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Critical Reviews in Oncology / Hematology

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CRITICAL REVIEWS in Oncology Hematology

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The management of adrenocortical carcinoma in the era of precision medicine

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F. Flauto, G. Ferone, M.C. De Martino, R. Pivonello, A. Colao, V. Damiano Department of Clinical Medicine and Surgery, University of Naples Federico II, Naples, Italy

ARTICLE INFO

ABSTRACT

Keywords: Adrenocortical carcinoma Epigenomics Precision medicine Multidisciplinary team Endocrine cancers

Adrenocortical carcinoma (ACC) is a rare but highly aggressive malignancy of the adrenal cortex, with hetero- geneous clinical behaviour and limited therapeutic efficacy in advanced disease. While surgical resection remains the mainstay of curative treatment for localized tumours, outcomes in metastatic ACC remain poor, with long- term disease control achieved only in a minority of patients. Recent multi-omic studies have uncovered distinct molecular subtypes of ACC, providing deeper insight into tumor biology and potential therapeutic vulnerabilities. However, the translation of these molecular findings into clinical decision-making remains limited. This review offers a comprehensive and clinically relevant synthesis of current knowledge on ACC, with a specific focus on how molecular profiling can refine diagnosis, prognosis, and treatment strategies. We discuss current gaps, implementation challenges, and future directions toward a precision oncology approach for ACC.

1. Introduction

Adrenocortical carcinoma (ACC) is a rare and highly aggressive malignancy of the adrenal cortex, with an annual incidence ranging from 0.5 to 2 cases per million individuals and a median overall survival (OS) of three to four years (Fassnacht et al., 2020a, 2018). The disease displays a bimodal age distribution, with peaks in early childhood (ages 1-6) and mid-adulthood (ages 46-55). While sex does not appear to significantly influence prognosis, the role of age at diagnosis remains debated (Tella et al., 2018; Kim et al., 2016; Luton et al., 1990; Crucitti et al., 1996; Schulick and Brennan, 1999). Prognosis is primarily determined by tumor stage, according to the European Network for the Study of Adrenal Tumours (ENSAT) classification, and by the Ki-67 proliferation index (Elhassan et al., 2022; Fassnacht et al., 2009; Beus- chlein et al., 2015; Libé et al., 2015). Radical surgical resection (RO) remains the only curative option for localized disease, yet recurrence rates remain high, ranging from 30 % to 75 % (Fassnacht et al., 2020a, 2018). More than half of patients are diagnosed at an advanced or metastatic stage, where five-year survival falls below 15 %.

In advanced disease, mitotane remains the cornerstone of systemic therapy, either as monotherapy or in combination with cytotoxic chemotherapy (Fassnacht et al., 2012). However, durable responses are uncommon. Pediatric ACCs often follow a distinct biological course and tend to have more favorable outcomes, although mitotane-based

regimens remain the standard of care (Riedmeier et al., 2022, 2024; Grisanti et al., 2021). Immunotherapy has shown partial responses in selected patients, particularly when combined with multi-kinase in- hibitors, but clinical benefit in terms of progression-free survival (PFS) or OS remains limited (Grisanti et al., 2020a).

Against this backdrop, advances in multi-omic profiling, including genomic, transcriptomic, epigenetic, and miRNA-based analyses, have substantially deepened our understanding of ACC biology. Large-scale studies, such as those led by ENSAT and The Cancer Genome Atlas (TCGA), have enabled the molecular subclassification of ACC into bio- logically and prognostically distinct subgroups, revealing key oncogenic pathways and potential therapeutic vulnerabilities.

Despite these advances, the incorporation of molecular profiling into routine clinical practice remains limited. Current treatment algorithms largely overlook tumor heterogeneity, contributing to suboptimal out- comes. There is a growing need to bridge the gap between translational research and therapeutic decision-making, particularly in identifying robust prognostic and predictive biomarkers that can guide patient stratification and therapy selection.

This review aims to provide an updated, comprehensive, and clini- cally oriented synthesis of current knowledge on ACC, with particular focus on the relevance of molecular profiling in diagnostic, prognostic, and therapeutic frameworks. By addressing both consolidated and emerging concepts in ACC biology and management, the article is

* Corresponding author.

