Immunophenotyping.

The expression of surface ROR1 and intracellular Bcl2 were determined by flow cytometry using viably stored samples from CLL patients as previously described (21). The expression of ROR1 and Bcl2 were detected using Alexa-647-conjugated anti-ROR1 mAb (4A5) and a phycoerythrin (PE)-conjugated anti-Bcl2 antibody (Becton Dickinson, Pharmingen). CLL cells also were analyzed for CD19, CD20, and CD23, using mAbs conjugated to APC, PerCp, FITC, and PE, respectively (Becton Dickinson, Pharmingen). Fluorochrome-conjugated, isotype control mAbs of irrelevant specificity were used in all experiments to monitor for nonspecific staining. The relative expression levels of ROR1 and intracellular Bcl2 are reported as AbMFI, which is determined by subtracting the mean fluorescence intensity of the CLL cells stained with a nonspecific antibody of the same isotype from the mean fluorescence intensity of the CLL cells stained with the anti-ROR1 or anti-Bcl2 antibody. All immunophenotyping data were acquired using the FACSCalibur (BD Biosciences) and analyzed with FlowJo (version 2.7.4). For this study, we selected 12 ROR1-positive samples showing ΔMFI (mean fluorescence intensity) values that exceeded the isotype control by at least twofold, and 12 ROR1 negative samples showing a ΔMFI values similar with the one observed in the isotype control (included in cohort A). Thirty-two samples from cohort B were also used for assessment of Bcl2 expression by flow cytometry.