Adrenocortical carcinoma (ACC) is a rare aggressive endocrine malignancy with an incidence of approximately one per million inhabitants per year.1 Patients diagnosed with ACC have a dismally poor prognosis. Even after complete resection, there is a strong propensity toward local recurrences and progression of distant metastases. Median overall survival in metastatic disease is less than 15 months. Complete surgical resection is the treatment of choice in localized ACC.2 The current recommended standard care for patients in advanced disease is mitotane or the combination of etoposide, doxorubicin, cisplatin, and mitotane.1–6

Activation of antitumorous immune response with immune checkpoint inhibitors has revolutionized the therapy of many cancer entities resistant to chemotherapy. Results of the first (small) trials with immunotherapy in ACC are modest, with a median progression-free survival times of 1.8, 2.1, 2.6 and 6.75 months, respectively.7–10 However, in one study with 39 patients, disease control rate was 52% and interestingly median overall survival reached almost 25 months clearly suggesting that at least a subset of patients benefits from this therapeutic approach.

One potential cause underlying these heterogeneous and partly disappointing results may be tumorous glucocorticoid secretion. Excess of steroid hormones is present in approximately 60% of patients. Cushing’s syndrome due to high glucocorticoid secretion has been associated with unfavorable prognosis even after complete resection.11–13 It is well known that glucocorticoids are immunosuppressive through regulation of circulating and tumor-infiltrating immune cells. They inhibit for instance T helper cells (CD3⁺CD4⁺) and cytotoxic T-lymphocytes (CTLs, CD3⁺CD8⁺) activation.14 Even without clinically apparent Cushing’s syndrome, high intratumorous glucocorticoid concentrations may lead to a local anergy of the antitumorous immune system.

CD3⁺CD4⁺ T cells are crucial for T cell receptor-mediated activation of the adaptive immune system by interacting with MHC class II-restricted antigen-presenting cells. Additionally, T helper cells mediate the production of antibody generating B lymphocytes and the activation and long-term maintenance of CD3⁺CD8⁺ CTLs through secretion of cytokines and chemokines such as interferon gamma and tumor necrosis factor alpha. CD3⁺CD8⁺ CTLs eradicate intracellular targets by direct cell-cell contact and ensure tumor surveillance.15 The presence of circulating and/or tumor-infiltrating CD3⁺CD4⁺− and CD3⁺CD8⁺ T lymphocytes (TILs) correlates with favorable outcome on patients’ overall survival in various human malignancies, for example, melanoma, lung cancer or head and neck squamous cell carcinoma.16–18

In contrast, regulatory T cells (Tregs, CD3⁺CD4⁺CD25⁺FoxP3⁺) maintain tolerance to self-antigens and prevent autoimmunity by modulating the induction of effector T cells. However, they actively impede antitumor immunity contributing to tumor progression in many cancers.19–21

By re-analyzing a ‘multiple omics’ approach using data from The Cancer Genome Atlas (TCGA) consortium, two distinct subgroups of ACC patients were identified, one with overexpression of genes related to steroidogenesis and one with overexpression of genes related to the immune system.22 Both phenotypes, the ‘steroid’ and the ‘immune’ phenotype, not only differ in terms of gene expression, but also regarding the prognosis.

In order to improve the understanding of the pathogenesis and prognostic of ACC, we focused on the interplay of steroid hormones and immune system to study the glucocorticoid-induced T cell depletion. A more profound analysis of the differences between the ‘steroid’ and ‘immune’ ACC phenotype will allow us to improve therapeutic immunogenic strategies resulting in a prognostic and clinical profit especially with regard to the immunotherapy of the ACC patients.

In this first large study on immune cell infiltration in ACC, we demonstrated a significant impact of CD3⁺−, CD3⁺CD4⁺− and CD3⁺CD8⁺ TILs on clinical outcome. In particular, in localized, non-metastatic ACC the effect of TILs on overall survival was independent of clinically relevant factors, like ENSAT stage, resection status and Ki67 index and remarkably with a relative risk reduction of about 70%. Additionally, our study suggests a relevant influence of tumor-induced glucocorticoids on intratumorous CD3⁺CD4⁺ T cells and overall prognosis.

