BCR‐ABL1 tyrosine kinase activity at diagnosis, as determined via the pCrkL/CrkL ratio, is predictive of clinical outcome in chronic myeloid leukaemia

Imatinib has become first-line therapy in chronic myeloid leukaemia (CML), but recent studies suggest that more than one-third of patients fail imatinib over 2–3 years. (de Lavallade et al, 2008; Lucas et al, 2008) It would be clinically helpful to prospectively identify patients unlikely to achieve a complete cytogenetic response (CCRe) following imatinib treatment. Prognostic scoring methods, such as the Sokal score, activity of the imatinib uptake transporter, hOCT1 (human organic cation transporter 1) (Wang et al, 2008) and BCR-ABL1 transcript type (Lucas et al, 2009) have been shown to correlate with clinical outcome. However, none of these parameters are sufficiently powerful to reliably prospectively predict patients destined to fare poorly. The phosphorylation status of CrkL has been identified as a surrogate marker of BCR-ABL1 tyrosine kinase activity (White et al, 2005), because BCR-ABL1 tyrosine kinase activity cannot be reliability detected in CML patient samples (Patel et al, 2007). White et al (2005) showed, by Western blotting, that changes in the level of CrkL phosphorylation after 1 month of imatinib treatment correlated with clinical outcome. In this prospective study we developed a fluorescenceactivated cell sorting (FACS) protocol to investigate the predictive value of measuring pCrkL, CrkL and the pCrkL/ CrkL ratio (Lucas et al, 2009) in fresh peripheral blood from 20 untreated chronic phase CML patients prior to commencing either imatinib or nilotinib treatment. For CrkL phosphorylation determination, cells were resuspended in 500 ll of 2% paraformaldehyde (VWR, Lutterworth, UK) fixed for 10 min at 37 C and chilled on ice for 1 min. Cells were harvested by centrifugation (770 g, 3 min), 500 ll 90% methanol (Fisher Scientific, Loughborough, UK) added, vortexed briefly and incubated on ice for 30 min. Cells were then washed (throughout with 1 ml of incubation buffer containing phosphate-buffered saline and 0Æ5% bovine serum albumin), harvested, and resuspended in 25 ll incubation buffer and incubated at room temperature for 10 min. Antibodies (pCrkL antibody Cat# 3181 lot 3 and 4, Cell Signalling Technology, Danvers, MA, USA; CrkL Cat#sc-319, Santa Cruz Biotechnology, Santa Cruz, CA, USA; control anti-normal-rabbit immunoglobulins Cat#AB-105-C, R&D Systems, Abingdon, UK) were added to a final concentration of 28 lg/ml, vortexed and incubated at room temperature for 40 min then washed twice, resuspended in fluorescein-labelled goat-anti-rabbit second layer antibody Alexa Fluor 488 (10 lg/ml; Invitrogen, Paisley, UK), and incubated at room temperature in the dark for 30 min. Twice washed cells were analysed using flow cytometry (FACScalibur; Becton Dickinson, Oxford, UK), with cellquest pro software (Becton Dickinson) for data analysis. The levels of pCrkL and CrkL present in the sample were determined as the geometric mean fluorescence intensity (MFI) minus the MFI value of the control sample. The pCrKL/CrKL ratio of a sample was determined via: