RESEARCH
Expression and clinical significance of VISTA and PD-L1 in adrenocortical carcinoma
Ziwei ZhangOD1,2,*, Menglian LiOD3,*, Jianjun Wang4, Mengsi Liu5, Huan Chen5, Yuan Lou2, Yijie Wang1, Qi Sun4, Dalong Zhu2, Ping LiDD2 and Yan BiDD1,2
1Department of Endocrinology, Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University, Nanjing, China
2Department of Endocrinology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, China
3Department of Endocrinology, Nanjing Drum Tower Hospital Clinical College of Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, China
4Department of Pathology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, China
5Department of Endocrinology, Nanjing Drum Tower Hospital, Chinese Academy of Medical Science & Graduate School of Peking Union Medical College, Nanjing, China
Correspondence should be addressed to P Li or Y Bi: li78321@yeah.net or biyan@nju.edu.cn
*(Z Zhang and M Li contributed equally to this work)
Abstract
Adrenocortical carcinoma (ACC) is a rare malignancy with a poor prognosis and challenging management. The present study aimed to investigate the expression of programmed death ligand-1 (PD-L1) and V-domain Ig-containing suppressor of T cell activation (VISTA) in ACC and their associations with clinicopathological features and survival outcomes. Immunohistochemistry was performed on formalin-fixed paraffin-embedded specimens from 54 ACC patients. Chi-square/Fisher’s exact tests or independent samples t/Mann-Whitney U tests were performed to assess correlations between immunoscores and clinicopathological parameters. The Kaplan-Meier method and Cox regression were conducted for survival analysis and to identify independent predictors of overall (OS) and disease-free (DFS) survival. Results showed that VISTA was expressed in tumor cells (TCs) and tumor-infiltrating immune cells (TICs) in 81.5% (44/54) and 40.7% (22/54) of the patients, respectively. PD-L1 positivity was found in either TCs or TICs in 11.1% (6/54) of the patients. Patients with positive VISTA expression in TCs had a higher tumor stage (56.9% vs 20%, P = 0.036) and Ki-67 index (30.50 ± 23.51% vs 14.76 ± 11.75%, P = 0.006). However, PD-L1 positivity in either TCs or TICs had no association with patient clinicopathological features. A higher VISTA expression intensity, a larger area and a higher immunoscore were associated with increased risks of disease progression and overall mortality, but PD-L1 expression in TCs or TICs was not associated with OS or DFS. In conclusion, positive TC VISTA expression was correlated with pathological parameters related to malignancy in ACC patients. This finding provides novel evidence of the value of VISTA, in addition to PD-L1, as an immunotherapeutic target in ACC.
Key Words
adrenocortical carcinoma
V-domain Ig suppressor of T-cell activation (VISTA)
programmed death ligand-1 (PD-L1)
immunohistochemistry
Endocrine-Related Cancer (2022) 29, 403-413
Introduction
Adrenocortical carcinoma (ACC) is a rare and highly aggressive malignancy with an annual incidence of 0.7-2.0 cases per million population (Crona & Beuschlein 2019). ACC has a dismal prognosis, with a 5-year survival ranging from 80% in patients with localized disease to 15% in those with advanced metastatic tumors (Karwacka et al. 2021). Radical surgery is the first-line therapy for localized disease. Nevertheless, the rate of relapse/recurrence remains nearly 50%, even with complete tumor resection (Altieri et al. 2020). Patients with advanced disease are treated with etoposide, doxorubicin and cisplatin chemotherapy regimens in combination with mitotane, an adrenolytic regimen, though adverse effects are frequent, as is limited response. Thus, new therapeutic approaches for ACC treatment are urgently needed.
In recent decades, immunotherapy has become a cutting-edge approach in cancer treatment. With this strategy, the antitumor immune response is mobilized, breaking tumor-related immunosuppression through active or passive means. To date, several studies have investigated the clinical significance of programmed death ligand-1 (PD-L1) expression in ACC (Fay et al. 2015, Liu et al. 2018, Billon et al. 2019, Parise et al. 2019). In one study, Billon et al. (2019) reported that PD-L1 mRNA was heterogeneously expressed in ACC patients and that high levels of expression were associated with longer disease- free survival (DFS). In contrast, Fay et al. (2015) employed immunohistochemistry to show PD-L1 expression in only 11% of ACC cases; overall, the expression of PD-L1 did not correlate with tumor stage, excessive hormone secretion or 5-year survival rate. Despite these inconsistent results for PD-L1 expression in ACC, five clinical trials have been published concerning the effects of PD-1 and PD-L1 antibodies in the treatment of advanced ACC, with objective response rates varying between 6 and 23% (Le Tourneau et al. 2018, Carneiro et al. 2019, Habra et al. 2019, Raj et al. 2020, Klein et al. 2021).
