EJE
Mitotane treatment of adrenocortical carcinoma induces tumoural secretion of GDF-15: impact on poor prognosis and impaired responsiveness to immunotherapy
Isabel Weigand,10 Alexandra S. Triebig,1 Tanja Maier,1 Tanja Anderlik,2 Hanna Remde,20D Laura-Sophie Landwehr,20D Otilia Kimpel,20D Miriam Reuter,2[D Jochen Schreiner,1 Florian Wedekink,3 Oliver Scherf-Clavel,40D Eva Hoster,5,6 Kai C. Wollert,70D Ilka Budde,2 Barbara Altieri,20D Paul Schwarzlmueller,10 Martin Reincke, 10D Jorg Wischhusen, 3,8,9[D Martin Fassnacht,2,8,9[D and Matthias Kroiss 1,2,6,8,*[D
1LMU University Hospital, Department of Medicine IV, Ludwig-Maximilians-University, 80336 Munich, Germany
2Department of Internal Medicine I, Division of Endocrinology and Diabetes, University Hospital, University of Wuerzburg, 97080 Wuerzburg, Germany
3Experimental Tumour Immunology, Department of Obstetrics and Gynecology, University Hospital, University of Wuerzburg,
97080 Wuerzburg, Germany
4Clinical Pharmacy and Pharmacotherapy, Ludwig-Maximilians-University, 80337 Munich, Germany
5Institute for Medical Informatics, Biometry and Epidemiology, Ludwig-Maximilians-University, 81337 Munich, Germany
6Comprehensive Cancer Center Munich, 80336 Munich, Germany
7Division of Molecular and Translational Cardiology, Department of Cardiology and Angiology, Hannover Medical School, 30625 Hannover, Germany
8Bavarian Center for Cancer Research, BZKF, 91012 Erlangen, Germany
9Comprehensive Cancer Center Mainfranken, 97080 Würzburg, Germany
*Corresponding author: Department of Internal Medicine IV, LMU University Hospital, Ludwig Maximilians Universität München, Ziemssenstr. 5, Munich 80336, Germany. Email: matthias.kroiss@med.Imu.de
Abstract
Purpose: Treatment options for adrenocortical carcinoma (ACC), where mitotane remains a mainstay of therapy, are unsatisfactory. Response rates of ACC to immune checkpoint inhibition (ICI) are disappointing, and immune cells are scarce in ACC. Growth/differentiation factor 15 (GDF-15) is a cytokine impairing tumoural immune infiltration. We here aimed to assess the value of serum GDF-15 for the prognosis of ACC and as a predictor of response to ICI.
Methods: GDF-15 was measured in serum samples of 151 patients and correlated with clinical data. Serum GDF-15 was analysed in a second cohort of 46 ACC patients who received ICI, including 14 responders. mRNA expression of GDF15 and genes related to immune response was quantified in 58 ACC tumour samples.
Results: We found GDF-15 induction in ACC cells and patients upon mitotane treatment. In ACC patients, serum GDF-15 concentration below the median was associated with significantly longer patient survival. GDF-15 levels in responders to ICI were significantly lower than in non-responders (P =. 0379), and patients with low GDF-15 levels had a significant longer progression-free survival than patients with higher GDF-15 serum levels (P =. 036). Expression of pro-inflammatory immune-related genes was lower in ACC tissue with GDF-15 expression above the median.
Conclusions: Mitotane increases GDF-15 levels and is associated with poor response to ICI. GDF-15 may mediate reduced infiltration with immune cells in ACC.
Keywords: tumour immune micro-environment, immune checkpoint inhibition, macrophage-inhibitory cytokine 1, adrenal cancer
Significance
Immune checkpoint inhibition (ICI) is only effective in approximately 15% of adrenocortical carcinoma (ACC) patients. Yet, there is no biomarker to identify responders prior to treatment. Mitotane remains the mainstay of ACC treatment. We studied a large cohort of ACC patients with and without mitotane and 46 patients receiving ICI. We discovered that mitotane therapy increases serum concentrations of the immunosuppressive cytokine growth/differentiation factor 15 (GDF-15). High serum GDF-15 is associated with shorter survival. High circulating GDF-15 at ICI start is associated with treatment failure and an anti-inflammatory gene expression profile in tissue. The combination of ICI with GDF-15 neu- tralising antibodies may be active in patients with ACC as currently successfully tested in other tumour entities.
Introduction
Adrenocortical carcinoma (ACC) is a rare endocrine malig- nancy which affects approximately 1 per 1 million people per year,1 and the majority of patients suffer from adrenal ster- oid excess.2 Based on retrospective studies,3 current guidelines recommend adjuvant treatment with mitotane in patients with intermediate and high risk of recurrence,4 but mitotane is not beneficial in low-risk patients.5 In advanced stages, mitotane is used as monotherapy or in combination with cytotoxic chemotherapy. Yet, the rate of objective tumour response to mitotane monotherapy is at best ~20% in selected patients6 and adverse events are manifold and common. Even the com- bination of mitotane with platinum-based chemotherapy only leads to an objective response rate of about 25%.7 Immune checkpoint inhibitors (ICI) have revolutionised the treatment of many cancer entities but not of ACC. The results of pub- lished studies are heterogeneous and objective response was observed in only 17 (13%) of 121 patients treated with differ- ent ICI across 5 clinical trials.8-12 A similar response rate was confirmed in 2 large retrospective analyses.13,14 In none of the trials, established response markers to ICI such as tumour mu- tational burden (TMB) or microsatellite instability (MSI) were associated with response. Serum markers of response to con- ventional treatment and immunotherapy have not been de- scribed in ACC.
