BRIEF REPORT
Check for updates
Hepcidin concentration and effect on iron homeostasis in patients with adrenocortical carcinoma and benign adrenal lesions - a pilot study
Aleksandra Krygier1D . Dorota Filipowicz1D . Barbara Brominska1D . Hanna Komarowska1D. Ewelina Szczepanek-Parulska1(D . Marek Ruchala1D
Received: 11 August 2025 / Accepted: 13 November 2025 @ The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2025
Abstract
Purpose The evaluation of hepcidin concentration and iron (Fe) homeostasis parameters in patients with: adrenocortical carcinoma (ACC) to compared to control subjects (CS) and in ACC patients before surgical or pharmacological treatment (ACC-nt) to ACC patients during mitotane therapy (ACC-t).
Methods We enrolled 33 adult patients with histopathologically confirmed non-adenoma of adrenal gland. Each patient underwent hepcidin, Fe metabolism parameters and biochemical evaluation. Hepcidin levels were measured using the Hep- cidin 25 (bioactive) hs ELISA, a highly sensitive enzyme immunoassay designed for the quantitative in vitro diagnostic determination (DRG Instruments GmbH, Germany). The study group was consisted of: 13 patients (10 females) with ACC following adrenal surgery undergoing mitotane therapy (ACC-t), 9 patients (6 females) with ACC who had not received prior pharmacological or surgical treatment (ACC-nt) and 11 patients (6 females) with histologically benign adrenal tumors who underwent subsequent surgery (CS).
Results The patients with ACC present higher median values of hepcidin compared to CS: [13.9 (7.6-24.3) vs. 12.3 (1.5- 20.9) ng/ml, p>0.052], while in ACC-nt group was lower than in ACC-t [11.1 (8.3-24.3) vs. 14.9 (7.6-24.3) ng/ml, p=0.053]. We noticed statistically significant differences between ACC and CS group: RBC-m, HGB, MCH, MCHC, Fe-m concentration and correlations between: hepcidinAcc vs. ferritin-male Acc, hepcidinAcc VS. ferritin-female ACC, hepcidin ACC-nt vs. ferritin-male ACC-nt, hepcidin ACC-nt VS. ferritin-female ACC-nt, hepcidincs vs. ferritin-femalecs and hepcidincs vs. MCV cs- Conclusions In patients with ACC the neoplastic process increases hepcidin level, which leads to decreased hematological parameters, while treatment improve Fe homeostasis.
Keywords Hepcidin . Adrenocortical carcinoma . Iron . Ferritin . Anemia . Adrenal
Introduction
Adrenocortical carcinoma (ACC) is a rare malignancy rep- resenting 0.4-4% of adrenal incidentalomas, affecting up to 12 people per million annually, with 2 new cases diag- nosed each year, predominantly in women aged 40-60 [1, 2]. ACCs often produce excessive glucocorticosteroids,
androgens, and rarely mineralocorticoids or estradiol, how- ever 40-50% are hormonally inactive and lack diagnostic biomarkers. Prognosis varies with tumour staging, Ki-67, mitotic rate, resection degree, and cortisol overproduc- tion. Overall survival is generally less than 4 years, ranging from 90% to below 15% in advanced disease over 5 years [3]. Patients with a high recurrence rate benefit from adju- vant mitotane therapy, followed by radiotherapy, cytotoxic drugs, or immunotherapy [4]. Mitotane exerts an adreno- lytic and anti-steroidogenic effect via mitochondrial dam- age. In addition to induction of adrenal insufficiency, its high toxicity frequently leads to liver dysfunction, neural damage, gonadal and thyrotropin axes dysregulation, altered
☒ Aleksandra Krygier akrygier@ump.edu.pl
1 Department of Endocrinology, Metabolism and Internal Medicine, Poznan University of Medical Sciences, Poznan, Poland
lipid metabolism, and disturbed myelopoiesis, causing side effects and poor tolerance in many patients [4].
Hepcidin, a liver-derived hormone, binds to the iron exporter ferroportin on enterocytes, hepatocytes, and macro- phages, triggering internalization and degradation, thereby reducing iron efflux into the plasma. During iron overload or inflammation, hepcidin is upregulated, limiting pathogen growth while promoting iron-restricted erythropoiesis. Con- versely, iron deficiency, anemia, hypoxia, and pregnancy suppress hepcidin, thereby enhancing erythropoietic and maternofetal iron supply [5]. Altered hepcidin metabolism is reported in hematologic diseases and other solid cancers. Moreover, decreased local ferroportin1 expression corre- lated with poor staging and prognosis of ACC [6], however serum hepcidin concentrations had not been studied. Hepci- din modulators are currently being tested in clinical trials.
To our knowledge, this is the first study assessing hep- cidin concentration in ACC patients. The study aims to evaluate hepcidin level in: ACC patients before surgery and mitotane treatment (ACC-nt), ACC patients during mitotane therapy (ACC-t), and all ACC patients compared to healthy control subjects (CS) with benign adrenal lesions, with an emphasis on hematologic parameters, liver and thyroid function.
Methods and results
We enrolled 33 adult patients with histopathologically con- firmed non-adenoma of adrenal gland. Each patient under- went evaluation of hepcidin, ferritin, Fe, complete blood count (CBC), creatinine, aminotransferases (ALT, AST), C-reactive protein (CRP), and thyroid-stimulating hormone (TSH). Hepcidin levels were measured using the Hepcidin 25 (bioactive) hs ELISA, a highly sensitive enzyme immu- noassay designed for the quantitative in vitro diagnostic determination (DRG Instruments GmbH, Germany).
The studied group consisted of: thirteen patients (10 females) with ACC following adrenal surgery undergoing mitotane therapy (ACC-t) and nine patients (6 females) with ACC who had not received prior pharmacological or surgical treatment (ACC-nt), in whom the diagnosis of ACC was later confirmed after surgery. TNM classification of ACC was between II-IV stage, and patients met criteria of mitotane treatment [3]. The CS consisted of 11 patients (6 females) with histologically benign adrenal tumours (6 oxyphilic tumours, three haemorrhagic cysts, one schwan- noma type B and one ganglioneuroma) who underwent sub- sequent surgery because of potential malignant image of computed tomography.
Strict exclusion criteria were applied across all groups: hemolysis, hemorrhage, inflammatory diseases, chronic
kidney or liver disease, pregnancy or breastfeeding, hemo- chromatosis, use of erythropoietin, and supplementation with Fe, vitamin B12, or folic acid up to three mounts enrolment.
Statistical analysis was conducted using STATISTICA software (StatSoft, Tulsa, Oklahoma, USA). Given the non- parametric nature of the data, results are presented as medi- ans with interquartile ranges (25th-75th percentile, IQR). The Mann-Whitney U test was used for comparisons among the ACC and CS groups. Spearman’s rank correlation coef- ficient was applied to assess relationships between hepcidin concentrations and the evaluated laboratory parameters [7]. For parameters with gender-dependent reference ranges; red blood cells (RBC), haemoglobin (HGB), hematocrit (HTC), Fe, and ferritin, analyses were conducted separately for males and females. Statistical significance was set at p < 0.05.
Comparisons of hepcidin levels and other parameters between patients with ACC and CS group are presented in Table 1. The median level of hepcidin in patients with ACC-nt is 11.1 (8.3-24.3) ng/ml and ACC-t is 14.9 (7.6- 24.3) ng/ml, p=0.053. A comparison of other parameters between ACC-t and ACC-nt groups is shown in Fig. 1. Ref- erence ranges and units are consistent with those presented in Table 1.
The following statistically significant correlations between hepcidin and other parameters were observed: hepcidinAcc Vs. ferritin-maleAcc (rho=0.900), hepcidi- nACC VS. ferritin-female Acc (rho=0.870), hepcidin ACC-nt VS. ferritin-male ACC-nt (rho=0.640), hepcidinACC-nt VS. ferritin- female ACC-nt (rho=0.860), hepcidincs vs. ferritin-femalecs (rho=0.900) and hepcidincs vs. MCVcs (rho =- 0.670).
Median levels of CRP, ALT, AST, creatinine, and TSH remained within reference ranges across in all groups.
Discussion
Recent studies have proposed a broader role for hepcidin in oncological context. Aberrant expression has been docu- mented in malignancies of the lung, pancreas, gastrointes- tinal tract, and breast, where elevated levels correlate with cancer-associated anemia [8-10]. In contrast, diminished hepcidin expression has been observed in hepatocellular carcinoma [11]. Both hypo- and hyper-hepcidinemia appear to be associated with adverse clinical outcomes, positioning hepcidin as a potential prognostic biomarker and therapeu- tic target [12].
