sequencing reaction as described previously¹⁰  to sequence the KD.

The loss or gain of a restriction site due to base substitution was used to confirm the coexistence of the 2 mutations in a single mutant subclone. These studies were performed on 450-bp amplicon from residues 220 to 370 amplified by nested PCR, using standard methods and endonuclease in accordance with manufacturer's recommendations.

We detected KD mutations by direct sequencing at the time of starting dasatinib in 12 of the 46 patients, of whom 6 were in CP and 6 in more advanced phases (Table 1). In the remaining34 patients no mutations were found at the time of starting dasatinib or thereafter. Of the 12 patients with mutations, 8 were still taking dasatinib at the time of this analysis for a median period of more than 11 months (range 6+ to 25+), including 5 of the 6 patients still in CP when they started dasatinib. The drug had been discontinued in 4 patients.

In these 12 patients we identified 4 patterns of evolution after starting dasatinib: (1) In 4 patients (nos. 1–4) the mutant clone identified before starting dasatinib became undetectable and remained so during the duration of follow-up. This was associated with a clinical response and a more than 2-log reduction in BCR-ABL transcripts compared with the predasatinib transcript level. (2) In 3 patients (nos. 5–7) the mutant clone identified before the start of dasatinib persisted during treatment or became transiently undetectable and then recurred. The occurrence of a mutation does not always precede clinical relapse. (3) In 3 patients (nos. 8–10) the original mutated clone disappeared, but a new mutant clone was acquired, which was F317L in each case (Figure 1A. (4) In 2 patients (nos. 11 and 12) a new mutation (T315I) appeared in addition to the original mutation in the same clone (Figure 1B,C).

In the 2 group 4 patients one mutation was identified in the earliest available sample, but the second mutation, T315I, was identified after initial response to dasatinib. The fact that in both cases the level of the second mutation paralleled closely the level of the first mutation provided strong evidence that the second mutation actually occurred in the subclone that carried the original mutation. This was confirmed by restriction fragment length polymorphism (RFLP) data, which yielded fragment sizes consistent with sequencing data showing that in each case the 2 mutations were within the same allele. It is interesting to note that Shah and colleagues also have described the coexistence of 2 or more mutations in the same BCR-ABL mRNA, a phenomenon that they term a “compound mutation,” in 4 of their 13 IM-resistant CML patients treated subsequently with dasatinib.¹¹  In contrast, Q-SNP kinetic data for the 2 mutations in patient number 10, while on IM, strongly suggested the presence of 2 distinct subclones.

Our findings support the notion that dasatinib, being appreciably smaller than imatinib, may have a greater affinity for the active kinase conformation and may therefore be able to inhibit a significant number of imatinib-resistant mutant clones.^12,,–15  Furthermore, the data support reports suggesting that contact site mutants, notably T315I and F317L, may both be especially resistant to dasatinib.^12,–14  These observations imply that in patients numbers 6 and 7 with mutant subclones G250R and M244V, respectively, neither of whom ever achieved MMR despite continuing dasatinib, dasatinib resistance was not directly attributable to the mutant subclone but to other undefined mechanism(s). In this series no patient who lacked a mutation when starting dasatinib subsequently acquired one, which suggests that a specific subgroup of CML patients may have a particular predisposition to acquire new BCR-ABL KD mutations.

These findings raise the issue of when precisely the T315I subclone originated in the leukemia cell population in patient numbers 11 and 12. The fact that the dasatinib-resistant clone appeared to develop abruptly in both patients implies either that the T315I mutation developed de novo after start of dasatinib or that it preexisted the dasatinib treatment but was held in check by another unidentified clonal population that was in turn then suppressed by dasatinib. Whichever the case, the 2 mutations may act in synergy to confer a major proliferative advantage over the unmutated leukemia cell population.

The precise mechanism that links a TKI-resistant KD mutation and subsequent events is unclear. First, what is the basis of the proliferative advantage of that subclone over the nonmutated clone, and second, what is the basis of the possible progression to advanced phase disease?¹⁶  One can only speculate that a given mutant clone is permitted to expand “selectively” when the choice, dose, and duration of TKI therapy are all “suboptimal.” This expansion would presumably allow an increase in the size of a population of leukemia cells especially susceptible to acquisition of additional genetic changes, either in the BCR-ABL gene or equally credibly in other genes, and this in turn would expedite progression to a more aggressive phenotype for the leukemia. This supposition highlights the need for early detection of an emerging resistant clone with the aim of eradicating it before it becomes refractory to further treatment.

We thank the various staff members responsible for the clinical care of the patients described in this report.

This work was supported by the funds for research and clinical service provided by the Hammersmith Hospitals Trust. Ms Mehta was undertaking a medical student elective and was supported by bursaries from Cancer Research UK, the Leukemia Research Fund (United Kingdom), the Pathological Society of Great Britain and Northern Ireland, and the Wellcome Trust.

Contribution: J.S. Khorashad, M.A., A.B., J.S. Kaeda, and S.G. performed RQ-PCR for BCR-ABL transcripts and quantitation of the size of the mutant subclones; A.G.R. and V.D.M. carried out FISH studies and contributed to molecular studies; H.d.L. and P.M. collected clinical data; J.F.A., D. Marin, E.O., and D. Milojkovic supervised care of patients in the clinic and wrote various parts of the manuscript; and J. S. Kaeda, J. S. Khorashad, D. Milojkovic, D. Marin, E.O., J.M.G., and J.F.A. prepared the final manuscript for submission.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Prof John M. Goldman, Department of Haematology, Imperial College - Hammersmith Hospital Campus, London W12 ONN, United Kingdom; e-mail: j.goldman@imperial.ac.uk.