CD25+FOXP3+CD45RA- regulatory T-cell infiltration as a prognostic biomarker for endometrial carcinoma

Abstract

Background: Regulatory T (Treg) cells reportedly play crucial roles in tumor angiogenesis as well as antitumor immunity. In order to explore their therapeutic potential, we investigated the precise prognostic impact of Treg markers in endometrial carcinoma.

Methods: We performed multiplexed immunofluorescence and quantitative image analyses of CD25, FOXP3, CTLA4, and CD45RA in tumor specimens from 176 consecutive patients treated at our institution for primary endometrial carcinomas. Bioinformatics analyses were further conducted to corroborate the findings.

Results: High CD25⁺, FOXP3⁺, and CD25⁺FOXP3⁺CD45RA^- stromal cell counts correlated with better overall survival (OS) (p = 0.00019, 0.028 and 0.0012) and MSI-high (p = 0.015, 0.016 and 0.047). High CD45RA⁺ stromal cell count was associated with superficial myometrial invasion (p = 0.0038). Bioinformatics survival analysis by Kaplan-Meier plotter showed that high CD25, FOXP3, CTLA4, and CD45RA mRNA expressions correlated with better OS (p = 0.046, 0.00042, 0.000044, and 0.0022). Univariate and multivariate analyses with various clinicopathologic prognostic factors indicated that high CD25⁺ or CD25⁺FOXP3⁺CD45RA^- stromal cell count was significant and independent for favorable OS (p = 0.0053 and 0.0015). We subsequently analyzed the correlations between the multiplexed immunofluorescence results and treatment-free interval (TFI) after primary chemotherapy in recurrent cases, finding no significant associations. Further analysis revealed that high ratio of CD25⁺ : CD8⁺ cell count or CD25⁺FOXP3⁺CD45RA^- : CD8⁺ cell count correlated with longer TFI (p = 0.021 and 0.021).

Conclusion: The current observations suggest that the balance between CD25⁺ or CD25⁺FOXP3⁺CD45RA^- cells and CD8⁺ cells, corresponding to promoting or inhibiting effect on tumor angiogenesis, affect tumor chemosensitivity leading to prognostic significance. CD25⁺FOXP3⁺CD45RA^- effector Treg tumor infiltration may serve as a useful prognostic biomarker and a potential target for immunotherapeutic manipulation of tumor chemosensitivity by novel management for advanced/recurrent endometrial carcinomas.

Keywords: CD25; CD45RA; Endometrial carcinoma; FOXP3; Survival; Treg cell.

Fig. 1

Representative images of multiplexed immunofluorescence analysis. Composite image, pathological views, and phenotype maps for CD25, FOXP3, CTLA4, and CD45RA. × 200

Fig. 2

Comparison of overall survival curves (days) of patients with endometrial carcinomas according to CD25⁺, FOXP3⁺, CTLA4⁺, CD45RA⁺, or CD25⁺FOXP3⁺CD45RA⁻ stromal cell counts, or CD8⁺ tumor-infiltrating immune cell (TIC) counts

Fig. 3

A, Comparison of CD8⁺ tumor-infiltrating immune cell (TIC) count of endometrial carcinoma tissues according to multiplexed immunofluorescence results. High (n = 154) vs. low (n = 22) CD25⁺ stromal cell count, high (n = 23) vs. low (n = 153) FOXP3⁺ stromal cell count, high (n = 49) vs. low (n = 127) CTLA4⁺ stromal cell count, high (n = 26) vs. low (n = 150) CD45RA⁺ stromal cell count, and high (n = 143) vs. low (n = 33) CD25⁺FOXP3⁺CD45RA⁻ stromal cell count. B, Comparison of treatment-free interval (TFI, days) of patients with endometrial carcinomas according to multiplexed immunofluorescence results. High (n = 16) vs. low (n = 4) CD25⁺ stromal cell count, high (n = 2) vs. low (n = 18) FOXP3⁺ stromal cell count, high (n = 4) vs. low (n = 16) CTLA4⁺ stromal cell count, high (n = 2) vs. low (n = 18) CD45RA⁺ stromal cell count, and high (n = 15) vs. low (n = 5) CD25⁺FOXP3⁺CD45RA⁻ stromal cell count. C, Comparison of TFI (days) of patients with endometrial carcinomas according to multiplexed immunofluorescence results. High (n = 2) vs. low (n = 18) ratio of CD25⁺ stromal cell count : CD8⁺ TIC count, high (n = 2) vs. low (n = 18) FOXP3⁺ stromal cell count : CD8⁺ TIC count, high (n = 7) vs. low (n = 13) CTLA4⁺ stromal cell count : CD8⁺ TIC count, high (n = 2) vs. low (n = 18) CD45RA⁺ stromal cell count : CD8⁺ TIC count, and high (n = 2) vs. low (n = 18) CD25⁺FOXP3⁺CD45RA⁻ stromal cell count : CD8⁺ TIC count

Fig. 4

A, Kaplan-Meier plotter analysis of overall survival curves in patients with endometrial carcinomas according to the mRNA expressions. Survival curves of patients (n = 542) with high vs. low CD25 expression, high vs. low FOXP3 expression, high vs. low CTLA4 expression, and high vs. low CD45RA expression. B, TIMER2.0 analysis of overall survival curves in patients with endometrial carcinomas (n = 545) based on CD8⁺ T-cell infiltration and the gene expression level of CD25, FOXP3, CTLA4, or CD45RA