ACC is a rare malignancy, and existing studies are lim- ited. The patient population is highly heterogeneous with respect to age, sex, disease stage, and treatment status. Moreover, the limited number of studies include patients at
| Parameter | Refer- ence range | Adreno- cortical carcinoma (n=22) | Control subjects (n=11) | p-value |
|---|---|---|---|---|
| Hepcidin -25 (ng/ | 13.9 | 12.3 | 0.052 | |
| mL) | (7.6-24.3) | (1.5-20.9) | ||
| *RBC - m (x10°/uL) | 3.2- | 4.4 (3.9-4.6) | 4.9 (4.8-5.6) | 0.001 |
| 5.4 | ||||
| *RBC - f (x10°/uL) | 3.5- | 4.1 (3.8-4.8) | 4.2 (4.0-4.7) | 0.074 |
| 5.2 | ||||
| *HGB- m (g/dL) | 14.0- | 12.4 | 15.3 | <0.001 |
| 18.0 | (11.2-14.0) | (13.1-15.6) | ||
| *HGB - f (g/dL) | 12.0 | 12.7 | 12.7 | 0.001 |
| -15.6 | (10.9-13.6) | (12.3-13.2) | ||
| *HTC - m(%) | 41-50 | 38 (35-42) | 46 (39-47) | 0.003 |
| *HTC-f (%) | 36-44 | 31 (34-42) | 39 (37-40) | 0.008 |
| MCV (fL) | 80-99 | 88 (85-90) | 85 (85-92) | 0.113 |
| MCH (pg) | 27-33 | 29 (27-30) | 28 (27-30) | 0.006 |
| MCHC (g/dL) | 31-38 | 33 (32-34) | 33 (32-33) | 0.002 |
| RDW-CV (%) | 11-16 | 15 (14-16) | 13 (13-14) | <0.001 |
| *Ferritin-m(ng/ml) | 30- | 169 | 330 | 0.921 |
| 400 | (131-273) | (225-348) | ||
| *Ferritin -f (ng/ml) | 20- | 112 (83-202) | 37 (25-179) | 0.640 |
| 200 | ||||
| *Fe -m (ug/dL) | 50- 158 | 77 (51-121) | 156 (67-169) | 0.031 |
| *Fe-f (ug/dL) | 37- | 79 (61-89) | 123 | 0.261 |
| 145 | (77-137) |
Values are expressed as median [IQR] for non-parametric tests (Mann-Whitney U-test)
RBC - red blood cells, HGB - haemoglobin, HTC - haematocrit, MCV - mean corpuscular volume, MCH - mean corpuscular haemoglobin, MCHC - mean corpuscular haemoglobin concentration, RDW-CV - red blood cell distribution width - coefficient of variation, Fe - iron *Parameters with different reference range in men and women; m - male, f - female
various stages of treatment-prior to, during, and follow- ing therapeutic intervention. Additional challenges include variability in systemic health status, the presence of comor- bidities, and potential complications arising from standard treatment, including surgical procedures and pharmacother- apy, such as mitotane [2].
To the best of our knowledge, the evaluation of hepcidin levels in patients with ACC has not yet been reported. There was only one study, which described that decreased ferro- portin1 expression correlated with higher stage and worser outcome in ACC, however hepcidin was not evaluated [6].
The evaluation of hepcidin and Fe homeostasis in ACC is particularly complex due to mitotane’s indirect myelotoxic effects on bone marrow function. Mitotane usage leads to anemia, leucopenia and thrombocytopenia. Moreover, mito- tane hamper the evaluation of Fe metabolism by influenc- ing thyroid physiology. Specifically, mitotane increases the levels of hormone-binding proteins [13], which may induce
hypothyroidism. It is worth noting that hepcidin levels have been shown to decrease in hypothyroid patients with Hashimoto’s thyroiditis, with normalisation following the restoration of euthyroidism [14]. Hepcidin expression is regulated by both stimulatory and inhibitory factors; hence, broad exclusion criteria were applied in the present study to reduce confounding factors.
Given the limited data on Fe homeostasis in ACC, we conducted a study to evaluate serum hepcidin levels in patients newly diagnosed with ACC as well as those receiv- ing treatment. We additionally compared these findings to individuals with benign adrenal tumors to delineate poten- tial disease-specific alterations in Fe regulation.
