In Vitro Sensitivity to Tyrosine Kinase Inhibitors Is One of Possible Predictive Parameters for Therapy Switch In Patients with Chronic Myeloid Leukemia

Tyrosine kinase inhibitors (TKI) are very effective in chronic myeloid leukemia (CML) suppression, however, the problem with development of resistance in some patients exists. It is necessary to find optimal methods for therapy response prediction and for detection of resistance. Many studies on the resistance to imatinib therapy were performed on cell lines or model systems. However, these systems are not fully consistent with CML situation in vivo. Sensitivity to imatinib and its predictivity to molecular response in patients with de novo CML were tested in vitro on patients′ leukocytes by White et al. [Blood 2005; 106: 2520]. They found that IC50 values could be predictive mainly in patients with low Sokal score.

To optimize in vitro method for evaluation of patients′ sensitivity to various TKIs and to test its predictivity for molecular response in therapy and/or after therapy change.

The sensitivity to TKIs: imatinib, nilotinib and dasatinib were studied on leukocytes isolated from CML patients at diagnosis and various responses to treatment. Cell lines were used as controls. Isolated leukocytes/cell lines were cultivated with/without TKIs. Optimization of cultivation was performed on cell lines (ML-2, K562, CML-T2, JURL-MK1) and on leukocytes from CML newly diagnosed patients (15) and healthy donors (6). Various incubation times (4, 24, 48 and 72h) were tested. Concentrations of TKI were used in values near to physiological levels: 2 –3 concentrations for each inhibitor (1uM, 10uM imatinib, 0,5uM and 2uM nilotinib and 1nM, 10nM and 100nM dasatinib). In given time-points the cells were harvested and lysed for protein and mRNA analyses. Sensitivity to TKIs was tested by BCR-ABL kinase inhibition – via Crkl phosphorylation (western blots) and also by WT1 transcript level kinetics [Cilloni et al, Cancer 2004; 101: 979]. Quality of cultivation was tested by apoptosis level (RNA degradation, Annexin staining – Agilent Bioanalyzer 2100).

We found 48 h to be the optimal time for in vitro cultivation. This time was long enough to see TKIs dependent changes on protein as well as mRNA level. At this time the intensity of apoptosis was relatively low and did not influence results. The predictive ability of cultivation with TKIs was tested on patients at diagnosis (15), with optimal (5) and suboptimal response (5) and patient with therapy failure (13). The disease state of all patients was further monitored in range from 6 to 21 months (median 12 months) after cultivation. Mostly all of newly diagnosed patients were in vitro sensitive to all three TKIs, 10 of them achieved MMR (median 7 months, range 5 – 16) on imatinib. In patients with resistance to imanitib therapy the good sensitivity to one of 2nd generation TKI on in vitro tests represented the good response to this inhibitor, 4 patients from 10 on dasatinib achieved MMR (within 4 months), the other responded to therapy with continual decrease of BCR-ABL transcript level. Thus, the cultivation test can help with the therapy switch. However, the prognosis of patients with additive chromosomal aberration was poor even if they were sensitive to TKIs in vitro. Only one of 3 patients with 8 trisomy sensitive to dasatinib in vitro achieved MMR at 4th month after starting of dasatinib. Two patients with T315I were not sensitive to any of TKIs in vitro and in vivo, as it was expected. We continue to follow up of all patients. In conclusion, the results from in vitro cultivations of patients′ leukocytes with TKIs can help with the choice of efficient inhibitor for individual patient′s therapy, however, it is necessary to take into consideration the results of cytogenetic analyses of patients and other factors influencing CML.

Supported by MZOUHKT2005.

No relevant conflicts of interest to declare.