In addition to PD-1 and PD-L1, several newly characterized immune checkpoint targets have been identified, with promising results in preclinical studies or clinical trials. V-domain Ig suppressor of T-cell activation (VISTA), which shares structural similarity with PD-L1, is a member of the B7 family that has become a promising target for overcoming resistance to current immune checkpoint inhibitors. VISTA is expressed on myeloid cells, inflammatory monocytes, dendritic cells and lymphocytes such as CD4+ and CD8+ T cells (Hosseinkhani et al. 2021). VISTA has been found to downregulate the immune system
by inducing quiescence in myeloid and naïve T cells and by inhibiting T cell activation and cytokine production (ElTanbouly et al. 2020).
Although VISTA was found to be expressed on either tumor cells (TCs) or tumor-infiltrating immune cells (TICs) in the tumor microenvironment (TME), its role in tumorigenesis and neoplastic prognosis in various diseases is intriguingly context dependent. For example, in hepatocellular carcinoma (Zhang et al. 2018), non-small cell lung cancer (Villarroel-Espindola et al. 2018) and esophageal adenocarcinoma (Loeser et al. 2019), higher expression of VISTA is associated with better patient survival. In triple-negative breast cancer, patients with VISTA-positive TICs show prolonged relapse-free and overall survival (OS) (Cao et al. 2020). Conversely, in multiple myeloma (Mutsaers et al. 2021), melanoma (Kakavand et al. 2017), advanced germ cell tumor (Pęksa et al. 2021) and extranodal natural killer/ T-cell lymphoma (He et al. 2021), high expression of VISTA correlates significantly with detrimental clinical characteristics and reduced survival, and it is reported to be an independent unfavorable prognostic factor. Although functional and mechanism-related validation are required to fully understand the different expression patterns of VISTA under various circumstances, there have been four clinical trials of VISTA blockade since 2016 aiming to reactivate T-cell proliferation and cytokine production and to stimulate antitumor immune responses in patients with advanced tumors or lymphomas (NCT02812875, CTRI/2017/12/011026, NCT02671955, and NCT04475523). Thus far, however, no studies have investigated the expression and clinical prognostic significance of VISTA in the TME of ACC.
Therefore, this study was performed to assess VISTA expression in combination with PD-L1 in ACC patients, analyze their associations with clinicopathological features and evaluate their value in predicting prognosis in ACC.
Materials and methods
Patient cohort and specimens
Fifty-four patients with a pathological diagnosis of ACC between 2003 and 2021 were included in this study. The standard pathological diagnosis was made according to the Weiss criteria (Weiss 1984) or Lin-Weiss-Bisceglia criteria (Bisceglia et al. 2004). The European Network for the Study of Adrenal Tumors (ENSAT) classification (Fassnacht et al. 2018) was used to define ACC tumor stage. The diagnosis of
hypercortisolism and primary aldosteronism followed the most recent guidelines (Nieman et al. 2015, Funder et al. 2016).
Patient clinical and pathological data were obtained from the integrated electronic medical record system (Zesing Software, Shanghai, China) of Nanjing Drum Tower Hospital and surgical pathology reports. Telephone interviews were conducted to obtain survival information from each patient. Formalin-fixed, paraffin-embedded (FFPE) tumor samples were collected from the Department of Pathology, and 2-um thick sections (HM325, Thermo Scientific) were prepared by a pathological technician. Written informed consent for the publication of their clinical details and images was obtained from each patient. The Ethics Committee of Nanjing Drum Tower Hospital approved the study protocol. All study methods and procedures were in accordance with the ethical standards of the Declaration of Helsinki.
Immunohistochemistry
FFPE sections were deparaffinized and rehydrated using standard methods. For antigen retrieval, the sections were treated with Tris-EDTA repair solution (pH 9.0) for 8 min at 100℃ using a microwave. The sections were then immersed for 25 min in 3% H2O2 to block endogenous peroxidase activity, followed by incubation for 30 min with 3% BSA to block nonspecific antibody binding sites. The sections were incubated at 4℃ overnight with an anti- VISTA MAB (MA5-24704, Invitrogen) diluted at 1:1000, an anti-PD-L1 MAB (ab213524, Abcam) diluted at 1:250 and then with horseradish peroxidase-conjugated secondary antibodies for 50 min at room temperature. Finally, staining was performed with 3,3-diaminobenzidine tetrahydrochloride, and counterstaining was achieved with Mayer’s hematoxylin. Human tonsils and placenta were employed as positive controls. Immunohistochemistry analysis was performed using a Nikon E100 microscope (NIKON CORPORATION). All sections were examined independently by two observers using a multiheaded light microscope (BX53, Olympus Corporation), and discrepant results were reviewed and discussed; a consensus was obtained.
