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1 Supplementary Tables Supplementary Tables and Figures Supplementary Table 1: Tumor types and samples analyzed. Supplementary Table 2: Genes analyzed here. Supplementary Table 3: Statistically significant single-codon hotspots. Supplementary Table 4: Statistically significant indel hotspots. Supplementary Figures Supplementary Figure 1: The frequency distribution of 1,165 mutational hotspots. Supplementary Figure 2: Hotspots enriched in metastatic disease. Supplementary Figure 3: Representative indel hotspots. Supplementary Figure 4: Structure of AKT1 with single-codon and indel hotspots. Supplementary Figure 5: Co-mutational pattern in effectors of MAPK and PI3K signaling. Supplementary Figure 6: Characteristics of the rate of hotspot identification. Supplementary Figure 7: Clinical actionability of hotspots in actionable genes.
2 Supplementary Tables Supplementary Table 1: Tumor types and samples analyzed. Table included as a separate file.
3 Supplementary Table 2: Genes analyzed here. Table included as a separate file.
4 Supplementary Table 3: Statistically significant single-codon hotspots. Table included as a separate file.
5 Supplementary Table 4: Statistically significant indel hotspots. Table included as a separate file.
6 Supplementary Figures Supplementary Figure 1: The frequency distribution of 1,165 mutational hotspots. The long right tail of the frequency distribution of mutational hotspots identified in this study.
7 Supplementary Figure 2: Hotspots enriched in metastatic disease. Mutational hotspots enriched in metastatic disease compared to primary cancers of a given cancer type. Plotted is the nominal p-value of each gene from its cancer type-specific analysis, with significant results (q-value 0.1) colored by affected cancer type.
8 Supplementary Figure 3: Representative indel hotspots. Shown are mutant proteins (or the indicated subset in residue number, gray line) with the position of single-codon hotspots (top, green arrows) versus indel hotspots (bottom, blue lines, each of which is a unique indel called in a tumor in the cohort). A subset of affected genes is shown and categorized as follows. a) Canonical tumor suppressor genes with very different patterns. b) Genes that only possess indel hotspots in regions spanning single-codon hotspots in either two-dimensional space or in the three dimensions of the folded protein. c) Genes with indels that span the position of established single-codon hotspots. d) Genes that possess indel hotspots distal in position to their single-codon hotspots in three dimensions. Arcing red lines reflect the distant in angstroms between the indels and single-codon hotspots. e) The lines of prior therapy received by two breast cancer patients harboring clonal ESR1 V422del mutations in their post-treatment biopsy specimens.
9 Supplementary Figure 4: Structure of AKT1 with single-codon and indel hotspots. The hotspot of duplication indels in AKT1 are shown in three dimensions (dark red) and lie adjacent to the L52 and Q79 single-codon hotspots in AKT1 (orange). The E17K hotspot is shown in red for reference.
10 Supplementary Figure 5: Co-mutational pattern in effectors of MAPK and PI3K signaling. The pattern of mutational co-occurrence among both single-codon and indel hotspots in effectors of MAPK (a) and PI3K (b) signaling reveals co-existing hotspot mutations in the same tumors. The number in each cell is the count of co-mutated specimens; the numbers in gray are the total co-mutated cases (row and column); and cell shading indicates increasing statistical significance of the association (inset legend).
11 Supplementary Figure 6: Characteristics of the rate of hotspot identification. a) Principal component analysis divided genes harboring one or more hotspots into four classes (green, purple, red, and blue). The first and second principal components are shown and selected cluster members are similarly colored at right. b) The clusters are independent of overall mutational burden by gene. c) Representative genes from their respective clusters indicates variability from gene to gene.
12 Supplementary Figure 7: Clinical actionability of hotspots in actionable genes. a) The number of hotspots per actionable cancer gene is shown and includes (left-facing) those with existing clinical evidence of activity to approved or investigational therapies (blue) or have been biologically studied but clinically uncharacterized (gold) versus the number of hotspots identified here for the first time and of unknown significance (red, right-facing). b) As in panel (a) but for individual mutant alleles.
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