Sensitivity and resistance of leukemia cells to imatinib and dasatinib in cml patients

Konference: 2008 IV. ročník DDPEO A I. ročník sympózia O cílené biologické léčbě

Kategorie: Onkologická diagnostika

Téma: Nové prediktivní faktory v klinické onkologii

Číslo abstraktu: 003

Autoři: Mgr. Jitka Veselovská, Ph.D.; Mgr. Renata Mojzíková (Solná), Ph.D.; Mgr. Šárka Rožmanová ; prof. MUDr. Edgar Faber, CSc.; Prof. RNDr. Mgr. Marie Jarošová, CSc.; RNDr. Milena Holzerová, Ph.D.; prof. MUDr. Karel Indrák, DrSc.; Doc.RNDr. Vladimír Divoký, Ph.D.

Background. Second generation Abl tyrosine kinase inhibitors (TKIs), such as dasatinib (DAS) or nilotinib, are currently available for the treatment of patients with chronic myeloid leukemia (CML) resistant or intolerant to imatinib (IM) therapy. The efficacy of TKIs can be evaluated in vitro by degree of inhibition of phosphorylation of selected signaling molecules downstream Bcr-Abl after incubation of leukocytes with the drug.

Aim. To evaluate functional assay that enables the clinicians to predict therapy responses and the degree of resistance/sensitivity of BcrAbl-positive leukemia patients to TKIs.

Methods. Quantitative real time RT-PCR (Q-RT-PCR) was performed to monitor the level of BCR-ABL transcripts. BCR-ABL mutational status was assessed using sequencing of the RT-PCR products. The in vitro test of sensitivity to TKIs was based on assessment of inhibition of phosphorylation of Crkl and Src Family of Kinases (SFK) after incubation of patient’s leukocytes with the drug in vitro. We used western blot with anti-Crkl 32H4 monoclonal antibody (Cell SignalingTechnology, Beverly, MA) and Phospho-Src Family (Tyr416) Antibody (Cell Signaling Technology Inc., Danvers, MA). Leukocytes were incubated one hour with or without 10µM IM or 250nM DAS and than processed for immunoblotting.

Results. Over 60 patients were analyzed using the test, some of them repeatedly. The extent of inhibition of phosphorylation of Crkl and SFK in this assay correlated with the clinical status and the numbers of BCR-ABL-positive cells assessed by FISH and/or Q-RT-PCR. The patients’ cells with Y253H and T315I mutations showed a complete or partial failure to inhibit Bcr-Abl TK by IM, based on the ratio of mutant and wild-type clones. DAS in T315I patients did not inhibit Bcr-Abl TK, but completely inhibited SFK. Patient with E255K mutation showed resistance to IM but sensitivity to DAS, which correlated with his in vivo responses to these drugs. Some IM-resistant patients lacking mutation of BCR-ABL proved sensitivity to higher dosage of IM or to DAS in this assay, which lead to appropriate therapeutical intervention. In one patient, BCR-ABL clone was eradicated on IM therapy; however, a Ph negative myeloproliferative disorder associated with a high activation of SFK persisted, suggesting activation of na alternative signaling pathway not inhibited by IM and independent of Bcr-Abl. The patient developed resistance to DAS, which corresponded with a failure to inhibit SFK in in vitro phosphorylation assay.

Conclusions. In vitro test of sensitivity of Bcr-Abl-leukemia cells to TKIs allows direct evaluation of the Bcr-Abl protein inhibition and reflects both the (possible) mutational status of Bcr-Abl as well as other mechanisms of resistance intrinsic to the cell. It could serve as a simple test for indication of these inhibitors in a patient-specific manner.

Acknowledgement. Supported by grant MSM 6198959205 and by Bristol-Myers Squibb.

Datum přednesení příspěvku: 26. 11. 2008