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. 2024 Jul 4;12(7):e009058.
doi: 10.1136/jitc-2024-009058.

Unique immune characteristics and differential anti-PD-1-mediated reinvigoration potential of CD8+ TILs based on BRCA1/2 mutation status in epithelial ovarian cancers

Junsik Park  1   2 , Jung Chul Kim  1   2 , Yong Jae Lee  1 , Sunghoon Kim  1 , Sang Wun Kim  1 , Eui-Cheol Shin  3 , Jung Yun Lee  4 , Su-Hyung Park  5
Affiliations

Unique immune characteristics and differential anti-PD-1-mediated reinvigoration potential of CD8+ TILs based on BRCA1/2 mutation status in epithelial ovarian cancers

Junsik Park et al. J Immunother Cancer. .

Abstract

Background: We aimed to investigate the distinct immunological characteristics of the tumor immune microenvironment in epithelial ovarian cancer (EOC) according to BRCA1/2 mutations status and differential PD-1 expression levels.

Methods: Tumor-infiltrating lymphocytes (TILs) were collected from patients with newly diagnosed advanced-stage EOC (YUHS cohort, n=117). This YUHS cohort was compared with The Cancer Genome Atlas (TCGA) data for ovarian serous cystadenocarcinoma (n=482), in terms of survival outcomes and immune-related gene profiles according to BRCA1/2 status. We used multicolor flow cytometry to characterize the immune phenotypes and heterogeneity of TILs with or without BRCA1/2 mutations. In vitro functional assays were conducted to evaluate the reinvigorating ability of CD8+ TILs on anti-PD-1 treatment.

Results: We found that EOC patients with BRCA1/2 mutations (BRCA1/2mt) exhibited better survival outcomes and significantly higher tumor mutation burden (TMB), compared with BRCA1/2 non-mutated (BRCA1/2wt) patients. Furthermore, CD8+ TILs within BRCA1/2mt tumors displayed characteristics indicating more severe T-cell exhaustion than their BRCA1/2wt counterparts. Notably, the capacity for anti-PD-1-mediated reinvigoration of CD8+ TILs was significantly greater in BRCA1/2wt tumors compared with BRCA1/2mt tumors. Additionally, within the BRCA1/2wt group, the frequency of PD-1highCD8+ TILs was positively correlated with the reinvigoration capacity of CD8+ TILs after anti-PD-1 treatment.

Conclusion: Our results highlight unique immune features of CD8+ TILs in EOC and a differential response to anti-PD-1 treatment, contingent on BRCA1/2 mutation status. These findings suggest that immune checkpoint blockade may be a promising frontline therapeutic option for selected BRCA1/2wt EOC patients.

Keywords: Immune Checkpoint Inhibitor; Ovarian Cancer; Tumor infiltrating lymphocyte - TIL.

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Conflict of interest statement

Competing interests: JYL reports grants and personal fees from AstraZeneca, Beigene, Bergenbio, Clovis Oncology, Immunogen, Janssen, Merck, MSD, Novartis, Roche, Seagen, Synthon, and Takeda. All other authors have no conflicts of interest to disclose.