We and others have reported low tumoural infiltration by T lymphocytes and natural killer (NK) cells in ACC, which is even lower in glucocorticoid-secreting tumours.15,16 Nonetheless, no association was found between glucocortic- oid secretion and response to ICI.8-12 In other tumour entities, growth/differentiation factor 15 (GDF-15) has emerged as a promising biomarker for predicting ICI failure.17 GDF-15 is a divergent member of the transforming growth factor ß super- family and acts as an endogenous immunosuppressive agent and hormone. Physiologically, GDF-15 is involved in feto- maternal tolerance.18 Serum GDF-15 reduces appetite19,20 and polymorphisms in the GDF15 gene are linked to hyperem- esis gravidarum.21 Circulating GDF-15 levels increase with age, after injury, in states of inflammation and in the presence of malignant neoplasias. Mechanistically, GDF-15 was shown to inhibit activity of NK cells during systemic inflammation and to favour regulatory T cell (Treg) differentiation.22 It has been demonstrated that GDF-15 promotes tumour growth, impairs effective intratumoural immune cell infiltration and potently blocks immune checkpoint inhibitor activity.23 Mitochondrial dysfunction, a hallmark of mitotane ac- tion,24,25 is a known inducer of GDF-15.26 Consistently, we have observed GDF-15 mRNA to be the single most highly up- regulated gene in ACC cells after mitotane treatment.27
Given the common use of mitotane in the treatment of ACC, the low tumour infiltration with immune cells and the upregu- lation of GDF-15 by mitotane in vitro, we investigated the im- pact of mitotane on serum GDF-15 in ACC patients. Based on the literature, we hypothesised that GDF-15 is associated with poor prognosis and impaired response to ICI treatment.
Methods
Clinical cohorts
This retrospective study was conducted as part of the European Network for the Study of Adrenal Tumours (ENSAT) registry study that was approved by the ethics committees at the
University Hospitals Würzburg (approval #88/11) and LMU Munich (approval 379-10) and conducted in accordance with the principles of the Declaration of Helsinki. All patients provided written informed consent.
Clinical and pathological data including sex, age at diagno- sis, date of diagnosis, tumour stage according to the ENSAT staging system, hormone secretion, Weiss score, Ki-67 prolif- eration index, and mitotane plasma concentrations at the time point of GDF-15 measurement were collected. Hormone ex- cess was defined as pathological glucocorticoid, androgen, al- dosterone, or mixed steroid hormone secretion at diagnosis. Tumour mass was quantified as the number of involved organs and number and total maximum diameter of all tumour le- sions using cross-sectional imaging from computer tomog- raphy (CT), 18-F-fluorodeoxyglucose (FDG) positron emission tomography (PET)-CT, or abdominal magnetic resonance imaging (MRI) at the time point of GDF-15 measurement.
Survival was determined from the time point of blood draw used to determine GDF-15 levels until ACC-related death or patient survival at the time point of last follow-up (September, 20, 2024).
Discovery cohort
Serum samples from 99 ACC patients before and 52 ACC pa- tients after mitotane treatment were collected to study the im- pact of mitotane on GDF-15 serum levels. Serum of 42 adrenocortical adenoma (ACA) patients served as controls. ACC patients were consecutively recruited at the University Hospitals in Würzburg and Munich. The clinical data of these ACC patients are shown in Table 1 (refer to Table S1 for ACA patients).
Immune checkpoint inhibitor treated cohort
GDF-15 serum levels of 46 ACC patients who received ICI treatment on a compassionate use basis were determined in se- rum samples collected at the time of ICI initiation ± approxi- mately 4 weeks. One patient with a pathogenic CTNNB1 mutation and inconsistent data was excluded.
Tumour control rate was defined as complete response (CR), partial response (PR) and stable disease (SD) by clinical judg- ment in analogy to RECIST 1.1 criteria.28 Progression was con- firmed by cross-sectional imaging, survival as ACC-related death or patient alive at last follow-up (September, 20, 2024). The clinical data for this cohort are displayed in Table 2.
Quantification of GDF-15
GDF-15 concentration in serum was determined with the Quantikine ELISA Human GDF-15 Kit (SGD150, R&D Systems) in the discovery cohort and Roche Cobas ECLIA in the ICI treatment cohort. A previous study showed that results obtained by either method correlate well.29 Healthy controls were measured with the Elecsys GDF-15 assay (Roche Diagnostics), raw data obtained by29 and matched 1 to 1 by age and sex with respective patient samples. Healthy controls were apparently healthy individuals with no adrenal, nor oth- er diseases.
Statistics
Descriptive statistics are given as median and range or SD; P <. 05 was considered statistically significant. Group
| Pre-mitotane | Post-mitotane | |
|---|---|---|
| Patients-n | 99 | 52 |
| Age at diagnosis in years: median (range) | 51 (19-80) | 50.5 (19-74) |
| Age at blood draw in years: median (range) | 53 (19-80) | 52 (19-76) |
| Sex-n (%) | ||
| Female | 57 (58) | 33 (63) |
| Male | 41 (42) | 19 (37) |
| ENSAT stage-n (%) | 97 | 52 |
| I | 5 (5) | 3 (6) |
| II | 42 (43) | 25 (48) |
| III | 23 (24) | 15 (29) |
| IV | 27 (28) | 9 (17) |
| R status-n {ENSATI-III} (%) | ||
| 0 | 53 (80) | 28 (78) |
| X | 6 (9) | 4 (11) |
| 1 | 6 (9) | 4 (11) |
| 2 | 1 (2) | 0 (0) |
| Ki-67-index-n (%) | ||
| <10 | 17 (23) | 7 (19) |
| ≥10 | 56 (77) | 30 (81) |
| Endocrine activity-n (%) | 66 | 34 |
| Hormone secretion | 43 (65) | 19 (56) |
| No hypersecretion | 23 (35) | 15 (43) |
| GDF-15 levels (ng/ml) median (range) | 0.81 (0.01-30.6) | 2.17 (0.5-12.5) |
| TTP from blood draw (months): median (range) (n =95) (n= 51) | 45 (0-125) | 34 (0-96) |
| Sum of tumour diameter (cm): median (range) (n = 93) (n= 46) | 5.0 (0-53) | 1.3 (0-53) |
| Mitotane level at blood draw (mg/dL) median (range) (n = 47) | 14.3 (0.3-28.7) |
comparisons were done as indicated for each result. Overall survival was defined as the time interval from blood sample collection to death or last follow-up. Recurrence-free survival in R0 resected patients was defined as the time interval be- tween blood sample collection and the first occurrence of re- lapse. The Kaplan-Meier method and log-rank test were performed to estimate and compare event-free survival. Cox proportional hazards regression modelling was used for the identification of clinical factors that independently influence patients’ survival. Proportional hazard ratio assumption was tested for time-dependent effects and Cox proportional hazards regression was only used when satisfied (>0.05). Known or potentially relevant prognostic factors (age, num- ber of affected sites to resemble localised and metastatic dis- ease and Ki-67 proliferation index) were analysed by univariate analysis. Since GDF-15 serum levels correlated with ENSAT stage, patients were stratified according to ENSAT stage before and during mitotane therapy. Receiver operating characteristics analyses were performed to calculate the area under the curve (AUC). To test for different con- founders, patients were divided into subgroups (eg, measur- able tumour mass) and sensitivity analyses were performed. Tumour control after immunotherapy was defined as either CR, PR, or SD and patients categorised into responders (tu- mour control at first staging) and non-responders (tumour progression at first staging or death prior to imaging). GDF-15 serum concentrations were determined around the
| Patients-n | 46 |
|---|---|
| Age at diagnosis in years: median (range) | 44.5 (13-78) |
| Age at blood draw in years: median (range) | 49.5 (18-82) |
| Sex n (%) | |
| Female | 28 (61) |
| Male | 18 (39) |
| ENSAT stage-n (%) | |
| I | 3 (7) |
| II | 14 (32) |
| III | 8 (19) |
| IV | 18 (42) |
| ICI compound-n (%) | |
| Pembrolizumab | 41 (89) |
| Nivolumab | 4 (9) |
| Ipilimumab + nivolumab | 1 (2) |
| Best response to ICI-n (%) | |
| CR | 0 (0) |
| PR | 7 (15) |
| SD | 7 (15) |
| PD | 32 (70) |
| Ki67-index-n (%) | |
| <10 | 4 (11) |
| ≥10 | 32 (89) |
| Endocrine activity-n (%) | |
| Hormone secretion | 26 (67) |
| Of which responders to ICI | 8 (31) |
| No hypersecretion | 13 (23) |
| Of which responders to ICI | 5 (38) |
| GDF-15 levels (ng/ml) at start ICI (n = 46) median | 4.2 (0.61-76.8) |
| TTP from blood draw (months): median (range) (n=46) | 3 (0-25) |
| Patients treated with mitotane before ICI: n (%) | 46 (100) |
| Mitotane parallel to ICI: n (%) | 6 (8) |
| Mitotane plasma levels at start ICI (mg/L) median (range) n = 34 | 1.9 (0-27.8) |
| Responders to ICI | 0.91 (0-23.4) |
| Non-responders to ICI | 2.85 (0-27.8) |
| Time (months) between end mitotane and start ICI: median (range) | 4 (0-136) |
start of ICI. The non-parametric Mann-Whitney U test was used to determine statistically significant differences.
Statistical analyses were performed using Prism (V.9.12, GraphPad Software Inc., La Jolla, CA, USA) and SPSS 27 (IBM Corporation, Armonk, New York, USA).
Nanostring nCounter gene expression analysis
Formalin-fixed paraffin-embedded (FFPE) ACC tissue sections (2-4 um) mounted on slides were subjected to RNA extraction with the AllPrep DNA/RNA FFPE Kit (Qiagen). RNA quan- tity and quality were assessed by NanoDrop2000 (Thermo Fisher Scientific). Samples that met predefined quality stand- ards were used for quantification of gene expression with NanoString nCounter. A customised panel (NanoString Technologies) consisting of 473 immune-related genes plus 11 reference genes was used according to the manufacturer’s instructions. Analysis was performed by nSolver Analysis Software 4.0 (NanoString Technologies). For samples that passed imaging quality controls (percentage of fields of view (FOV) read > 75%, binding density 0.05-2.25, positive con- trol linearity > 0.95, and positive control limit of detection >2), raw gene counts were normalised to technical controls and housekeeping genes after positive control normalisation (geometric mean as normalisation factor). Normalised counts were log2 transformed and expression compared between
| GDF-15 low | GDF-15-high | |
|---|---|---|
| Patients-n | 30 | 28 |
| Age at diagnosis in years: median (range) | 50 (18-74) | 46 (20-77) |
| Sex-n (%) | ||
| Female | 21 (70) | 16 (57) |
| Male | 9 (30) | 12 (43) |
| ENSAT stage-n (%) | ||
| I | 2 (8) | 0 (0) |
| II | 9 (35) | 9 (35) |
| III | 5 (19) | 8 (30) |
| IV | 10 (38) | 9 (35) |
| R status-n {ENSAT I-III} (%) | ||
| 0 | 18 (95) | 10 (59) |
| X | 0 (0) | 3 (18) |
| 1 | 1 (5) | 4 (23) |
| 2 | 0 (0) | 0 (0) |
| Ki-67 index-n (%) | ||
| <10% | 9 (32) | 3 (12) |
| ≥10% | 19 (68) | 22 (88) |
| Endocrine activity-n (%) | ||
| Hormone secretion | 23 (77) | 21 (75) |
| No hypersecretion | 7 (23) | 7 (25) |
| Sum of tumour diameter (cm): median (range) (n =28) (n =26) | 9 (4.5-18) | 10 (3-22) |
GDF-15 high and low groups were defined as below or above median GDF-15 mRNA expression.
groups of below and above median GDF-15 mRNA expres- sion. For each comparison, the P value (1-tailed Student t-test) and false discovery rate (FDR) obtained by the Benjamini-Yekutieli method were calculated. Finally, genes were ranked on the basis of fold change (FC) and FDR setting (FC≥2 and FDR<0.1). Bioinformatic analyses on gene-expression profiling were conducted by R Software v3.3.2. Clinical data of this tissue cohort are shown in Table 3. For GSEA pathway analysis, the WebGestalt analysis platform and the Reactome gene set were used (May, 16, 2024).
Results
Serum GDF-15 in ACC vs benign adrenal tumours
Tumoural GDF-15 secretion is a hallmark of tumour aggres- siveness in many tumours. Physiologically, serum GDF-15 lev- els are below 1.035 ng/mL.29 We found a trend for circulating GDF-15 to be lower in 42 patients with benign tumours (ACA) compared to 95 mitotane-naïve patients with ACC, yet within the physiological range of GDF-15 (0.65; range: 0.13-5.62 vs 0.81; range: 0.01-35.05 ng/ml, P =. 1, Figure 1A). Patients with localised ENSAT stage I-III ACC had significantly lower GDF-15 serum concentrations com- pared to advanced ENSAT stage IV (0.6; range: 0.01-30.61; 1.65; range: 0.38-35.05; P <. 0001; Figure 1B). For a sub- group of ACC patients (n =27), the sum of tumoural diam- eters as a measure of tumour burden was available at the time point of blood sampling. Circulating GDF-15 concen- trations correlated significantly with larger tumour mass (r = 0.44, Figure 1C). Age in ACA and ACC did not differ (ACA: 54.5 years vs ACC: 53.5 years, P = . 52, Figure S1A) and serum GDF-15 in mitotane-naïve patients with ACC did not differ significantly between age groups either (cut-off:
median 53 years; 0.73; range: 0.19-30.61 vs 0.90; range: 0.01-35.05 ng/L; P = . 34; Figure S1B).