Evaluation of VISTA and PD-L1 immunohistochemical expression
VISTA and PD-L1 expression on TCs and TICs were evaluated separately, and samples were divided into ‘negative’ and ‘positive’ groups. TCs were considered VISTA-positive
if at least 5% of TCs per core had membranous and/or cytoplasmic staining. TC VISTA expression intensity scores were as follows: no membranous or cytoplasmic staining, 0; light yellow staining in these areas, 1; yellow staining, 2; brown or dark brown staining, 3. VISTA expression histochemistry scores were calculated as the H-score=positive TC intensity score x percentage of positive TC area. Scoring of TC PD-L1 expression was based on the percentage of PD-L1-expressing TCs with respect to the total tumor area. VISTA and PD-L1 expression on TICs was defined as positive when the expression percentage was ≥1% and negative when the expression percentage was less than 1%.
Statistical analysis
Statistical analyses were conducted using SPSS 26.0 (IBM Corp.), and graphs were generated with GraphPad Prism 9.0 (GraphPad Software, Inc.). Continuous variables with symmetrical distributions are summarized as mean ± S.D. Quantitative variables with skewed distributions are presented as medians and interquartile ranges and categorical variables as frequencies and percentages. To compare clinicopathological features with VISTA and PD-L1 expression levels, independent samples t-tests or Mann-Whitney U tests were employed to compare continuous variables. Chi-square tests or Fisher’s exact tests were administered for categorical variables. OS was defined as the time from first diagnosis to death of any cause. DFS was defined as the time from diagnosis to the occurrence of relapse or death from any cause. OS and DFS rates were analyzed using the Kaplan-Meier method, and log-rank tests were employed to evaluate differences between survival curves. Univariate proportional hazard models were conducted to assess the effects of any single variable contributing to survival. In addition, variables that were statistically significant in the univariate models were added sequentially in multivariate proportional hazard models. For all analyses, P < 0.05 was considered statistically significant.
Results
Patient characteristics
As shown in Supplementary Table 1 (see section on supplementary materials given at the end of this article), this study enrolled 54 ACC patients. The median age at diagnosis was 50 (15-83) years old, and the female to male ratio was 1.7:1, showing a slight female predominance.
| Endocrine-Related | Z Zhang, M Li et al. | VISTA and PD-L1 in | 29:7 | 406 |
|---|---|---|---|---|
| Cancer | adrenocortical carcinoma |
Medical history of hyperglycemia, hypertension and hypokalemia was found in 11 (20.4%), 23 (42.6%) and 14 (25.9%) of the patients, respectively. Among 30 patients who had complete biomedical records, 17 (56.7%) individuals exhibited excessive hormone secretion. Among them, 1 had isolated mineralocorticoid excess; 16 patients had glucocorticoid excess with the diagnosis of Cushing’s syndrome. Regarding pathological characteristics, the average diameter of ACC tumors was 10.07 ± 5.09 cm. Eight (14.8%) patients had low Weiss scores of 1 to 3, whereas 46 (85.2%) patients had high Weiss scores of 4 to 9. Of the four patients with Weiss scores less than 3, one was diagnosed with adrenocortical carcinoma according to Lin-Weiss- Bisceglia criteria and the other three were considered to have malignant disease with clinical features including tumors with migratory tendencies, large tumors, high-grade Ki-67 indexes and malignant radiological imaging findings. According to ENSAT stage classification, the proportions of advanced stage III (n= 16) and stage IV (n= 11) cases were 29.6 and 20.4%, respectively. Regarding therapeutic regimens, 50 (92.6%) patients underwent surgical resection of local lesions; 10 (18.5%) patients received adjuvant mitotane or platinum-based chemotherapy alone or after surgery.
During a median follow-up of 79 months, tumor relapse occurred in 17 (31.5%) patients and 28 (51.9%)
patients experienced distant metastasis. According to Kaplan-Meier survival analyses, the median overall survival time was 35 (95% CI: 24.34-45.66) months and 1-, 3- and 5-year rates were 73.2, 46.9 and 41.7%, respectively (Supplementary Fig. 1A). The median DFS time was 22 (95% CI: 5.49-38.52) months and 1-, 3- and 5-year rates were 57.1, 38.4 and 34.2% (Supplementary Fig. 1B).
VISTA and PD-L1 expression and clinicopathological features
Immunohistochemistry revealed a cytoplasmic/ membranous staining pattern for VISTA (Fig. 1A and B). VISTA expression in TCs from ACC patients was observed in 44 (81.5%) samples, though its expression in TILs was detectable in only 22 (40.7%) samples (Table 1). PD-L1 tended to be expressed in the cell membrane (Fig. 1C and D), and its expression was found in a small proportion of TCs (n= 6, 11.1%) and TILs (n= 6, 11.1%) of ACC samples (Table 2).