Since CD3⁺CD4⁺− and CD3⁺CD8⁺ T cells are powerful assistants in preventing and combating cancer, their presence in the tumor microenvironment and within the tumor mass is crucial. The fraction of lymphocytes infiltrated within tumors varied considerably across tumor types and depends on different factors. Highly immune infiltrated tumors like lung adenocarcinoma and cutaneous melanoma are highly responsive to immune checkpoint therapies.24 In contrast, ACC is after uveal melanoma and prostate adenocarcinoma the tumor with the lowest leukocyte fraction according to Thorsson et al, who analyzed the composition of tumor immune infiltration within the TCGA cohort on the level of RNA expression.24

Here, we quantified CD3⁺−, CD3⁺CD4⁺−, CD3⁺CD8⁺− and CD3⁺CD4⁺FoxP3⁺ TILs in a cohort of 146 ACC patients and studied the influence of their presence on patients’ survival. Thereby, we provided evidence that—in contrast to a widespread perception—the majority of ACC samples (>85%) contained tumor-infiltrating T lymphocytes; as recently shown in childhood ACC.25 However, we have to acknowledge that the number of TILs is rather low in comparison to other tumors.26 27 Nevertheless, we could demonstrate a significant impact of TILs on ACC patients’ overall and recurrence-free survival. Many studies have shown the strong influence of TILs on patients’ prognosis, also independently of the choice of treatment. For instance, Zhang et al demonstrated already in 2003 an improved clinical outcome in advanced ovarian carcinoma depending on the presence or absence of lymphocytes. Immune depleted ovarian tumors show a median progression-free survival of only 7.6 months, while intratumorous immune infiltration is associated with 74.5 months until recurrence.27 A similarly favorable impact of tumor infiltration on overall and recurrence-free survival was observed in the present study of ACC. Accordingly, CD3^+-, CD3⁺CD4^+- and CD3⁺CD8⁺ TIL number was associated with a risk reduction of 53% to 61% for death and 57% to 69% for recurrence. In particular, TILs in localized, non-metastatic ACC may serve as a prognostic marker independently of clinically established factors, like ENSAT stage, resection status, and Ki67 index leading to a risk reduction for death of 70% to 81%. Furthermore, our study indicates that TILs are even less frequent in metastatic lesions in comparison to primary tumors. Similar observations were made in other tumors like metastatic breast cancer that is characterized by lower immune cell infiltration relative to its paired primary tumor.28

Several clinical studies on immune checkpoint inhibitors (ICIs), which flare up antitumor immune responses, showed major therapeutic improvements in many tumor entities. The first approved cytotoxic T-lymphocytes antigen-4 (CTLA-4) inhibitor, ipilimumab, demonstrated enormous success in advanced melanoma.29 Other ICIs targeting programmed cell death-1 (PD-1), nivolumab and pembrolizumab, exhibit very promising clinical benefit in non-small cell lung carcinoma, melanoma, Hodgkin's lymphoma, and other tumor entities;30–32 the combination of CTLA-4 and PD-1 targeting drugs is even more potent.31 However, so far, four small studies with a total of 115 patients have been published in ACC and overall the results were disappointing; only 15 patients experienced partial response and 12 long-term disease control for more than 12 months.7–10

Our study may shed some light, why strong immune infiltration is rarely seen in ACC and why current immunological therapeutic options were of limited efficacy. The fact that we found a negative correlation of tumor-associated glucocorticoid excess and T helper cells supports an expected role of steroids in this context. Anti-inflammatory impact of glucocorticoids was especially observed towards CD3⁺CD4⁺ TILs, which play a major role in immune activation and regulation of immune response. As indicated by our large cohort, ACC patients without hypercortisolism, but with CD3⁺CD4⁺ TILs may benefit from a major survival advantage compared to patients with hypercortisolim with or—even more pronouncedly—without CD3⁺CD4⁺ T cell infiltrated tumors (121 vs. 75 vs. 27 months). These observations might also explain why hypercortisolism has a significant effect on survival.11–13 In these patients, antitumorous immune response may be diminished which may lead to a higher rate of recurrence and ACC-related deaths.