Steroid hormone synthesis involves discrete mitochondrial steps and GDF-15 secretion has been reported to be induced by mitochondrial stress. In mitotane-naïve ACC patients with hormonal data available at the time of diagnosis (n = 64), we found patients with hormonally active ACC to have significantly higher mean GDF-15 serum levels compared to patients with inactive ACC (3.1±6.8 vs 0.9±0.6 ng/ml; P <. 05, Figure S1C). In ACA, we did not find significant high- er mean GDF-15 serum levels in cortisol-producing adenomas compared to endocrine inactive adenomas with 1.2 ±1.3 vs 0.7±0.3 ng/ml; (P =. 31, Figure S1D).
Mitotane induces GDF-15 secretion in vitro and in vivo
Next, we demonstrated a dose-dependent induction of GDF-15 expression in 4 ACC cell lines by mitotane treatment but not by other cytotoxic drugs (Supplementary Results and Figure S2).
Then we measured GDF-15 in ACC patients treated with mitotane (see Table S1 for clinical characteristics). We ex- cluded patients with adjuvant mitotane and no tumour mass present at the time point of blood collection. Circulating GDF-15 levels during mitotane therapy (n = 33) were signifi- cantly higher compared to samples collected before initiation of mitotane treatment (n =25, 2.0; range: 0.50-5.78 vs 0.6; range: 0.16-1.8 ng/ml; P <. 0001, Figure 1D). We additional- ly analysed 40 paired samples of patients before and during mitotane therapy (Figure 1E) and observed an increase in GDF-15 serum levels in almost all samples investigated (prior mitotane treatment 0.66; range: 0.1-13.05 vs during mitotane treatment 2.3; range: 0.50-19.97 ng/ml, P <. 0001). Mitotane plasma concentrations are known to vary widely, and some pa- tients never reach therapeutically effective blood concentrations. In line with a dose-dependent effect of mitotane on GDF-15, mi- totane plasma concentration and serum GDF-15 showed a strong positive correlation (r =0.58, P <. 005, Figure 1F). In addition, we analysed GDF-15 serum concentrations in ACC patients with adjuvant disease, before and during mitotane treat- ment. Prior mitotane treatment, GDF-15 levels were comparable to healthy controls in ACC patients (0.60 vs 0.61 ng/ml, respect- ively), yet increased significantly during mitotane treatment (2.0 ng/ml, P <. 0001, Figure S3).
High GDF-15 levels are associated with reduced overall survival in ACC patients
GDF-15 is known to be up-regulated in different malignancies and high expression is associated with more unfavourable out- comes.30,31 We therefore investigated the association of GDF-15 serum concentrations with survival in patients with ACC from the time when the blood sample was taken. To dis- criminate the actual impact of mitotane induced vs basal GDF-15 secretion, we analysed mitotane-naïve patients and patients treated with mitotane separately. Mitotane-naïve pa- tients with basal serum GDF-15 below the median of 0.81 ng/ mL had a significantly longer survival of 63 months compared to patients with high levels of GDF-15 (21 months, P <. 001; Figure 2A). As we observed higher levels of GDF-15 in higher ENSAT stages, we stratified for ENSAT stage (ENSAT stage I-III vs ENSAT stage IV), in a sensitivity analysis and again ob- served a significant longer survival in patients with serum GDF-15 below the median before mitotane therapy (stratified
A
ns
B
C
40
:
40
60
p=0.016
serum GDF-15 [ng/ml]
30
20
serum GDF-15 [ng/ml]
30
20
tumor diameter [cm]
15
10
40
10
5-
20
5
0
0
0
ACA (n=42)
ACC (n=95)
I-III
IV (n=26)
0
5
10
15
(n=72)
ENSAT stage (prior mitotane)
serum GDF-15 [ng/ml]
D
E
F
8
15
40
serum GDF-15 [ng/ml]
p=0.0005
6
?
serum GDF-15 [ng/ml]
10
plasma mitotane [mg/l]
4
5
4
3
30-
3
2
20
2
.
1
10-
1
0
n=40
0
during
0
pre
mitotane mitotane (n=33) (n=25)
pre mitotane
during mitotane
0
5
10
15
serum GDF-15 [ng/ml]
A
B
1.0
GDF-15 low
1.0
survival from blood sample
GDF-15 high
survival from blood sample
GDF-15 low GDF-15 high
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
n=91
n=48 p=0.008
p<0.001
0.0
0.0
0
20
40
60
80
100
120
0
20
40
60
80
100
time [months]
time [months]
no. at risk
no. at risk
GDF-15 low
45
41
30
20
10
4
1
GDF-15 low
24
20
12
6
1
GDF-15 high
46
24
15
7
3
1
1
GDF-15 high
24
8
6
3
P =. 049) (Figure S4A, B). In a univariate analysis, tumour burden (adjuvant, 0 affected sites; localised disease, 1 affected site; and metastatic disease, >1 affected site) and Ki-67 index were significantly associated with survival prior to mitotane treatment in line with their known prognostic role in ACC (Table 4). After adjustment for both factors in the group prior mitotane GDF-15 did not retain statistically significant with overall survival (HR: 1.47; 95% CI: 0.66-3.29; P =. 34)
(Table 4). Additionally, when only patients with tumour mass present were analysed, we did no longer find significant differences in overall survival in 34 patients before mitotane treatment (log-rank: P = . 288), Figure S5A.
Patients with ACC who received mitotane and had GDF-15 serum concentrations below the median of 2.17 ng/ml sur- vived for a median time of 55 months. Median survival of pa- tients with high GDF-15 serum concentrations was only
| Pre-mitotane | ||||||
|---|---|---|---|---|---|---|
| Survival | Univariate analysis | Multivariate analysis | ||||
| Variables | HR | 95% CI | P | HR | 95% CI | P |
| Sex | ||||||
| Female (n= 58) | ||||||
| Male (n=41) | 0.64 | 0.36-1.13 | 0.13 | |||
| Affected tumour sites | ||||||
| 0 | ||||||
| 1 | ||||||
| >1 | 2.39 | 1.66-3.45 | <0.001* | 2.29 | 1.48-3.55 | <. 001* |
| Ki67 | ||||||
| <10 (n=17) | ||||||
| ≥10 (n=52) | 3.33 | 1.29-8.59 | 0.013* | 3.56 | 1.17-10.84 | .03* |
| Age | ||||||
| <50 years (n = 35) | 0.99-3.34 | |||||
| >50 years (n = 59) | 1.82 | 0.06 | ||||
| GDF-15 | ||||||
| Low <0.81 ng/ml (n=45) | 0.66-3.29 | |||||
| High > 0.81 ng/ml (n=46) | 3.05 | 1.73-5.40 | <0.001* | 1.47 | .34 | |
Only significant parameters at univariate analysis were adjusted at multivariable analysis.