In terms of pathological characteristics, compared to the VISTA-negative group, many more patients with stage III and IV disease (56.9% vs 20%, P =0.036) and a higher Ki-67 index (30.50 ± 23.51% vs 14.76 ± 11.75%, P =0.006) exhibited TCs-expressing VISTA. However, no
A
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D
| Endocrine-Related | Z Zhang, M Li et al. | VISTA and PD-L1 in | 29:7 | 407 |
|---|---|---|---|---|
| Cancer | adrenocortical carcinoma |
| Characteristics | Vista expression (TCs) | P | Vista expression (TICs) | P | ||
|---|---|---|---|---|---|---|
| Negative (n = 10) | Positive (n = 44) | Negative (n = 32) | Positive (n = 22) | |||
| Age, median | 49.5 ± 14.09 | 48.11 ± 17.09 | 0.813 | 49.25 ± 16.24 | 47.09 ± 17.07 | 0.640 |
| Sex | 1.000 | 0.932 | ||||
| Female | 6 (60%) | 28 (63.6%) | 20 (62.5%) | 14 (63.6%) | ||
| Male | 4 (40%) | 16 (36.4%) | 12 (37.5%) | 8 (36.4%) | ||
| Diabetes | 3 (30%) | 8 (18.2%) | 0.408 | 6 (18.8%) | 5 (22.7%) | 0.743 |
| Hypertension | 2 (20%) | 21 (47.7%) | 0.161 | 16 (50%) | 7 (31.8%) | 0.184 |
| Hormone secretion excess | 1 (10%) | 16 (36.4%) | 0.144 | 12 (37.5%) | 5 (22.7%) | 0.251 |
| ENSAT stage | 0.036 | 0.268 | ||||
| I-II | 8 (80%) | 19 (43.2%) | 18 (56.3%) | 9 (40.9%) | ||
| III-IV | 2 (20%) | 25 (56.9%) | 14 (43.8%) | 13 (59.1%) | ||
| Tumor size (cm) | 7.66 ± 4.15 | 10.40 ± 4.99 | 0.120 | 9.11 ± 4.90 | 11.95 ± 5.68 | 0.064 |
| Weiss score | 1.000 | 0.449 | ||||
| 1-3 | 1 (10%) | 7 (15.9%) | 6 (18.8%) | 2 (9.1%) | ||
| 4-9 | 9 (90%) | 37 (84.1%) | 26 (81.3%) | 20 (90.9%) | ||
| Ki-67 index (%) | 14.76 ±11.75 | 30.50 ± 23.51 | 0.006 | 19.03 ± 16.50 | 18.86 ± 16.46 | 0.777 |
| Relapse/recurrence | 3 (30%) | 14 (31.8%) | 1.000 | 13 (40.6%) | 4 (18.2%) | 0.081 |
| Metastasis | 2 (20%) | 23 (52.3%) | 0.086 | 17 (53.1%) | 11 (50%) | 0.821 |
| More than 2 metastasis sites | 4 (40%) | 17 (38.6%) | 1.000 | 12 (37.5%) | 9 (40.9%) | 0.801 |
| OS (months), median (95% Cl) | 7 (1.16-12.84) | 35 (24.79-45.21) | 0.689 | 52 (8.12-95.88) | 31 (12.11-49.89) | 0.681 |
| DFS (months), median (95% Cl) | 5 (0.82-9.18) | 21 (6.18-31.82) | 0.395 | 23 (12.37-33.63) | 10 (0-28.79) | 0.863 |
ACC, adrenocortical carcinoma; DFS, disease-free survival; ENSAT, European Network for the Study of Adrenal Tumors; OS, overall survival; PD-L1, programmed death ligand-1; TCs, tumor cells; TICs, tumor-infiltrating immune cells; VISTA, V-domain Ig suppressor of T-cell activation. Bold indicates statistical significance.
significant differences in tumor size or Weiss score were found between the groups (Table 1 and Supplementary Fig. 2A). No statistically significant differences between TIC VISTA expression and pathological variables were observed, though the VISTA-positive TICs group had a
larger average tumor size than the negative group (Table 1 and Supplementary Fig. 2B).
