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Additional file 1: Fig. S1. Validation of novel somatic mutations from targeted NGS sequencing by Sanger Sequencing. A Somatic UBR5 mutation detected in sample LGS106. B Somatic TP53 mutation detected in sample LGS105. C Somatic ATRX and EPHA3 mutations were detected in sample LGS119. Fig. S2. Validation of DNM3 somatic nonsynonymous mutations in two LGSOCs by Sanger Sequencing. Fig. S3. Twenty differentially expressed proteins shared between two different cohorts (LGSOC_MDACC_DISOVERY and LGSOC_INOVA_VALIDATION). LGSOC_MDACC: n = 7 long (median = 146 months), n = 7 short (median = 24 months; 531 protein alterations, LIMMA p < 0.05). LGSOC_INOVA: n = 4 long (median = 102 months), n = 2 short (median = 33 months), 294 protein alterations, LIMMA p < 0.05). 20 co-altered, Spearman rho = 0.48 for protein abundance; all alterations trends are concordant except for 3 proteins. Fig. S4. Protein expression associated with patients’ survival. Up-regulation of GTF2F1 and TRIM27 transcripts correlate with better survival. Up-regulation of HBA1 correlates with poor survival. Figures were generated at KMplot website ( https://kmplot.com/analysis/index.php?p=service&cancer=ovar ). Fig. S5. Differentially expressed phosphosites between long-term and short-term survivors. A Heatmap of differentially expressed phosphoproteins between long-term and short-term survivors; LIMMA p < 0.01, fold-change ± 1.5. B Long-term and short-term survivors were separated by principal component analysis (PCA) with differentially expressed phosphoproteins PCA of protein alterations shown in A served to explain 59.1% and 10.4% of the variance between short and long-term survivors. Fig. S6. Correlation of 62 significantly differentially expressed genes with co-quantified proteins.

Figure S8. Characterization of JAK1 neoepitope-specific CD4+ T-cells. (a) Peptide reactivity of a JAK1 neoepitope-specific CD4+ T-cell line. IFN-γ and GM-CSF production on CD4+ T-cells against JAK1 mutated (IEILRNLYHEIIV) or wild-type (IEILRNLYHENIV) peptide-pulsed autologous EBV-B-cells were determined by intracellular cytokine staining. (b) TCR usage of JAK1 neoepitope-specific CD4+ T-cell line. T-cells were stained with TCR Vβ subtype-specific antibodies and analyzed by flow cytometry. (c) Purity of Vβ13.6+ cells after magnetic-beads sorting. (d) Avidity of JAK1 neoepitope-specific T-cell clone. CD4+ T-cell clones were stimulated with autologous EBV-B-cells pulsed with the indicated concentration of mutated or wild-type peptide for 6 h in the presence of Golgi stop. IFN-γ production from Vβ13.6+ cells were determined by flow cytometry. The data represents mean ± s.d. of duplicate wells. (e) Recognition of autologous tumor-derived cells by Vβ13.6+ T-cell clone. PBMC or AMC were co-cultured with Vβ13.6+ JAK1 neoepitope-specific CD4+ T clones or without T-cells (−) for 24 h. AMC: ascites-derived mononuclear cells. IFN-γ production was measured by ELISA. The data represent mean + s.d. of duplicate wells. *p