*P values are stated and significant P values additionally emphasised.
13 months (P =. 008; Figure 2B). When stratified for ENSAT stage, we observed a significant longer survival in patients with serum GDF-15 below the median during mitotane ther- apy (stratified P = . 047) (Figure S4C, D).
Due to non-satisfied hazard assumption, we did not perform hazard regression and multivariate analysis in patients during mitotane treatment. Yet, also here, log-rank test revealed a sig- nificant longer survival in patients with serum GDF-15 below the median (P =. 008). When only patients with tumour mass present were analysed, we did no longer find significant differ- ences in overall survival in 23 patients during mitotane treat- ment (log-rank: P = . 08), Figure S5B.
Serum GDF-15 and response to immunotherapy
GDF-15 is an endogenous immunosuppressant18 and leads to impaired T cell extravasation by inhibiting the lymphocyte function-associated antigen 1/intercellular adhesion molecule 1 (LFA-1/ICAM-1) axis in T cells.23 It is well established that ICI efficacy depends on T cells infiltrating the tumour which prompted us to assess GDF-15 in ACC patients prior to treatment with ICI.
In the 46 ACC patients (Table 2) with serum samples avail- able, GDF-15 serum concentrations ranged from 0.61 to 76.8 ng/mL and were significantly higher than in age- and sex- matched healthy controls (0.4 to 2.9 ng/ml). Median serum GDF-15 was 2.5; range: 0.64-12.99 ng/ml in responders vs 4.7; range: 0.61-76.80 ng/mL in non-responders (P =. 037; Figure 3A). Responders to immunotherapy had a significantly longer progression-free survival, with all responders being still alive at manuscript submission (P <. 001; Figure 3B). Furthermore, patients with GDF-15 serum levels below the median of 4.2 ng/ml at the start of ICI showed a significant longer progression-free survival compared to patients with high GDF-15 serum levels (P =. 036) (Figure 3C). We calcu- lated the AUC to discriminate between responders and non- responders and found an AUC of 0.69; P =. 038 (Figure S6A). For mitotane plasma concentration, we found no significant value to discriminate responders and non- responders to ICI (AUC: 0.58, P =. 40, Figure S6B). In this
cohort, serum GDF-15 and tumour burden at imaging were not significantly intercorrelated (Figure S7A) but again mito- tane plasma concentration significantly correlated with serum GDF-15 (Figure S7B).
In line with an immunosuppressive action of GDF-15 in the tumour immune micro-environment, we found a trend for lower expression of RNA encoding immune-related genes, such as CD3E, HLA-DRB1 and CXCL9 in 58 ACC tissue sam- ples from treatment-naïve patients with high vs low expression of GDF-15 and GSEA pathway analysis revealed a significant downregulation of immune-related pathways in GDF-15 high samples (Figure 3D; Table S2).
Discussion
GDF-15 is an endogenous immunosuppressive cytokine up- regulated in different tumour entities. It affects intratumoural immune cell infiltration. GDF-15 therefore reduces response to immune checkpoint blockade.23 Most ACC are immuno- logically cold with minimal infiltration by lymphocytes and NK cells15,16 and response to ICI is infrequent.8-13
Our study demonstrates that GDF-15 is elevated in serum of patients with ACC. We show that GDF-15 serum concentra- tions are significantly lower in patients who benefit from pro- grammed cell death 1 (PD-1)-directed immunotherapy. We therefore suggest GDF-15 as a parameter worthwhile to inves- tigate further for its value to inform selection of patients for anti-PD-1 immunotherapy. GDF-15 is readily measurable by a commercially available assay.2
Serum GDF-15 generally increases with tumour mass and is associated with poor prognosis in several cancer types.18 In ACC, there are hardly any tumour markers for use in clinical routine. Current guidelines recommend tumour stage and tis- sue Ki-67 labelling index to inform treatment decisions. 4,32,33 Molecular tissue markers of prognosis have been developed but have not changed clinical practice yet.34,
We here found no significant difference in serum GDF-15 between ACC without prior to treatment with mitotane vs ACA. We did not observe higher serum GDF-15 in steroid
A
B
1.0
#
p=0.0379
overall survival from ICI start
responder
non-responder
128
0.8
p<0.001
64
log2 GDF-15 serum [ng/I]
32
0.6
16
0.4
8
0.2
4
2
0.0
1
0
10
20
30
40
no. at risk
time [months]
0.5
0.25
responder 14
12
9
5
7
responder (n=14)
non- responder (n=32)
healthy control (n=46)
non-responder 32
D
FDR ≤ 0.05
FDR > 0.05
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
2.0
C
PI3K/AKT Signaling in Cancer Metabolism of carbohydrates
1.0
GDF-15 low GDF-15 high
* Metabolism
Cell surface interactions at the vascular wall
progression-free survival
Metabolism of proteins
0.8
Metabolism of lipids
Cellular responses to external stimuli
Growth hormone receptor signaling
p=0.036
Cellular responses to stress
Post-translational protein modification
0.6
Regulation of TP53 Activity
PPARA activates gene expression
Regulation of lipid metabolism by Peroxisome proliferator-activated receptor alpha (PP …
Circadian Clock
0.4
GPCR downstream signaling
TCR signaling
Downstream TCR signaling
Antigen processing-Cross presentation
G alpha (1) signalling events
0.2
Signaling by GPCR
ER-Phagosome pathway
Infectious disease
Cytokine Signaling in Immune system *
0.0
Neutrophil degranulation *
Interferon Signaling ·
0
5
10
15
20
25
30
Adaptive Immune System *
Innate Immune System *
time [months]
Peptide ligand-binding receptors .
Interleukin-10 signaling *
no. at risk
Class A/1 (Rhodopsin-like receptors) .
Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell *
GDF-15 low
23
7
5
4
3
1
GPCR ligand binding *
GDF-15 high
Interferon alpha/beta signaling *
23
4
2
2
1
Immune System *
-2.0
-1.5
-1.0
-0.5
Normalized Enrichment Score
0.0
0.5
1.0
1.5
2.0
hormone-producing vs endocrine inactive ACA which might be due to the even smaller sample size of ACA.
In treatment-naïve patients with adrenal tumours, serum GDF-15 might appear to depend on tumour mass, which is usually higher in stage IV vs localised tumour stages. This is possibly the reason why GDF-15 does not retain statistical sig- nificance after adjustment for tumour burden. It is conceivable that the involvement of mitochondrial steroidogenic enzymes that are active even in cases without clinical hormone excess36 leads to mitochondrial stress resulting in tumoural induction of GDF-15 in ACC. This is likely the reason why GDF-15 se- rum levels also increase in patients with no measurable tumour mass. Systemically, glucocorticoids suppress GDF-15 secre- tion as shown in patients with adrenal insufficiency,37 which argue in favour of a tumoural origin of circulating GDF-15 in ACC patients.
Mitotane is the treatment standard for advanced ACC alone or in combination with chemotherapy. It is useful as an inhibi- tor of steroidogenesis and strongly perturbs mitochondrial in- tegrity. At the molecular level, the GDF-15 promoter has been shown to comprise binding sequences for several transcription factors including C/EBP homologous protein (CHOP)38 that in turn is strongly induced by mitotane.27 Across all ACC
cell lines tested, a striking induction of GDF-15 by mitotane but not by other cytotoxic chemotherapy drugs confirmed our previous observation (Figure S2B).
Mitotane-dependent tumoural induction of GDF-15 was re- flected in serum of ACC patients treated with mitotane. The significant correlation of serum GDF-15 and plasma mitotane supports a direct effect of mitotane.
Given the known activity of GDF-15 in favouring immune cell exclusion23 and in inducing regulatory Treg cells,22 patients with high GDF-15 likely have an immunologically un- favourable tumour micro-environment which may contribute to their poor outcome. For ACC it is known that metastases exhibit lower T lymphocyte infiltration than the correspond- ing primary tumours.15 Standard mitotane therapy preceding resection of metastases may contribute to this immunological- ly cold tumour phenotype in ACC metastases and thus to the overall low response rate to PD-1 inhibition. In our ACC co- hort of 46 patients who received ICI, responders had signifi- cantly lower GDF-15 blood levels. This has previously been shown in non-small cell lung cancer patients, where patients with low serum GDF-15 had significantly higher objective re- sponse rates to anti-PD-1 and its ligand (PD-L1) therapy than patients with high serum GDF-15.17 Adverse immunological
effects of GDF-15 induction in ACC are further supported by lower expression of immune cell pathways in primary ACC tissues tumours with higher GDF-15 expression. For pathway analysis, the Nanostring nCounter panel of pre-selected genes and the limited number of available samples did not allow for a sufficiently powered analysis to statistically exclude possible false positives. Still, of 13 pathways with uncorrected P values of <. 05, 10 are related to immune regulation or tumour- immune interaction, and 1 to metabolic effects known to be associated with GDF-15. As such a result could hardly be ob- tained through accidental false positive discoveries, these data further support an immunomodulatory role of GDF-15 in ACC.
Our study has some limitations. First, all samples were retrospectively analysed from prospectively collected biologic- al specimens. Prospective studies are required for confirmation but will require long time periods to be completed in this orphan malignancy. Second, tissue samples were not available to directly study the impact of prior mitotane treatment on the tissue im- mune cell infiltration. In tissues post-mitotane treatment, we would have expected higher GDF-15 expression and likely great- er differences in the expression of immune-related genes. Finally, only a small number of serum samples and data from patients treated outside of clinical trials with PD-1 inhibition were avail- able and follow-up for some of these patients was short.
A strength of the study is the application of a validated and routinely available serum GDF-15 assay, and the high number of patients with sufficient follow-up available for the assess- ment of overall survival.
Beyond the validation of GDF-15 as biomarker in ACC, our study has important implications for the treatment of ACC. Although clinical trials have not found significant negative ef- fects of mitotane on response rates to ICI, numbers of included patients were low.8-11,13 Our finding of mitotane-induced GDF-15 in ACC raises the possibility that continuation of mi- totane beyond progression may affect later therapy with ICI. While we found high serum GDF-15 to be associated with un- responsiveness to ICI, it is noteworthy that plasma mitotane concentration is not significantly associated with treatment outcome of ICI (Figure S6B). To disentangle the relevance of mitotane and serum GDF-15, a clinical trial is needed. However, studies in hepatocellular carcinoma bearing mice have demonstrated that anti-GDF-15 treatment could reduce the number of tumoural Treg cells in combination with PD-1 in- hibition.22 Similarly, anti-GDF-15 and anti-PD-1 therapy re- duced the tumour burden and expression of FoxP3, a marker of Tregs, in a murine pancreatic cancer model while expression of granzyme B from activated cytotoxic T cells increased, indi- cating a more inflamed tumour micro-environment.23 A study supporting the idea of clinically targeting GDF-15 to overcome resistance to ICI was very recently published. The authors com- bined the GDF-15 neutralising antibody visugromab with the anti-PD-1 antibody nivolumab and could demonstrate response to immune checkpoint inhibition in several solid tumour entities previously refractory to ICI.39
Targeting GDF-15 in mitotane-treated ACC may therefore enhance the efficacy of ICI treatment in ACC, similar to effects recently demonstrated in other malignant tumours.39
Conclusions
In conclusion, GDF-15 is a tumour-secreted anti-inflammatory cytokine released from ACC after mitotane treatment and is
associated with poor prognosis. Serum GDF-15 may be used as a marker to select patients likely to benefit from immuno- therapy. Most importantly, GDF-15 is a druggable target in combination, with immunotherapy of mitotane-treated ACC.
Supplementary material
Supplementary material is available at European Journal of Endocrinology online.
Funding
This research was supported by the German Research Foundation (project number 314061271), the BMBF 01KT2310 TRANSCAN-3 (ERA-NET: Sustained collaboration of national and regional programmes in cancer research-JTC 2021) iParaCyts to J.W. This work has been carried out with the help of the Interdisciplinary Bank of Biomaterials and Data of the University Hospital of Würzburg and the Julius- Maximilians-University of Würzburg (IBDW), supported by the Federal Ministry for Education and Research (grant number FKZ: 01EY1102).