Regarding ACC patient follow-up data, no significant difference was detected between the TC and TIC-positive and -negative VISTA expression groups. However, those
| Characteristics | PD-L1 expression (TCs) | P | PD-L1 expression (TICs) | P | ||
|---|---|---|---|---|---|---|
| Negative (n = 48) | Positive (n = 6) | Negative (n = 48) | Positive (n = 6) | |||
| Age, median | 48.81 ± 16.75 | 44.83 ± 14.74 | 0.581 | 48.94 ± 15.78 | 43.83 ± 22.43 | 0.479 |
| Sex | 0.395 | 0.659 | ||||
| Female | 29 (60.4%) | 5 (83.3%) | 31 (64.6%) | 3 (50%) | ||
| Male | 19 (39.6%) | 1 (16.7%) | 17 (35.4%) | 3 (50%) | ||
| Diabetes | 9 (18.8%) | 2 (33.3%) | 0.590 | 10 (20.8%) | 1 (16.7%) | 1.000 |
| Hypertension | 20 (41.7%) | 3 (50%) | 1.000 | 21 (43.8%) | 2 (33.3%) | 1.000 |
| Hormone secretion excess | 15 (31.3%) | 2 (33.3%) | 1.000 | 16 (33.3%) | 1 (16.7%) | 0.652 |
| ENSAT stage | 0.669 | 1.000 | ||||
| I-II | 25 (52.1%) | 2 (33.3%) | 24 (50%) | 3 (50%) | ||
| III-IV | 23 (47.9%) | 4 (66.7%) | 24 (50%) | 3 (50%) | ||
| Tumor size (cm) | 9.08 ± 3.44 | 6.70 ± 4.60 | 0.940 | 9.86 ± 4.91 | 14.33 ± 1.53 | 0.070 |
| Weiss score | 0.575 | 0.213 | ||||
| 1-3 | 8 (16.7%) | 0 | 6 (12.5%) | 2 (33.3%) | ||
| 4-9 | 40 (83.3%) | 6 (100%) | 42 (87.5%) | 4 (66.7%) | ||
| Ki-67 index | 19.89 ± 16.82 | 11.67 ± 9.89 | 0.249 | 19.25 ± 16.23 | 16.20 ± 18.98 | 0.695 |
| Relapse/recurrence | 17 (35.4%) | 0 | 0.161 | 16 (33.3%) | 1 (16.7%) | 0.652 |
| Metastasis | 25 (52.1%) | 3 (50%) | 1.000 | 24 (50%) | 4 (66.7%) | 0.670 |
| More than 2 metastasis sites | 18 (37.5%) | 3 (50%) | 0.667 | 18 (37.5%) | 3 (50%) | 0.667 |
| OS (months), median (95% Cl) | 35 (10.38-59.62) | 36 (0-72.5) | 0.785 | 35 (24.81-45.19) | 78 (0-191.77) | 0.574 |
| DFS (months), median (95% Cl) | 22 (3.56-40.44) | 19 (9.04-28.96) | 0.956 | 22 (11.12-32.88) | 78 (0-181.29) | 0.306 |
ACC, adrenocortical carcinoma; DFS, disease-free survival; ENSAT, European Network for the Study of Adrenal Tumors; OS, overall survival; PD-L1, programmed death ligand-1; TCs, tumor cells; TICs, tumor-infiltrating immune cells.
with VISTA, expression by TCs had more metastases and were more likely to experience relapse than those whose TCs were negative for VISTA expression (52.3% vs 20%, P=0.086) (Table 1). Overall, no statistically significant associations between any clinicopathological features and PD-L1 expression were found for TCs or TICs (Table 2 and Supplementary Fig. 2C, D).
Prognostic significance of VISTA and PD-L1 expression
When analyzing the prognostic significance of TC and TIC VISTA expression in terms of OS and DFS, the results showed a median survival time of 35 months in the VISTA TC-positive group and 7 months in the VISTA TC-negative
group. DFS was 21 months in the VISTA TC-positive group and 5 months in the VISTA TC-negative group (Table 1). Although the VISTA TC-positive group had a slightly increased median survival time and DFS, there was no significant difference compared with the negative group (Fig. 2A and B). Moreover, no associations between VISTA TIC expression and OS or DFS were detected (Fig. 2E and F). Positive PD-L1 expression by TCs or TICs was not associated with better OS or DFS (Fig. 2C, D, G and H).