Figure S8. Characterization of JAK1 neoepitope-specific CD4+ T-cells. (a) Peptide reactivity of a JAK1 neoepitope-specific CD4+ T-cell line. IFN-γ and GM-CSF production on CD4+ T-cells against JAK1 mutated (IEILRNLYHEIIV) or wild-type (IEILRNLYHENIV) peptide-pulsed autologous EBV-B-cells were determined by intracellular cytokine staining. (b) TCR usage of JAK1 neoepitope-specific CD4+ T-cell line. T-cells were stained with TCR Vβ subtype-specific antibodies and analyzed by flow cytometry. (c) Purity of Vβ13.6+ cells after magnetic-beads sorting. (d) Avidity of JAK1 neoepitope-specific T-cell clone. CD4+ T-cell clones were stimulated with autologous EBV-B-cells pulsed with the indicated concentration of mutated or wild-type peptide for 6 h in the presence of Golgi stop. IFN-γ production from Vβ13.6+ cells were determined by flow cytometry. The data represents mean ± s.d. of duplicate wells. (e) Recognition of autologous tumor-derived cells by Vβ13.6+ T-cell clone. PBMC or AMC were co-cultured with Vβ13.6+ JAK1 neoepitope-specific CD4+ T clones or without T-cells (−) for 24 h. AMC: ascites-derived mononuclear cells. IFN-γ production was measured by ELISA. The data represent mean + s.d. of duplicate wells. *p

Figure S6. Recurrent somatic copy number alternations by the GISTC2.0 algorithm. GISTIC deletion (left) and amplification (right) plots using data from the five patients with mutant-specific T-cell response (top), and data from the five patients without mutant-specific T-cell response (bottom). The genome is oriented vertically from top to bottom, and GISTIC q-values at each locus are plotted from left to right on a log scale. The green line represents the default significance threshold (q-valueâ =â 0.25). For each plot, known or interesting cancer genes are highlighted. (PPTX 278 kb)

Figure S6. Recurrent somatic copy number alternations by the GISTC2.0 algorithm. GISTIC deletion (left) and amplification (right) plots using data from the five patients with mutant-specific T-cell response (top), and data from the five patients without mutant-specific T-cell response (bottom). The genome is oriented vertically from top to bottom, and GISTIC q-values at each locus are plotted from left to right on a log scale. The green line represents the default significance threshold (q-valueâ =â 0.25). For each plot, known or interesting cancer genes are highlighted. (PPTX 278 kb)

Figure S2. Comparison of somatic mutation and neoantigen between primary and local invasive tumors. (a) Somatic mutation. (b) Neoantigen. Pt #02 on the left and Pt #10 on the right. X-axis: variant allele frequency (VAF) in WES of primary tumor. Y-axis: VAF in WES of local invasive tumor. (PPTX 260 kb)

Figure S10. Analysis of TRPC4 neoepitope-specific CD8+ T-cell clone. (a) IFN-γ and GM-CSF production on CD8+ T-cells against TRPC4 mutated (QSLFWSIFV) or wild-type (QSLFWSIFG) peptide-pulsed autologous EBV-B-cells were determined by intracellular cytokine staining. (b) Avidity of TRPC4 neoepitope-specific T-cell clone. CD8+ T-cell clone was co-cultured with autologous EBV-B-cells pulsed with the different concentration of mutated or wild-type peptide for 24 h. IFN-γ level in the culture supernatant was measured by ELISA. The data represents mean ± s.d. of duplicate wells. (c) IFN-γ production from TRPC4 neoepitope-specific TCR-transduced T-cells against EBV-B pulsed with or without TRPC4 mutated or wild-type peptide was determined by intracellular cytokine staining. (PPTX 321 kb)

Figure S2. Comparison of somatic mutation and neoantigen between primary and local invasive tumors. (a) Somatic mutation. (b) Neoantigen. Pt #02 on the left and Pt #10 on the right. X-axis: variant allele frequency (VAF) in WES of primary tumor. Y-axis: VAF in WES of local invasive tumor. (PPTX 260 kb)

Figure S11. The expression level of APPM signature from the tumor RNA-Seq data. The APPM expression is derived from the median expression value (in RPKM) of the 31 genes within the APPM signature (Methods). The patients shown here include Pt #3, Pt #15, Pt #19 and Pt #5. (PPTX 50 kb)

Figure S4. Spontaneous CD8+ T-cell response against mutant-specific epitopes from autologous tumor-infiltrating lymphocytes (TIL) and/or peripheral blood lymphocytes (PBL). T-cell reactivity was measured by IFN-Îł ELISpot assay (Methods). (â ): no peptide; Wt: wildtype peptide; Mt.: Mutant peptide. (PPTX 84 kb)

