(A) RNA was isolated from 55 freshly frozen human GISTs and analyzed for ETV4 and ETV1 using real-time PCR. Prim/UT–primary, untreated GIST, Met/Res – metastatic, imatinib-resistant GIST, HPF – high power field. Horizontal bars represent the median (left and right). Student’s t test, *P < 0.05. A scatter plot shows the correlation between ETV4 and mitotic count (middle) (Spearman’s rho = 0.67 with P = 0.001 per 2-tailed test). (B) Protein extracts were prepared from freshly frozen human GISTs that were either primary, untreated tumors with a mitotic rate of ≤5/50 HPF or >5/50 HPF, and metastatic, imatinib-resistant GISTs with a mitotic rate of >10/50 HPF and immunoblotted with anti-human ETV4 IgG followed by anti-GAPDH IgG. (C) Representative ETV4 staining from 46 paraffin-embedded human GISTs. Scale bar, 20 μm. Inset is 40x magnification to show nuclear staining. (D) Freshly isolated KIT- and KIT+ cells from 3 metastatic, imatinib-resistant human GISTs with high ETV4 expression were analyzed for ETV4 mRNA by real-time PCR. Bars, mean ± SEM. Student’s t test, ***P <0.001.