As Kaplan-Meier survival analyses do not consider possible prognostic variables, we also carried out proportional hazards analyses. Based on univariate analysis, excessive hormone secretion (HR=2.66, P=0.044), Ki-67 index (HR=1.03, P=0.005), tumor
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p = 0.689
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p = 0.785
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p = 0.956
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p = 0.681
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@ 2022 Society for Endocrinology Published by Bioscientifica Ltd. Printed in Great Britain
| Endocrine-Related | Z Zhang, M Li et al. | VISTA and PD-L1 in | 29:7 | 409 |
|---|---|---|---|---|
| Cancer | adrenocortical carcinoma |
| Variables | Univariate analysis | Multivariate analysis | ||
|---|---|---|---|---|
| HR (95% CI) | P | HR (95% CI) | P | |
| Age | 1.01 (0.99-1.04) | 0.270 | ||
| Sex (male vs female) | 0.96 (0.47-1.96) | 0.837 | ||
| Hypertension (yes vs no) | 2.30 (0.93-5.69) | 0.071 | ||
| Diabetes (yes vs no) | 0.55 (0.24-1.24) | 0.148 | ||
| Hormone secretion excess (yes vs no) | 2.66 (0.96-7.73) | 0.044 | 30.53 (1.61-580.71) | 0.023 |
| Tumor size (high vs low) | 1.00 (0.90-1.12) | 0.948 | ||
| Weiss score (high vs low) | 1.12 (0.92-1.37) | 0.260 | ||
| ENSAT stage | ||||
| 2 vs 1 | 0.45 (0.10-2.06) | 0.301 | ||
| 3 vs 1 | 0.90 (0.19-4.15) | 0.890 | ||
| 4 vs 1 | 4.69 (0.59-37.52) | 0.146 | ||
| Ki-67 index | 1.03 (1.01-1.05) | 0.005 | 0.99 (0.94-1.04) | 0.676 |
| Surgery (yes vs no) | 0.13 (0.03-0.31) | 0.003 | 4.48 (0.58-34.40) | 0.150 |
| Relapse (yes vs no) | 5.09 (2.15-12.04) | 0.000 | 3.03 (1.00-7.05) | 0.003 |
| Metastasis (yes vs no) | 9.83 (2.91-33.92) | 0.000 | 5.04 (2.00-9.60) | 0.020 |
| VISTA intensity on TCs | ||||
| 1 vs 0 | 0.37 (0.13-1.05) | 0.061 | ||
| 2 vs 0 | 48.08 (0.12-194.96) | 0.020 | 0.52 (0.20-1.40) | 0.196 |
| 3 vs 0 | 83.89 (0.12-197.45) | 0.036 | 0.63 (0.16-2.57) | 0.522 |
| VISTA area (TCs) | 1.98 (1.97-2.00) | 0.049 | 0.99 (0.98-1.00) | 0.071 |
| VISTA H-score (TCs) | 2.47 (1.03-5.90) | 0.043 | 1.00 (0.99-1.01) | 0.558 |
| VISTA expression on TICs (positive vs negative) | 1.31 (0.64-2.69) | 0.454 | ||
| PD-L1 expression on TCs | 1.01 (0.97-1.06) | 0.585 | ||
| PD-L1 expression on TICs (positive vs negative) | 2.27 (0.31-16.71) | 0.421 | ||
ACC, adrenocortical carcinoma; DFS, disease-free survival; ENSAT, European Network for the Study of Adrenal Tumors; HR, hazard ratio; PD-L1, programmed death ligand-1; TCs, tumor cells; TICs, tumor-infiltrating immune cells; VISTA, V-domain Ig suppressor of T-cell activation. Bold indicates statistical significance.
relapse (HR=5.09, P=0.000) and distant metastasis (HR=9.83, P =0.000) were associated with increased risks of disease progression, though patients who underwent surgical treatment (yes vs no: HR=0.13, P=0.003) had decreased risks. For immune-related molecules, VISTA expression on TCs, including expression intensity (2 vs 0: HR=48.08, P=0.020; 3 vs 0: HR=83.89, P =0.036), expression area (HR=1.98, P=0.049) and H-score (HR=2.47, P =0.043) also contributed to increased risks of disease progression, whereas VISTA expression on TICs and PD-L1 expression on TCs/TICs did not appear to correlate with DFS. In multivariate analysis, excessive hormone secretion (HR=30.53, P =0.023), tumor relapse (HR=3.03, P=0.003) and metastasis (HR=5.04, P =0.020) remained significant independent factors for DFS (Table 3).
When we considered OS as an endpoint, the Ki-67 index (HR=4.03, P=0.004), tumor relapse (HR=4.68, P=0.000) and distant metastasis (HR=8.98, P=0.000) correlated with increased risks of overall mortality, but surgical treatment (yes vs no: HR=0.16, P =0.002) favored OS. The VISTA expression area on TCs (HR=2.95, P =0.025) seemed to be related to increased risks of overall mortality, whereas PD-L1 expression did not exhibit any correlations
with OS. In multivariate analysis, surgical therapy (yes vs no: HR=0.25, P =0.037) and tumor metastasis (HR=5.14, P =0.001) independently predicted OS (Table 4). Moreover, we performed a multivariate analysis using all the variables identified in the univariate analyses, and the results were similar.
Discussion
The present study is the first to report that VISTA is expressed on both TCs and TICs of ACC. Furthermore, high levels of VISTA expression on TCs were associated with worse ACC tumor classification, whereas PD-L1 expression on TCs or TICs did not show any prognostic value.