Figure S9. Reactivity of NUP214 neoepitope-specific TCR-transduced CD8+ and CD4+ T-cells. IFN-γ production (a) and GM-CSF (b) production from Vβ13.1+ or Vβ2−Vβ13.1− TCR- transduced CD8+ and CD8− (CD4+) T-cells against EBV-B-cells pulsed with or without NUP214 mutated or wild-type peptide were determined by intracellular cytokine staining. (PPTX 683 kb)

Figure S5. The expression level of a panel of 10 immune inhibitory molecules from the tumor RNA-Seq data. These immune inhibitory molecules include PD1, PDL1, CTLA4, CD80, CD86, LAG3, TIM3, LAGLS9, MYC and FOXP3. The patients shown here include Pt #2, Pt #8, Pt #16 and Pt #5. (PPTX 78 kb)

Figure S7. TCR Vβ usage of NUP214 neopeptide-specific CD4+ T-cell line. (a) TCR Vβ usage of NUP214 neopeptide-specific CD4+ T-cell line was determined by flow cytometry. (b) NUP214 neopeptide-specific IFN-γ production from Vβ2+, Vβ13.1+, or Vβ2−Vβ13.1− T-cells was examined by intracellular cytokine staining. (PPTX 221 kb)

Figure S3. Spontaneous CD4+ T-cell response against mutant-specific epitopes from autologous tumor-infiltrating lymphocytes (TIL) and/or peripheral blood lymphocytes (PBL). T-cell reactivity was measured by IFN-Îł ELISpot assay (Methods). (â ): no peptide; Wt: wildtype peptide; Mt.: Mutant peptide. (PPTX 85 kb)

Figure S3. Spontaneous CD4+ T-cell response against mutant-specific epitopes from autologous tumor-infiltrating lymphocytes (TIL) and/or peripheral blood lymphocytes (PBL). T-cell reactivity was measured by IFN-Îł ELISpot assay (Methods). (â ): no peptide; Wt: wildtype peptide; Mt.: Mutant peptide. (PPTX 85 kb)

Figure S11. The expression level of APPM signature from the tumor RNA-Seq data. The APPM expression is derived from the median expression value (in RPKM) of the 31 genes within the APPM signature (Methods). The patients shown here include Pt #3, Pt #15, Pt #19 and Pt #5. (PPTX 50 kb)

Figure S5. The expression level of a panel of 10 immune inhibitory molecules from the tumor RNA-Seq data. These immune inhibitory molecules include PD1, PDL1, CTLA4, CD80, CD86, LAG3, TIM3, LAGLS9, MYC and FOXP3. The patients shown here include Pt #2, Pt #8, Pt #16 and Pt #5. (PPTX 78 kb)

Figure S4. Spontaneous CD8+ T-cell response against mutant-specific epitopes from autologous tumor-infiltrating lymphocytes (TIL) and/or peripheral blood lymphocytes (PBL). T-cell reactivity was measured by IFN-Îł ELISpot assay (Methods). (â ): no peptide; Wt: wildtype peptide; Mt.: Mutant peptide. (PPTX 84 kb)

Figure S7. TCR Vβ usage of NUP214 neopeptide-specific CD4+ T-cell line. (a) TCR Vβ usage of NUP214 neopeptide-specific CD4+ T-cell line was determined by flow cytometry. (b) NUP214 neopeptide-specific IFN-γ production from Vβ2+, Vβ13.1+, or Vβ2−Vβ13.1− T-cells was examined by intracellular cytokine staining. (PPTX 221 kb)

Figure S1. The protein view of selected genes with somatic mutations in the 20 EOC patients studied. (a) TP53. (b) BRCA1. (c) BRCA2. (d) IL27RA. Note that splicing site mutations (c.673-2Aâ >â G in TP53 and c.4987-1Gâ >â T in BRAC1) are not shown. (PPTX 130 kb)