Interplay between glucocorticoids and tumor-infiltrating lymphocytes on the prognosis of adrenocortical carcinoma

Abstract

Background: Adrenocortical carcinoma (ACC) is a rare endocrine malignancy. Tumor-related glucocorticoid excess is present in ~60% of patients and associated with particularly poor prognosis. Results of first clinical trials using immune checkpoint inhibitors were heterogeneous. Here we characterize tumor-infiltrating T lymphocytes (TILs) in ACC in association with glucocorticoids as potential explanation for resistance to immunotherapy.

Methods: We performed immunofluorescence analysis to visualize tumor-infiltrating T cells (CD3+), T helper cells (CD3+CD4+), cytotoxic T cells (CD3+CD8+) and regulatory T cells (Tregs; CD3+CD4+FoxP3+) in 146 ACC tissue specimens (107 primary tumors, 16 local recurrences, 23 metastases). Quantitative data of immune cell infiltration were correlated with clinical data (including glucocorticoid excess).

Results: 86.3% of ACC specimens showed tumor infiltrating T cells (7.7 cells/high power field (HPF)), including T helper (74.0%, 6.7 cells/HPF), cytotoxic T cells (84.3%, 5.7 cells/HPF) and Tregs (49.3%, 0.8 cells/HPF). The number of TILs was associated with better overall survival (HR for death: 0.47, 95% CI 0.25 to 0.87), which was true for CD4+- and CD8+ subpopulations as well. In localized, non-metastatic ACC, the favorable impact of TILs on overall and recurrence-free survival was manifested even independently of ENSAT (European Network for the Study of Adrenal Tumors) stage, resection status and Ki67 index. T helper cells were negatively correlated with glucocorticoid excess (Phi=-0.290, p=0.009). Patients with glucocorticoid excess and low TILs had a particularly poor overall survival (27 vs. 121 months in patients with TILs without glucocorticoid excess).

Conclusion: Glucocorticoid excess is associated with T cell depletion and unfavorable prognosis. To reactivate the immune system in ACC by checkpoint inhibitors, an inhibition of adrenal steroidogenesis might be pivotal and should be tested in prospective studies.

Keywords: immunity; immunotherapy; lymphocytes, tumor-infiltrating; t-lymphocytes; tumor microenvironment.

Conflict of interest statement

Competing interests: None declared.

© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1

Immunofluorescence staining of tumor-infiltrating lymphocytes in adrenocortical carcinoma, n=146, (A–D). ACC, adrenocortical carcinoma; HPF, high power field; TILs, tumor-infiltrating T lymphocytes.

Figure 2

Comparison of tumor-infiltrating lymphocytes in primary tumors and metastases of ACC (median 95% CI). A−C, CD3+− (A), CD4+− (B) and CD8+− (C) T cells infiltrated in adrenocortical tumor samples from primary localized tumor tissue (n=107) or distant metastases (n=23) of entire cohort. Per sample analysis (D–F), CD3+− (D), CD4+− (E) and CD8+− (F) T cells quantified in primary tumor and metastasis of the same patient (n=14). ACC, adrenocortical carcinoma; HPF, high power field.

Figure 3

Overall survival (A–C) and recurrence-free survival (D–F) in patients with adrenocortical carcinoma according to CD3+−, CD4+− and CD8+ TILs, determined by immunofluorescence. Kaplan-Meier overall survival of all ACC patients with primary tumor samples (n=107) influenced by CD3+− (A), CD4+− (B) and CD8+− (C) T cell infiltration. In a subgroup, patients after complete surgical resection and in localized ACC, a Kaplan-Meier recurrence-free survival analysis was performed regarding CD3+− (D), CD4+− (E) and CD8+− (F) tumor infiltration (n=59). Multivariate Cox regression, overall survival (G–I) in patients with localized, non-metastatic ACC according to different influencing factors; ENSAT stage, resection status, Ki67 proliferation index and TILs. Overall survival of all ACC patients with localized, non-metastatic primary tumor samples (n=67) influenced by CD3+− (G), CD4+− (H) and CD8+− (I) T cell infiltration independently of different factors. ACC, adrenocortical carcinoma; ENSAT, European Network for the Study of Adrenal Tumors; TILs, tumor-infiltrating T lymphocytes.

Figure 4

Correlation of glucocorticoids and CD3+CD4+ T cells. (A) An unpaired non-parametric Mann-Whitney test was performed for correlation of the number of CD3+CD4+ TILs with glucocorticoid excess or hormone inactive primary ACC (box/whiskers 10 to 90 percentile; p=0.0007). (B) Overall survival in patients with CD3+CD4+ T cell-infiltrated or T cell-depleted primary ACC considering presence or lack of glucocorticoid excess (n=80). Primary tumors with available hormone secretion status were classified as ‘lymphocytes-infiltrated’ depending on positive immune infiltration (blue) and in case of absent tumor-infiltrating immune cells as ‘lymphocytes-depleted’ (red). Additionally, further subdivision of phenotypes according to glucocorticoid excess or hormonal inactivity. ACC, adrenocortical carcinoma; HPF, high power field; TILs, tumor-infiltrating Tlymphocytes.