E-mail address: vdamiano@unina.it (V. Damiano).

https://doi.org/10.1016/j.critrevonc.2025.104839

intended for oncologists, endocrinologists, and translational researchers engaged in the multidisciplinary care of patients with adrenal malig- nancies. Through this approach, we seek to promote a shift toward precision medicine strategies capable of addressing the biological complexity and unmet clinical needs in ACC.

2. Genetics and molecular classification

Recent years have brought significant advances in understanding ACC’s molecular basis. Large-scale studies have mapped its genomic and transcriptomic landscape, defined molecular subtypes, and identified therapeutic targets. Weiss et al. first proposed a two-grade pathological classification based on mitotic rate and clinical outcomes (Weiss et al., 1989). Subsequent research, on North American and Brazilian paediatric cohorts (Juhlin et al., 2015; Pinto et al., 2015), ENSAT (Assié et al., 2014), and TCGA (Zheng et al., 2016), used integrated molecular plat- forms to validate key genetic alterations driving ACC tumorigenesis and progression (Table 1.)

2.1. Genomic and transcriptomic profiling

ACC exhibits marked chromosomal instability, contributing to its aggressive nature and complex molecular pathology (Lerario et al., 2014a). This instability, seen as hypodiploidy or hyperdiploidy, results from recurrent chromosomal amplifications and deletions (Zheng et al., 2016). Whole-genome doubling (WGD) is common, as shown by DNA copy number analyses (Zheng et al., 2016). Amplifications frequently affect oncogenes like MDM2 and CDK4 on chromosome 12q, driving tumorigenesis through unregulated cell cycle progression (Assié et al., 2014; Zheng et al., 2016). In contrast, deletions often impact tumour suppressor regions such as TP53 (17p13) and CDKN2A (9p). TP53 loss, found in over 80 % of ACCs, impairs DNA repair and apoptosis, strongly correlating with poor prognosis (Assié et al., 2014; Zheng et al., 2016).

B-catenin (CTNNB1) gain-of-function mutations are present in ~25 % of both benign and malignant adrenocortical tumours, more frequently in nonfunctioning adenomas (ACAs) (Tissier et al., 2005). A transcriptomic analysis of 51 ACCs revealed that TP53 and CTNNB1 pathways dysregulation significantly contributed to tumour

aggressiveness and poor prognosis. The study found that mutations in CTNNB1 and TP53 alterations were mutually exclusive, covering 52 % of mutation in poor-prognosis (Ragazzon et al., 2010).

The insulin-like growth factor II gene (IGF-II) plays a key role in both sporadic and familial ACCs, including Beckwith-Wiedemann syndrome (BWS) (Li et al., 1998; Ilvesmäki et al., 1993; Gicquel et al., 1994). IGF-II and the tumour suppressor H19 are located at 11p15.5 and regulated by genomic imprinting (Brannan et al., 1990; Hao et al., 1993; Feinberg, 1999). In ~80 % of ACCs, loss of heterozygosity (LOH) leads to IGF-II overexpression and H19 silencing, creating an autocrine loop that pro- motes proliferation and survival (Gicquel et al., 1997; Logie et al., 1999).

Recent genomic studies have identified potential driver genes, including Zinc and Ring Finger 3 (ZNRF3), Telomerase Reverse Tran- scriptase (TERT), and Telomeric Repeat Binding Factor 2 (TERF2), and has revealed molecular subgroups with distinct clinical outcomes (Assié et al., 2014; Hao et al., 2012). ZNRF3 mutations (found in ~20 % of ACCs) impair Wnt/ß-catenin signaling regulation (Assié et al., 2014; Zheng et al., 2016). TERT promoter mutations (C228T, C250T) and increased expression are linked to WGD and telomere maintenance in aggressive ACCs (Assié et al., 2014; Zheng et al., 2016). TERF2 dysre- gulation similarly supports telomere stability in advanced disease (Zheng et al., 2016).

Furthermore, DNA sequencing of sporadic ACAs recently uncovered a recurrent activating L206R mutation in the catalytic subunit of cAMP- dependent protein kinase A (PKA) (PRKACA), leading to constitutive PKA activity by disrupting the interaction between PRKACA and its regulatory subunits, including PRKAR1A (Goh et al., 2014; Calebiro et al., 2014).

