(A) Gradually increased cellularity and mitotic rates are observed in representative low-risk (left, intermediate-risk (middle), and high-risk (right) GISTs of the independent validation set (upper row). No amplification, low-level amplification, and high-level amplification are detected by FISH assay targeting the AMACR gene, with the ratio of the red signal to the green signal being 1, 4, and ≥5 in the corresponding cases, respectively (middle row). Granular cytoplasmic immunoexpression of AMACR is consistent with the subcellular distribution of mitochondria and peroxisomes and is classified as absent, increased, and overexpressed in the representative low-risk, intermediate-risk, and high-risk GISTs, respectively (lower row). (B) Comparison of mitotic activity shows significantly higher mitotic rates in GISTs with AMACR gene amplification (left) and protein overexpression (right) than in those without.(C) Mutation analysis of the KIT gene shows a favorable genotype (exon 11 point mutation) in one representative GIST featuring neither AMACR amplification nor AMACR overexpression (left) and an unfavorable genotype (exon 11 deletion at codons 557-558) in a representative AMACR-amplified GIST with protein overexpression (right).