Authors’ contributions
Isabel Weigand (Formal analysis [lead], Methodology [equal], Validation [equal], Visualization [lead], Writing-original draft [lead]), Alexandra S. Triebig (Methodology [equal], Writing-review & editing [equal]), Tanja Maier (Formal analysis [supporting], Methodology [equal], Visualization [supporting], Writing-review & editing [supporting]), Tanja Anderlik (Methodology [supporting]), Hanna Remde (Resources [supporting], Writing-review & editing [support- ing]), Laura-Sophie Landwehr (Investigation [supporting], Resources [supporting], Writing-review & editing [equal]), Otilia Kimpel (Resources [equal], Writing-review & editing [supporting]), Miriam Reuter (Resources [equal], Writing- review & editing [supporting]), Jochen Schreiner (Resources [equal], Writing-review & editing [supporting]), Florian Wedekink (Methodology [equal], Writing-review & editing [supporting]), Oliver Scherf-Clavel (Methodology [equal], Resources [equal], Writing-review & editing [supporting]), Eva Hoster (Formal analysis [supporting], Validation [sup- porting], Writing-review & editing [supporting]), Kai C. Wollert (Resources [equal], Writing-review & editing [supporting]), Ilka Budde (Resources [equal]), Barbara Altieri (Resources [supporting]), Paul Schwarzlmueller (Formal analysis [supporting], Resources [supporting], Writing-review & editing [supporting]), Martin Reincke (Resources [supporting], Writing-review & editing [support- ing]), Jörg Wischhusen (Formal analysis [supporting], Investigation [supporting], Methodology [supporting], Supervision [supporting], Writing-review & editing [sup- porting]), Martin Fassnacht (Project administration [support- ing], Resources [supporting], Writing-original draft [supporting]), and Matthias Kroiss (Conceptualization [lead], Formal analysis [supporting], Funding acquisition [lead], Investigation [equal], Methodology [supporting], Resources [equal], Supervision [lead], Visualization [support- ing], Writing-original draft [supporting], Writing-review & editing [supporting])
Conflict of interest: J.W. is a collaborator of CatalYm, a company developing a therapeutic GDF-15 antibody and
co-inventor of patents held by the University of Würzburg on anti-GDF-15 based therapies. J.W. and M.K. received travel support from CatalYm. K.C.W. is a collaborator of Roche Diagnostics and holds the following planned patents: EP 2 047 275 B1; US 8,951,742 B2, US 11,199,552 B2.
References
1. Else T, Kim AC, Sabolch A, et al. Adrenocortical carcinoma. Endocr Rev. 2014;35(2):282-326. https://doi.org/10.1210/er. 2013-1029
2. Fassnacht M, Libé R, Kroiss M, Allolio B. Adrenocortical carcin- oma: a clinician’s update. Nat Rev Endocrinol. 2011;7(6): 323-335. https://doi.org/10.1038/nrendo.2010.235
3. Terzolo M, Angeli A, Fassnacht M, et al. Adjuvant mitotane treat- ment for adrenocortical carcinoma. N Engl J Med. 2007;356(23): 2372-2380. https://doi.org/10.1056/NEJMoa063360
4. Fassnacht M, Assie G, Baudin E, et al. Adrenocortical carcinomas and malignant phaeochromocytomas: ESMO-EURACAN Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol. 2020;31(11):1476-1490. https://doi.org/10.1016/j.annonc. 2020.08.2099
5. Terzolo M, Fassnacht M, Perotti P, et al. Adjuvant mitotane versus surveillance in low-grade, localised adrenocortical carcinoma (ADIUVO): an international, multicentre, open-label, randomised, phase 3 trial and observational study. Lancet Diabetes Endocrinol. 2023;11(10):720-730. https://doi.org/10.1016/S2213-8587(23)00 193-6
6. Megerle F, Herrmann W, Schloetelburg W, et al. Mitotane mono- therapy in patients with advanced adrenocortical carcinoma. J Clin Endocrinol Metab. 2018;103(4):1686-1695. https://doi.org/ 10.1210/jc.2017-02591
7. Fassnacht M, Terzolo M, Allolio B, et al. Combination chemother- apy in advanced adrenocortical carcinoma. N Engl J Med. 2012;366(23):2189-2197. https://doi.org/10.1056/NEJMoa1200966
8. Le Tourneau C, Hoimes C, Zarwan C, et al. Avelumab in patients with previously treated metastatic adrenocortical carcinoma: phase 1b results from the JAVELIN solid tumor trial. J Immunother Cancer. 2018;6(1):111. https://doi.org/10.1186/s40425-018-0424-9
9. Carneiro BA, Konda B, Costa RB, et al. Nivolumab in metastatic adrenocortical carcinoma: results of a phase 2 trial. J Clin Endocrinol Metab. 2019;104(12):6193-6200. https://doi.org/10. 1210/jc.2019-00600
10. Raj N, Zheng Y, Kelly V, et al. PD-1 blockade in advanced adreno- cortical carcinoma. J Clin Oncol. 2020;38(1):71-80. https://doi.org/ 10.1200/JCO.19.01586
11. Habra MA, Stephen B, Campbell M, et al. Phase II clinical trial of pembrolizumab efficacy and safety in advanced adrenocortical car- cinoma. J Immunother Cancer. 2019;7(1):253. https://doi.org/10. 1186/s40425-019-0722-x
12. Klein O, Senko C, Carlino MS, et al. Combination immunotherapy with ipilimumab and nivolumab in patients with advanced adreno- cortical carcinoma: a subgroup analysis of CA209-538. Oncoimmunology. 2021;10(1):1908771. https://doi.org/10.1080/ 2162402X.2021.1908771
13. Remde H, Schmidt-Pennington L, Reuter M, et al. Outcome of im- munotherapy in adrenocortical carcinoma: a retrospective cohort study. Eur J Endocrinol. 2023;188(6):485-493. https://doi.org/10. 1093/ejendo/lvad054
14. Ababneh O, Ghazou A, Alawajneh M, et al. The efficacy and safety of immune checkpoint inhibitors in adrenocortical carcinoma: a systematic review and meta-analysis. Cancers (Basel). 2024;16(5): 900. doi:10.3390/cancers16050900