Transcriptomic clustering by de Reyniès et al. identified two major malignant groups: C1 (ACCs) and C2 (ACAs). C1 tumours were further divided into C1A and C1B subgroups. C1A tumours had poor outcomes, while C1B tumours had better outcomes, still displaying malignant features (de Reyniès et al., 2009).

This study also revealed that among the malignant tumours, the combined expression of BUB1 Mitotic Checkpoint Serine/Threonine Kinase B (BUB1B) and PTEN-induced Kinase 1 (PINK1) was the best predictor of OS (de Reyniès et al., 2009).

Table 1 Genetic alterations in ACCs. POLD1: DNA Polymerase Delta 1, Catalytic Subunit; AURKA: Aurora Kinase A; KIF23: Kinesin Family Member 23.

Gene/ Feature	Locus	Protein function	Alteration	Prevalence	Other Features
TP53	17p13	Tumour suppressor, regulates DNA repair and apoptosis	Loss of function mutations, deletions	~25 % in ACC	Correlates with poor prognosis and chromosomal instability
CTNNB1	3p22.1	Component of Wnt signaling, regulates cell adhesion and transcription	Gain of function mutations	~25 % in ACC and ACA	Often mutually exclusive with TP53 mutations
IGF-II	11p15.5	Growth factor, promotes proliferation and survival	Overexpression due to 11p15.5 LOH	~80-90 % in ACC	Creates autocrine stimulatory loop promoting tumour growth
CDK4	12q14	Cyclin-dependent kinase, regulates cell cycle progression	Amplification	Frequent in ACC	Leads to unchecked cell cycle progression
CDKN2A	9p21	Tumour suppressor, inhibits CDK4/6 to regulate cell cycle	Deletion, loss of function	Frequent in ACC	Loss results in unregulated cell division
MDM2	12q15	E3 ubiquitin ligase, regulates p53 degradation	Amplification	Frequent in ACC	Enhances tumorigenesis by degrading p53
ZNRF3	22q12.1	E3 ubiquitin ligase, negative regulator of Wnt/ ß-catenin pathway	Loss of function mutations	~20 % in ACC	Key regulator of tumour progression via Wnt/ ß-catenin
TERT	5p15.33	Telomerase reverse transcriptase, maintains telomere length	Promoter mutations (C228T, C250T)	Frequent in aggressive ACC	Associated with whole genome doubling in ACC
TERF2	16q22.1	Telomeric repeat binding factor, protects telomeres from degradation	Deregulation, increased expression	Frequent in aggressive ACC	Linked to telomere maintenance in aggressive tumours
PRKAR1A	17q24.2	Regulatory subunit of PKA, involved in cAMP signaling	L206R activating mutation	Frequent in ACC	Disrupts regulatory interactions leading to constitutive PKA activation
POLD1	19q13.33	DNA polymerase delta catalytic subunit, involved in DNA replication and repair	Mutations affecting polymerase proofreading activity	Associated with aggressive ACC subtypes	Loss of function mutations may lead to genomic instability
AURKA	20q13.2	Aurora kinase A, regulates mitosis and cell cycle progression	Overexpression, amplifications	Highly expressed in proliferative ACCs	Targeted by Aurora kinase inhibitors in other cancers
KIF23	15q23	Kinesin family member 23, involved in cytokinesis and cell division	Overexpression, transcriptional dysregulation	Upregulated in aggressive ACCs	Linked to poor prognosis and high proliferation rates in ACC

Further genomic and transcriptomic profiling has better refined ACC classification into two distinct subgroups (Assié et al., 2014; Giordano et al., 2009). Poor-outcome C1A tumours carried TP53 mutations (C1A-p53), CTNNB1 mutations (C1A-ß-catenin), or unidentified alter- ations (C1A-x), exhibited high mutation burden, chromosomal insta- bility, and a CpG Island Methylator Phenotype (CIMP), with a 5-year survival rate of ~20 % (Ragazzon et al., 2010; de Reyniès et al., 2009; Giordano et al., 2009). Conversely, the good-outcome C1B tumours display non-CIMP status and deregulated miRNA clusters, including MIR483 and DLK1-MEG3, achieving a 5-year survival rate of 91 %.