Figures

Figure 1
Figure 1
Differences in survival outcomes and tumor microenvironments in epithelial ovarian cancer, according to BRCA1/2 mutations status. (A) Patient distribution according to BRCA1/2 status in the YUHS cohort. (B) Comparison of PFS and OS between patients with BRCA1/2 mutation (BRCA1/2mt) and BRCA1/2 non-mutation (BRCA1/2wt) in the YUHS cohort. (C) Patient distribution according to BRCA1/2 status in the TCGA cohort. (D) Comparison of PFI and OS between BRCA1/2mt and BRCA1/2wt patients from the TCGA data. (E) Comparison of tumor mutation burden (TMB) and neoantigen load between ovarian cancers with BRCA1/2mt and BRCA1/2wt from the TCGA data. (F) Comparison of gene expression profiles between ovarian cancers with BRCA1/2mt and BRCA1/2wt from the TCGA data. (G) Hallmark pathway enrichment analysis, and (H) GSEA of T-cell exhaustion (TEX)-associated genes, according to BRCA1/2 status in TCGA data. *p<0.05; **p<0.01; ***p<0.001. GSEA, gene set enrichment analysis; NES, normalized enrichment score; ns, not significant; OS, overall survival; PFI, progression-free interval; PFS, progression-free survival; TCGA, The Cancer Genome Atlas.
Figure 2
Figure 2
Comparison of tumor-infiltrating CD8+ T cells according to BRCA1/2 mutations status in advanced epithelial ovarian cancer. (A) Representative dot plots and comparison of the frequencies of PD-1+ cells among CD8+ TILs between patients with BRCA1/2 mutation (BRCA1/2mt) and BRCA1/2 non-mutation (BRCA1/2wt). (B, C) Representative dot-plots and comparison of the frequencies of CD39+, CD39+CD103+ (B), TCF-1+, and TOX+ cells (C) among PD-1+CD8+ TILs between patients with BRCA1/2mt and BRCA1/2wt. (D, E) Representative dot plots and the frequencies of CTLA-4+, TIM-3+, and TIGIT+ cells (D) and 4-1BB+, and CD226+ cells (E) among CD8+ TILs between patients with BRCA1/2mt and BRCA1/2wt. ns, not significant; *p<0.05; **p<0.01; ***p<0.001. TILs, tumor-infiltrating lymphocytes.
Figure 3
Figure 3
Immune checkpoint inhibitor-induced reinvigoration of tumor-infiltrating CD8+ T cells is significantly greater in BRCA1/2wt than in BRCA1/2mt patients. CTV-labeled TILs of EOC patients were treated with anti-PD-1 ± anti-CTLA-4 or isotype control in the presence of anti-CD3 stimulation for 96 hours. Proliferative capacity was measured as the percentage of proliferated CTVlowCD8+ TILs and the fold changes. (A, D) Representative data. (B, C) The anti-PD-1 induced proliferative capacity was analyzed in BRCA1/2wt (B) and BRCA1/2mt (C) patients. (E, F) The anti-PD-1+ anti-CTLA-4-induced proliferative capacity was analyzed in BRCA1/2wt (E) and BRCA1/2mt (F) patients. (G) Patient distribution with or without front-line immune checkpoint blockade (ICB) therapy in the YUHS cohort. (H) Comparison of PFS between patients treated with or without ICB among BRCA1/2wt and BRCA1/2mt patients. *p<0.05; **p<0.01; ***p<0.001. IO therapy, Immuno-oncology therapy; ns, not significant; PFS, progression-free survival; TILs, tumor-infiltrating lymphocytes.
Figure 4
Figure 4
Among BRCA1/2wt, high PD-1high expressers are promising candidates for immune checkpoint blockade therapy in advanced stage ovarian cancer. (A) TIL samples were grouped as “low responding TILs” (n=14) and “highly responding TILs” (n=15), based on the proliferative responses (median fold changes of CTVlowCD8+ = 1.13). (B) Expression of PD-1 on CD8+ TILs was analyzed in the “highly responding TILs” and “low responding TILs” groups. (C) The percentage of PD-1high among CD8+ TILs was analyzed for a correlation with the relative fold changes of CTVlowCD8+ (anti-PD-1) using Pearson’s correlation coefficients (r). (D) Performance power of PD-1highCD8+ TILs for predicting responses to anti-PD-1. (E) Distribution of low and high PD-1high expressers, divided using the optimal cut-off value of PD-1highCD8+ TILs in the BRCA1/2wt YUHS cohort. (F) Percentages of PD-1high cells among CD8+ TILs in the “low PD-1high expresser” and “high PD-1high expresser” groups. (G) Percentages of TOX+cells among PD-1+CD8+ TILs in the “low PD-1high expresser” and “high PD-1high expresser” groups. (H) Percentages of CD39+, CD103+, and CD39+CD103+ cells among PD-1+CD8+ TILs in the “low PD-1high expresser” and “high PD-1high expresser” groups. (I) Percentages of TCF-1+, 4-1BB+, and CD226+ cells among PD-1+CD8+ TILs in the “low PD-1high expresser” and “high PD-1high expresser” groups. *p<0.05; **p<0.01; ***p<0.001. ns, not significant; TILs, tumor-infiltrating lymphocytes.
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