Our analysis revealed elevated hepcidin levels (border significance) in the ACC cohort if compared to controls (Table 1). This observation aligns with existing evidence indicating that hepcidin is upregulated in various malig- nancies, a phenomenon frequently attributed to the pro- inflammatory cytokine interleukin-6 (IL-6). IL-6 activates the JAK/STAT3 signalling pathway, leading to enhanced transcription of the hepcidin gene (HAMP), thereby con- tributing to Fe sequestration and hypoferremia commonly observed in cancer-related anemia [15].
Comparing patients treated to untreated, the hepcidin concentrations exhibited a trend to higher value (p=0.053). While hepcidin expression is typically downregulated in the context of anemia, this potential confounder was eliminated through the exclusion of anemic patients from the study cohort. An alternative explanation may involve a mitotane- induced upregulation of hepatic biosynthetic activity, result- ing in increased hepcidin production via mechanisms other than from erythropoietic drive or Fe status.
Furthermore, a positive correlation between serum hepci- din and ferritin levels was observed across all study groups, consistent with previously published findings [14, 16-18]. Both hepcidin and ferritin are recognized as acute-phase reactants, and their concentrations typically increase in response to systemic inflammation [18]. Notably, our data demonstrated a lower ferritin level in patients after thera- peutic intervention, suggesting a possible attenuation of inflammatory activity. In male patients with ACC, serum Fe levels were significantly lower compared to those in the CS (p = 0.031). Although not reaching statistical significance, treatment was associated with a trend toward improvement in several erythrocyte indices, including RBC count, HGB concentration, HCT, mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH). Particular attention should be given to red cell distribution width (RDW), a marker of anisocytosis. In the ACC cohort, RDW showed a non-significantly higher value in ACC-nt than ACC-t. Importantly, RDW values were consistently higher in ACC patients compared to controls, irrespective of treatment
ACC-nt
ACC-t
261
169
131
108
87
86
96
86
51
64
42
38
11.2
12.8
11.9
35
35
14
29
33
33
30
3.9
4.4
4.1
15
14
4
RBC-M
RBC-K
HGB-M
HGB-F
HTC-M
HTC-F
MCV
MCH
MCHC
RDV
FERRITIN-M
FERRITIN-F
FE-M
FE-F
status, potentially reflecting persistent erythropoietic stress or ineffective erythropoiesis. RDW has emerged as a sig- nificant prognostic biomarker in various malignancies [19], however to date there are no studies concerning ACC.
In summary, therapeutic intervention in ACC may con- tribute to partial normalization of Fe metabolism and eryth- ropoiesis. However, the observed trend toward increased hepcidin concentrations following treatment appears para- doxical, as it may reflect persistent or treatment-induced inflammatory responses. Importantly, mitotane therapy itself could contribute to hepatic stimulation of hepcidin synthe- sis, independent of systemic inflammation or Fe demand. Conversely, elevated hepcidin levels post-therapy may not necessarily indicate a pathological process. Analogous to hepatocellular carcinoma, increased hepcidin expression in ACC could potentially signify a favourable treatment response, as hepcidin restricts the mobilization of intracel- lular Fe stores and limits Fe availability to neoplastic cells, thus exerting a tumor-suppressive effect.
The limitation of the research is heterogenicity and lim- ited size of study group. However, it might be attributed to the rarity of the disease.
In conclusion, the patients with ACC present higher median values of hepcidin and slightly decreased
hematological parameters compared to CS. Additionally, we observe better results of Fe homeostasis in ACC-t in com- parison to ACC-nt despite higher median level of hepcidin in ACC-t group. The neoplastic process increases hepcidin level, which leads to decreased hematological parameters, while treatment improve Fe homeostasis. Our observations need to be further investigated and study additional factors influencing Fe homeostasis and hepcidin regulation in the context of ACC. Future research is warranted to evaluate whether hepcidin could serve as prognostic factor in strati- fying ACC outcomes.
Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by A.K., D.F. and B.B. The first draft of the manuscript was written by A.K. and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Data availability No datasets were generated or analysed during the current study.
Declarations
Ethics approval The studies involving human participants were re- viewed and approved by the Bioethical Committee of Poznan Univer- sity of Medical Sciences. The patients provided their written informed consent to participate in this study.