A 2024 integrated genomic analysis of 162 ACC patients identified a 45-gene signature linked to poor prognosis (Sun-Zhang et al., 2024). Many of these genes, such as POLD1, AURKA, and KIF23, are involved in DNA replication and mitosis, and several interact with TP53 (Sun-Zhang et al., 2024). Mutations in these genes correlated with significantly worse survival, independent of tumour stage or patient age (Sun-Zhang et al., 2024). These findings suggest that multi-gene panels may improve risk stratification and uncover novel therapeutic targets, such as AURKA inhibitors for tumours with AURKA overexpression.

2.2. Epigenetic modifications: CpG island methylator phenotype

CpG islands are DNA regions rich in CpG sites, cytosine and guanine separated by a phosphate, commonly found in gene promoters. These sites are prone to epigenetic modification, particularly DNA methyl- ation, which leads to chromatin condensation and gene silencing (Kulis and Esteller, 2010). The CIMP, first described in colorectal cancer, in- volves hypermethylation of tumour suppressor and immune-regulatory genes (Toyota et al., 1999).

In a genome-wide methylation study of 51 ACCs and 84 ACAs, car- cinomas were classified into CIMP-high, CIMP-low, and non-CIMP groups (Barreau et al., 2013). Hypermethylation was more frequent in ACCs and associated with poor prognosis. An inverse correlation be- tween methylation and gene expression was observed, with H19 being most affected.

Methylation patterns varied across transcriptome-based subgroups. Nearly all tumours in the poor-outcome C1A-p53 and C1A-x subgroups were CIMP-high, whereas C1A-ß-catenin and C1B tumours showed non- CIMP profiles. This distribution of hypermethylation across different carcinoma subgroups likely contributes to the prognostic significance of hypermethylation. However, since some poor-prognosis tumours from the C1A-B-catenin subgroup were not hypermethylated, the prognostic value of DNA methylation-based ACC classification is limited, under- scoring the need for integrated analyses (Ragazzon et al., 2010; de Reyniès et al., 2009; Giordano et al., 2009; Barreau et al., 2013).

The mechanisms driving CIMP in ACC remain unclear. However, Kerdivel et al. demonstrated that overexpression of DNA methyl- transferase (DNMT1 and DNMT3A) contributes to CIMP, despite no specific mutations being identified in ENSAT or TCGA cohorts (Assié et al., 2014; Zheng et al., 2016; Kerdivel et al., 2023).

2.3. microRNA expression and molecular stratification

In the ENSAT study, miRNA expression was analyzed in 45 ACC and 3 normal adrenal gland samples, identifying three distinct clusters: Mi1, Mi2, and Mi3 (Assié et al., 2014). The Mil cluster showed the most significant alterations, marked by upregulation of 11 miRNAs from the miRNA-506-514 cluster (Xq27.3) and downregulation of 38 miRNAs from the imprinted DLK1-MEG3 cluster (14q32.2).

Mi1 tumours exhibited LOH on chromosome 14q and hyper- methylation of the MEG3 promoter, silencing the DLK1-MEG3 cluster and contributing to tumorigenesis.

Mi2 tumours showed weaker miRNA-506-514 overexpression, pre- viously linked to melanoma development (Streicher et al., 2012).

Mi3 tumours had no significant miRNA changes compared to normal samples and were characterized by hypomethylation at non-island CpG

sites.

Transcriptome-based clustering in the ENSAT study closely aligned with methylation and miRNA expression profiles. The poor-prognosis C1A subgroup mostly included CIMP-high and Mi3 tumours, while the better-outcome C1B subgroup was typically non-CIMP and aligned with Mi1 or Mi2 clusters. Notably, exome and single nucleotide poly- morphisms (SNPs) analyses revealed that most gene alterations occurred in C1A tumours, while miRNA dysregulation was more prominent in C1B.

2.4. Integrated genomic approaches: subtypes and clinical implications

The TCGA study integrated multi-omics data, including DNA muta- tions, copy number, methylation, mRNA, miRNA, and protein expres- sion, for comprehensive tumour profiling (Zheng et al., 2016). Using a cluster-of-clusters (CoC) method, three molecular subtypes were defined: CoC I, II, and III.

CoC I tumours, enriched with previously defined indolent C1B types, exhibited low proliferation, strong immune-related gene expression, low-CIMP, and mutations in genes such as ZNRF3 and MEN1. These tumours had the best prognosis, with only ~7 % showing progression and median event-free survival (EFS) not reached in the TGCA cohort (Zheng et al., 2016).