It is well established that glucocorticoids execute anti-inflammatory, pro-apoptotic effects and have a strong impact on multiple physiological processes, that is, cell differentiation, proliferation, migration and apoptosis. Furthermore, it has been shown that hypersecretion of glucocorticoids enhances tumor cell proliferation in vitro and in vivo.33 Additionally, glucocorticoids also hamper peripheral T lymphocyte function, reducing their potential to eradicate tumor cells in the case of active ACC. The tumor-associated elevation of glucocorticoids has been observed in many cancers and associated with impaired prognosis and metastatic spread. For instance, in renal cell carcinoma, high levels of serum cortisol were positively correlated with tumor size and impaired prognosis.34 Inversely, a study concerning optimal application of steroids in anticancer therapy showed glucocorticoids to enhance tumorous PD-1 expression resulting in immune evasion.35 Thus, ACC-induced hypercortisolism might be a major contributor to the ‘immunological coldness’ of ACC.36

Our study might provide at least three potential explanations for the disappointing results of the first trials with checkpoint inhibitors in ACC.7–10 (i) Many ACC are probably infiltrated by too few or no helper and cytotoxic T cells that would allow flaring up an antitumor response by checkpoint inhibitors. (ii) The fact that metastatic lesions are in average even ‘colder’ than the primary tumors suggests that immune escape mechanisms are even more developed in advanced ACC increasing the challenge for immunotherapies.36 (iii) The likelihood that glucocorticoids are—at least in part—responsible for the poor immunogenicity is high and further supported by the demonstrated negative correlation of hypercortisolism and T helper cells in ACC. However, one has to bear in mind that intratumorous glucocorticoid concentrations might be also much higher in clinically non-functioning ACC (without systemically measurable cortisol levels) in comparison to other solid tumors.

The main limitations of our study are the relatively low number of patients and tumor samples, especially of metastatic material compared with other tumor entities. However, the extraordinary rarity of ACC renders the collection of a larger series of clinically annotated cases. This retrospective nature of the study made the availability of certain clinical data challenging.

It might also be interesting to correlate TILs and glucocorticoid excess in a quantitative manner or with intratumorous steroid excess. However, this requires also a prospective approach.

Our study has several clinical implications: It is obvious that future immunotherapy trials in ACC require selection of patients. In addition to parameters like PD-1/PD-L1 (programmed cell death-ligand-1) expression or tumor mutational burden, the presence of TILs could be a promising predictive marker that should be explored. Instead of analyzing primary tumor samples, metastatic lesions could be the better and more reliable context. In addition, it is most likely useful to reduce the effect of glucocorticoids by inhibiting steroidogenesis37 or blocking glucocorticoid receptors.38 Failure to reduce the intratumorous immune suppressive potential of glucocorticoids, even lead to inefficacy of immunotherapy in ACC without detectable cortisol excess. Finally, adoption of concepts investigated in other tumor entities to transform ‘cold’ tumors in ‘hot’ tumors36 39 are probably required in addition to the measures mentioned above.

In conclusion, tumor-infiltrating CD3⁺−, CD3⁺CD4⁺− and CD3⁺CD8⁺ T cells are of major prognostic and clinical relevance in ACC patients. In addition, the interplay of glucocorticoids and immune cells are most likely crucial for ACC patients’ survival. Consequently, a combined therapy of at least immune activating approaches and blockers of glucocorticoid synthesis or action might be required to improve clinical outcome in ACC. However, this hypothesis requires prospective investigations.