This one-center study cohort comprised 54 ACC patients from Nanjing Drum Tower Hospital. The demographic features, including sex predominance, age distribution and hormone excess phenotypes, of the cohort were consistent with those of previous studies (Berruti et al. 2021). The median survival time of 35 months and 5-year survival rate of 41.7% were also typical for ACC patients.
| Endocrine-Related | Z Zhang, M Li et al. | VISTA and PD-L1 in | 29:7 | 410 |
|---|---|---|---|---|
| Cancer | adrenocortical carcinoma |
| Variables | Univariate analysis | Multivariate analysis | ||
|---|---|---|---|---|
| HR (95% CI) | P | HR (95% CI) | P | |
| Age | 1.01 (0.99-1.04) | 0.176 | ||
| Sex (male vs female) | 1.13 (0.54-2.37) | 0.740 | ||
| Hypertension (yes vs no) | 1.78 (0.81-3.93) | 0.152 | ||
| Diabetes (yes vs no) | 2.23 (0.97-5.14) | 0.060 | ||
| Hormone secretion excess (yes vs no) | 0.38 (0.14-1.04) | 0.059 | ||
| Tumor size | 1.02 (0.91-1.15) | 0.742 | ||
| Weiss score | 2.02 (1.91-3.27) | 0.100 | ||
| ENSAT stage | ||||
| 2 vs 1 | 0.51 (0.11-2.29) | 0.377 | ||
| 3 vs 1 | 1.15 (0.25-5.29) | 0.854 | ||
| 4 vs 1 | 5.34 (0.68-41.93) | 0.111 | ||
| Ki-67 index | 4.03 (2.64-8.19) | 0.004 | 1.01 (0.98-1.04) | 0.488 |
| Surgery (yes vs no) | 0.16 (0.05-0.51) | 0.002 | 0.25 (0.07-0.92) | 0.037 |
| Relapse (yes vs no) | 4.68 (1.98-11.05) | 0.000 | 0.53 (0.20-1.44) | 0.212 |
| Metastasis (yes vs no) | 8.98 (2.65-30.46) | 0.000 | 5.14 (1.00-6.46) | 0.001 |
| VISTA intensity on TCs | ||||
| 1 vs 0 | 1.00 (0.22-4.46) | 0.997 | ||
| 2 vs 0 | 0.66 (0.11-3.93) | 0.643 | ||
| 3 vs 0 | 0.30 (0.01-8.02) | 0.470 | ||
| VISTA area (TCs) | 2.95 (0.94-5.49) | 0.025 | 0.99 (0.98-1.01) | 0.269 |
| VISTA H-score (TCs) | 1.02 (0.99-1.05) | 0.285 | ||
| VISTA expression on TICs (positive vs negative) | 1.40 (0.68-2.90) | 0.364 | ||
| PD-L1 expression on TCs | 1.01 (0.97-1.06) | 0.585 | ||
| PD-L1 expression on TICs (positive vs negative) | 1.99 (0.27-14.80) | 0.501 | ||
ACC, adrenocortical carcinoma; ENSAT, European Network for the Study of Adrenal Tumors; HR, hazard ratio; OS, overall survival; PD-L1, programmed death ligand-1; TCs, tumor cells; TICs, tumor-infiltrating immune cells; VISTA, V-domain Ig suppressor of T-cell activation. Bold indicates statistical significance.
Based on these cohort characteristics, this population was representative.
VISTA is a type I transmembrane protein that shares homology with PD-L1. VISTA is expressed by myeloid cells, inflammatory monocytes, dendritic cells and lymphocytes (Tagliamento et al. 2020); it may induce peripheral tolerance by promoting quiescence in myeloid and naïve T cells, thus inhibiting the activation of T cells and cytokine production (Tagliamento et al. 2020). Huang et al. (2020) first investigated the profiles of VISTA gene expression in 30 major human cancer types and showed it to be expressed at higher levels in cholangiocarcinoma, glioblastoma multiform, kidney renal clear cell carcinoma, acute myeloid leukemia, brain lower grade glioma and pancreatic adenocarcinoma. Furthermore, VISTA expression levels correlate positively with almost all types of tumor- infiltrating lymphocytes and critical immunomodulators or major histocompatibility complexes (MHCs), including critical immune checkpoints, such as programmed cell death 1 (PD-1), PD-L1, CD80 and CD86. In terms of protein profiling, VISTA expression is higher on TCs in hepatocellular carcinoma, gastric cancer, prostate cancer, ovarian cancer and endometrial cancer (Böger et al. 2017, Gao et al. 2017, Zhang et al. 2018, Mulati et al. 2019,
Zong et al. 2020). Conversely, VISTA is highly expressed on TICs in melanoma, non-small-cell lung cancer, acute myeloid leukemia, esophageal adenocarcinoma and renal cell carcinoma (Villarroel-Espindola et al. 2018, Wang et al. 2018, Hong et al. 2019, Loeser et al. 2019, Choi et al. 2020). To our knowledge, this is the first study to evaluate the expression of VISTA in ACC. In our study, immunohistochemistry showed that in accordance with previous studies, VISTA is expressed in the cell membrane and/or cytoplasm but mostly expressed by TCs (81.5%) instead of TICs (40.7%) in ACC. In contrast to Andre P Fay’s study (Fay et al. 2015), in which PD-L1 was found to be mainly expressed on tumor-infiltrating mononuclear cells, in our ACC cases, PD-L1 was expressed on a small proportion of TCs and TICs.
