(A) Left: Human chromosome 3 is shown with the spike in amplification (in blue) that occurred in 6 of 13 human CCS tumors. Right: Copy number microarray data showing that the recurrent, focal amplification occurred at the MITF locus in all 6 tumors. (B) Mouse DNA sequences depicting the Mitf vitiligo (Mitf ^vit) mutation (top left panel, in blue) and the normal Mitf sequence. The BsiEI restriction enzyme was used to confirm Mitf ^vit after PCR, because it recognizes and cuts WT Mitf but not Mitf ^vit (right panel). (C) Mouse breeding schematic showing the strategy to generate EA1 heterozygotic mice with homozygous mutant or homozygous WT Mitf littermates, which we injected with TATCre to induce EWS-ATF1 expression. (D) Kaplan-Meier curve shows the achievement of morbidity of Mitf ^WT/WT mice (solid line) and Mitf ^vit/vit mice (dotted line) following TATCre injection at 28 days of age (n = 37 Mitf ^WT/WT mice and n = 15 Mitf ^vit/vit mice). The 50% median survival time to morbidity for Mitf ^vit/vit mice was 102 days compared with 87 days for WT mice. A log-rank test was performed, and the difference was deemed statistically significant (P < 0.0001, z score = 4.52). (E) Tumor mass measurements for Mitf ^WT/WT mice (black dots) and Mitf ^vit/vit mice (gray circles). The tumors were not significantly different in size (P = 0.6, by 2-tailed Student’s t test). (F) Graph of the blinded quantitation of histological features of tumors developing from EA1 expression in either Mitf ^WT/WT mice (black diamonds, n = 37) or Mitf ^vit/vit mice (gray diamonds, n = 15), with human CCSs on a tissue microarray (n = 20, blue diamonds). P = 0.05, by 2-tailed Student’s t test (none of these comparisons between the 2 mouse groups reached statistical significance). (G) Representative histomorphologies in H&E-stained tissue sections of EA1-expressing tumors. Each photomicrograph is a 100 μm square obtained with a 60× original magnification objective lens.