Conflict of interest The authors declare no competing interests.
References
1. M. Fassnacht et al., European society of endocrinology clinical practice guidelines on the management of adrenal incidentalo- mas, in collaboration with the European network for the study of adrenal tumors. Eur. J. Endocrinol. 189(1), G1-G42 (2023)
2. M. Fassnacht, O. Dekkers, T. Else, E. Baudin, European society of endocrinology clinical practice guidelines on the management of adrenocortical carcinoma in adults, in collaboration with the European network for the study of adrenal tumors. Eur. J. Endo- crinol. 179(4), G1-G46 (2018)
3. M. Fassnacht, G. Assie, E. Baudin, Adrenocortical carcinomas and malignant phaeochromocytomas: ESMO-EURACAN clini- cal practice guidelines for diagnosis, treatment and follow-up. Ann. Oncol. 31(11), 1476-1490 (2020)
4. D. Hadkiewicz-Junak, M. Dedecjus, U. Ambroziak, Polish diag- nostic and therapeutic recommendations for adrenocortical carci- noma. Endokrynol Pol. 5(4), 339-358 (2024)
5. E. Nemeth, T. Ganz, Hepcidin and iron in health and disease. Annu. Rev. Med. 27, 74:261-277 (2023)
6. B. Zhu, Q. Zhi, Q. Xie, Reduced expression of ferroportin1 and ceruloplasmin predicts poor prognosis in adrenocortical carci- noma. J. Trace Elem. Med. Biol. 56, 52-59 (2019)
7. J. Nowak, J. Walkowiak, Study designs in medical research and their key characteristics. JMS. 92(4), e928 (2024)
8. R. Szabo, C. Petrisor, S. Tranca, Vitamin D and iron levels cor- relate weakly with Hepcidin levels in postoperative patients with digestive neoplasms undergoing open abdominal surgery. Eur. Rev. Med. Pharmacol. Sci. 5(9), 3530-3535 (2021)
9. T. Shibabaw, B. Teferi, M.D. Molla, B. yelign, Inflammation mediated Hepcidin-Ferroportin pathway and its therapeutic win- dow in breast cancer. Breast Cancer. 12, 165-180 (2020)
10. Y. Fan, B. Liu, F. Chen, Z. Song, Hepcidin upregulation in lung cancer: A potential therapeutic target associated with immune infiltration. Ront Immunol. 12, 612144 (2021)
11. J.H. Joachim, K. Metha, J. Hepcidin in hepatocellular carcinoma. Br. J. Cancer. 27, 185-192 (2022)
12. P. Ruchala, E. Nemeth, The pathophysiology and Pharmacology of Hepcidin. Trends Pharmacol. Sci. 35(3), 155-161 (2014)
13. M. Bianchi, G. Puliani, A. Chiefari, Metabolic and endocrine tox- icities of mitotane: A systematic review. Cancers. 13(19), 5001 (2021)
14. A. Hernik, E. Szczepanek-Parulska et al., The Hepcidin con- centration decreases in hypothyroid patients with hashimoto’s thyroiditis following restoration of euthyroidism. Sci. Rep. 9(1), 16222 (2019)
15. F. Lin, A. Tuffour, G. Hao, Distinctive modulation of Hepcidin in cancer and its therapeutic relevance. Front. Oncol. 13, 1141603 (2023)
16. A. Krygier et al., Iron homeostasis and Hepcidin concentration in patients with acromegaly. Front. Endocrinol. 2, 788247 (2022)
17. A. Krygier, E. Szczepanek-Parulska, D. Filipowicz, M. Ruchala, Changes in serum Hepcidin according to thyrometabolic status in patients with graves’ disease. Endocr. Connect. 9(3), 234-242 (2020)
18. A. Hernik et al., Hepcidin and iron homeostasis in patients with subacute thyroiditis and healthy subjects. Mediators Inflamm. 019, 5764061 (2019)
19. P .- F. Wang, S .- Y. Song, H. Guo, Prognostic role of pretreatment red blood cell distribution width in patients with cancer: A meta- analysis of 49 studies. J. Cancer. 10(18), 4305-4317 (2019)
Publisher’s note Springer Nature remains neutral with regard to juris- dictional claims in published maps and institutional affiliations.
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.