With respect to association between VISTA and clinical parameters, VISTA gene expression correlates with tumor stage in lung adenocarcinoma and ovarian serous cystadenocarcinoma and is associated with tumor grade in head and neck squamous cell carcinoma and stomach adenocarcinoma (Huang et al. 2020). In our study, ACC patients with positive VISTA expression on TCs had a significantly higher ENSAT stage and Ki-67 index. Because pathologic stage and the Ki-67 index both indicate the
| Endocrine-Related | Z Zhang, M Li et al. | VISTA and PD-L1 in | 29:7 | 411 |
|---|---|---|---|---|
| Cancer | adrenocortical carcinoma |
degree of malignancy, these results suggest that VISTA may play a role in ACC tumorigenesis. The possible underlying mechanisms include VISTA overexpression downregulating the immune system by suppressing T-cell proliferation, blunting cytokine secretion and CD3+CD4+ Foxp3+ Treg recruitment and promoting the persistence of the regulatory T-cell pool, thus contributing to tumor progression (Yuan et al. 2021). Additionally, VISTA overexpression has been associated with the activation of beta-catenin (Yuan et al. 2021), and activation of WNT/ beta-catenin signaling has been recognized as an oncogenic driver in a large subset of ACC patients (Shimada et al. 2021). Nonetheless, the specific mechanisms by which VISTA promotes tumorigenesis in ACC require further exploration.
In contrast to the study by Billon et al. (2019), which showed that PD-L1 mRNA expression is heterogeneous in ACC, high expression was associated with longer DFS. Our study and Fay’s study (Fay et al. 2015) did not find any associations between clinicopathological features and PD-L1 expression on either TCs or TICs of ACC. Possible explanations for this discordance are related to interpretative subjectivity, the small size and heterogeneity of ACC samples and imperfect correlation between mRNA and protein expression levels.
Researchers have evaluated the prognostic value of VISTA in different cancers. In advanced germ cell tumors, low VISTA-expressing TICs were identified as predictors of shorter event-free survival, independent of tumor histology and the location of metastases (Pęksa et al. 2021). With regard to human non-small-cell lung cancer, VISTA expression in the tumor microenvironment predicts longer 5-year survival (Villarroel-Espindola et al. 2018). In multiple myeloma, moreover, the combination of abundant VISTA+ and CD11b+ cells in the tumor together with a low abundance of CD8+ T cells in the same tissue compartment is significantly associated with shorter OS (Mutsaers et al. 2021). In our study, VISTA and PD-L1 expression on TCs displayed no statistically significant associations with OS or DFS. In addition to classic prognostic factors, including excessive hormone secretion, the Ki-67 index, radical surgery, tumor relapse and metastasis, VISTA expression indexes such as the H-score, the expression intensity, the area and TIC VISTA expression showed no significant associations with the OS or DFS rate. Similar results to those observed for VISTA were observed for the PD-L1 expression index. These findings indicate that using VISTA or PD-L1 alone as an immune biomarker has limited value in predicting the prognosis of ACC patients.
There were several limitations in our study. First, since the sample size was small, and the patients were only from
a single center, which might lead to selection bias, the statistical power of comparisons may be limited, which may explain why the large differences in median OS and DFS survival rates were not statistically significant. Additionally, we did not perform a combined analysis to evaluate the impact of VISTA and PD-L1 on TCs and TICs together due to the small sample sizes. Therefore, large and heterogeneous samples from various clinics are needed to reach statistically representative conclusions. Secondly, this was a retrospective study spanning 17 years, which may have led to missing data and scarce tissue samples; thus, it was difficult to validate the immunohistochemical results at other levels, and in vivo studies are needed to elucidate the underlying mechanisms within the ACC tumor microenvironment.
In conclusion, our study for the first time demonstrates VISTA expression in the ACC tumor microenvironment. As positive VISTA expression correlates with higher tumor pathological grading, VISTA is considered a potentially novel target for ACC treatment. Further studies on the immunoregulatory mechanisms of these immune molecules are needed to provide more evidence for ACC immunotherapy.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/ ERC-22-0066.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by the National Natural Science Foundation of China (81970689), the Natural Science Foundation of Jiangsu Province (BK20181116) and the National Key Research and Development Program of China (2021YFC2501603, 2021YFC2501600).
Acknowledgement
The authors thank all the patients who gave consent for the use of their postoperative samples in this study.
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Received in final form 7 April 2022 Accepted 25 April 2022 Accepted Manuscript published online 25 April 2022