15. Landwehr LS, Altieri B, Schreiner J, et al. Interplay between gluco- corticoids and tumor-infiltrating lymphocytes on the prognosis of adrenocortical carcinoma. J Immunother Cancer. 2020;8(1): e000469. https://doi.org/10.1136/jitc-2019-000469
16. Greenstein AE, Habra MA, Wadekar SA, Grauer A. Adrenal tumors provide insight into the role of cortisol in NK cell activity. Endocr
Relat Cancer. 2021;28(8):583-592. https://doi.org/10.1530/ERC- 21-0048
17. Hong G, Sun P, Chung C, et al. Plasma GDF15 levels associated with circulating immune cells predict the efficacy of PD-1/PD-L1 in- hibitor treatment and prognosis in patients with advanced non- small cell lung cancer. J Cancer Res Clin Oncol. 2023;149(1): 159-171. https://doi.org/10.1007/s00432-022-04500-5
18. Wischhusen J, Melero I, Fridman WH. Growth/differentiation factor-15 (GDF-15): from biomarker to novel targetable immune checkpoint. Front Immunol. 2020;11:951. https://doi.org/10. 3389/fimmu.2020.00951
19. Hsu JY, Crawley S, Chen M, et al. Non-homeostatic body weight regulation through a brainstem-restricted receptor for GDF15. Nature. 2017;550(7675):255-259. https://doi.org/10.1038/nature 24042
20. Johnen H, Lin S, Kuffner T, et al. Tumor-induced anorexia and weight loss are mediated by the TGF-beta superfamily cytokine MIC-1. Nat Med. 2007;13(11):1333-1340. https://doi.org/10. 1038/nm1677
21. Fejzo M, Rocha N, Cimino I, et al. GDF15 linked to maternal risk of nausea and vomiting during pregnancy. Nature. 2024;625(7996): 760-767. https://doi.org/10.1038/s41586-023-06921-9
22. Wang Z, He L, Li W, et al. GDF15 induces immunosuppression via CD48 on regulatory T cells in hepatocellular carcinoma. J Immunother Cancer. 2021;9(9):e002787. https://doi.org/10. 1136/jitc-2021-002787
23. Haake M, Haack B, Schafer T, et al. Tumor-derived GDF-15 blocks LFA-1 dependent T cell recruitment and suppresses responses to anti-PD-1 treatment. Nat Commun. 2023;14(1):4253. https://doi. org/10.1038/s41467-023-39817-3
24. Sescot S, Slama A, Lombes A, et al. Mitotane alters mitochondrial respiratory chain activity by inducing cytochrome c oxidase defect in human adrenocortical cells. Endocr Relat Cancer. 2013;20(3): 371-381. https://doi.org/10.1530/ERC-12-0368
25. Poli G, Guasti D, Rapizzi E, et al. Morphofunctional effects of mi- totane on mitochondria in human adrenocortical cancer cells. Endocr Relat Cancer. 2013;20(4):537-550. https://doi.org/10. 1530/ERC-13-0150
26. Montero R, Yubero D, Villarroya J, et al. GDF-15 is elevated in chil- dren with mitochondrial diseases and is induced by mitochondrial dysfunction. PLoS One. 2016;11(2):e0148709. https://doi.org/10. 1371/journal.pone.0148709
27. Sbiera S, Leich E, Liebisch G, et al. Mitotane inhibits sterol-O-acyl transferase 1 triggering lipid-mediated endoplasmic reticulum stress and apoptosis in adrenocortical carcinoma cells. Endocrinology. 2015;156(11):3895-3908. https://doi.org/10.1210/en.2015-1367
28. Eisenhauer EA, Therasse P, Bogaerts J, et al. New response evalu- ation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer. 2009;45(2):228-247. https://doi.org/10.1016/j. ejca.2008.10.026
29. Wollert KC, Kempf T, Giannitsis E, et al. An automated assay for growth differentiation factor 15. J Appl Lab Med. 2017;1(5): 510-521. https://doi.org/10.1373/jalm.2016.022376
30. Weide B, Schafer T, Martens A, et al. High GDF-15 serum levels in- dependently correlate with poorer overall survival of patients with tumor-free stage III and unresectable stage IV melanoma. J Invest Dermatol. 2016;136(12):2444-2452. https://doi.org/10.1016/j.jid. 2016.07.016
31. Vocka M, Langer D, Fryba V, et al. Growth/differentiation factor 15 (GDF-15) as new potential serum marker in patients with meta- static colorectal cancer. Cancer Biomark. 2018;21(4):869-874. https://doi.org/10.3233/CBM-170792
32. Beuschlein F, Weigel J, Saeger W, et al. Major prognostic role of Ki67 in localized adrenocortical carcinoma after complete resec- tion. J Clin Endocrinol Metab. 2015;100(3):841-849. https://doi. org/10.1210/jc.2014-3182
33. Libe R, Borget I, Ronchi CL, et al. Prognostic factors in stage III-IV adrenocortical carcinomas (ACC): an European network for the
study of adrenal tumor (ENSAT) study. Ann Oncol. 2015;26(10): 2119-2125. https://doi.org/10.1093/annonc/mdv329
34. Assie G, Jouinot A, Fassnacht M, et al. Value of molecular classifi- cation for prognostic assessment of adrenocortical carcinoma. JAMA Oncol. 2019;5(10):1440-1447. https://doi.org/10.1001/ jamaoncol.2019.1558
35. Assie G, Letouze E, Fassnacht M, et al. Integrated genomic charac- terization of adrenocortical carcinoma. Nat Genet. 2014;46(6): 607-612. https://doi.org/10.1038/ng.2953
36. Arlt W, Biehl M, Taylor AE, et al. Urine steroid metabolomics as a biomarker tool for detecting malignancy in adrenal tumors. J Clin Endocrinol Metab. 2011;96(12):3775-3784. https://doi.org/10. 1210/jc.2011-1565
37. Melvin A, Chantzichristos D, Kyle CJ, et al. GDF15 is elevated in conditions of glucocorticoid deficiency and is modulated by gluco- corticoid replacement. J Clin Endocrinol Metab. 2020;105(5): 1427-1434. https://doi.org/10.1210/clinem/dgz277
38. Baek SJ, Wilson LC, Eling TE. Resveratrol enhances the expression of non-steroidal anti-inflammatory drug-activated gene (NAG-1) by increasing the expression of p53. Carcinogenesis. 2002;23(3): 425-434. https://doi.org/10.1093/carcin/23.3.425
39. Melero I, de Miguel Luken M, de Velasco G, et al. Neutralizing GDF-15 can overcome anti-PD-1 and anti-PD-L1 resistance in solid tumours. Nature. 2025;637(8048):1218-1227. https://doi.org/10. 1038/s41586-024-08305-z