SH2 profiling of 74 primary CLL patients and clinical correlation. (A) Combined unsupervised cluster analysis of SH2 profiles of 74 primary CLL samples with the SHP2 SH2 domain. Far western blots (WBs) were scanned, images were digitalized, and lanes were horizontally subdivided into bins according to the molecular weight of the phosphoproteins. Hierarchical cluster analysis revealed 2 different clusters of low and high SHP2 SH2 binding. (B) TTFT Kaplan-Meier analysis according to SHP2 SH2 patient profiles (SHP2 SH2 high cluster: TTFT 96.2 ± 11.6 months; SHP2 SH2 low cluster: TTFT 50.9 ± 7.1 months; P < .001). (C) SHP2 activity determined after immunoprecipitation (IP) of SHP2 from 20 CLL samples of the high and low SHP2 SH2 clusters using 6,8 difluro-4-methylumbelliferyl phosphate as fluorogenic substrate. Fluorescence signals were normalized to SHP2 signals determined by immunoprecipitation and western blot analysis. Mean values of SHP2 activity are given as horizontal lines; level of significance was determined by the Wilcoxon rank-sum test. (D) SHP2 western blot analysis of the 20 CLL samples subsequent to SHP2 immunoprecipitation.