CoC II tumours showed intermediate-CIMP, moderately high prolif- eration, and mutations in TP53, CTNNB1, and ZNRF3. They had an in- termediate prognosis, with ~56 % progression and median EFS of 38 months. This group was more heterogeneous, suggesting it might contain a spectrum of tumour behaviours (Zheng et al., 2016).

CoC III tumours, largely overlapping with aggressive C1A subtypes, were characterized by high-CIMP and mutations in CDK4, RB1, and TERT. These had the poorest prognosis, with 96 % progression rate, and median 8 months EFS. Many of these were advanced-stage tumours, though notably even some early-stage tumours that fell into CoC III behaved aggressively (Zheng et al., 2016).

These molecular subtypes demonstrated prognostic value beyond traditional staging. To facilitate clinical use, researchers derived a simplified DNA methylation signature using 68 probes, accurately classifying tumours into the three groups with ~92 % accuracy. Methylation profiling can be performed on formalin-fixed tissue and is more accessible than full multi-platform analysis, offering a feasible route for integrating molecular classification into routine practice (Zheng et al., 2016).

2.5. Hereditary predisposition and genetic testing

For adults diagnosed with ACC, a comprehensive genetic assessment is recommended, focusing on family history and signs of hereditary syndromes. ACC is associated with several inherited conditions, including multiple endocrine neoplasia type 1 (MEN1), familial adeno- matous polyposis (FAP), neurofibromatosis type 1 (NF1), Li-Fraumeni syndrome (LFS), and Lynch syndrome (LS) (Table 2) (Else et al., 2014; Raymond et al., 2013). Germline TP53 mutations are relatively common in ACC and show significant variability, resulting from low-penetrance alleles or de novo mutations. Notably, up to 20 % of new LFS di- agnoses arise from de novo cases (Wasserman et al., 2015). Given the mutation prevalence, TP53 testing, including deletion/duplication analysis, is recommended for all ACC patients, regardless of family history, aligning with Chompret criteria (Chompret et al., 2001). In Southern Brazil, the p.R337H TP53 founder mutation is highly prevalent and accounts for up to 90 % of pediatric ACC cases in the region, sup- porting the effectiveness of targeted screening and surveillance (Wasserman et al., 2015). The American College of Medical Genetics and Genomics recommends including TP53, mismatch repair genes (MSH2, MSH6, MLH1, PMS2), and MEN1 in genetic testing panels (Custódio et al., 2013; Scatolini et al., 2024; Petr and Else, 2018).

Table 2 Syndromic associations with ACCs. NF1: Neurofibromin 1. FLNC: Folliculin.

Syndrome	Gene/locus	Mechanism	Prevalence	Prevalence of ACC	Other Features
Li-Fraumeni Syndrome	TP53	Impaired tumour suppression	1:20,000 to	50-80 % of children.	Breast cancer, brain cancer, sarcoma, lung cancer,
	17p13		1:1000,000	3-7 % of adults	leukemia
Lynch Syndrome (Raymond	MSH2,	Microsatellite instability	1:440	3 % of adults	Colorectal, endometrial, small bowel, ureteral cancer, pancreatic, prostate cancer
et al., 2013)	MLH1, PMS2,
	MSH6,
	EPCAM
Multiple Endocrine Neoplasia Type 1 (Gatta-Cherifi et al., 2012)	MEN1	Loss of menin	1:30,000	1-2 % of adults	Pituitary adenomas, primary hyperparathyroidism, pancreatic neuroendocrine tumours, other foregut neuroendocrine tumours
Familial Adenomatous Polyposis (Smith et al., 2000)	APC	Wnt/ß-catenin dysregulation	1:30,000	Rare, case reports	Colon polyps, colorectal cancer, thyroid cancer, duodenal adenoma
Beckwith-Wiedemann	11p15	Overexpression of	1:13,000	Rare, case reports;	Cancers in childhood, Wilms tumour, hepatoblastoma,
Syndrome (Lapunzina, 2005)	Imprinting	IGF2		occur in childhood only	rhabdomyosarcoma, neuroblastoma
Carney Complex (Anselmo	PRKAR1A	Dysregulated cAMP signaling	Rare >700	Rare, case reports	Pituitary and thyroid tumours, cardiac myxomas, schwannomas and other tumours
et al., 2012)			patients in the world
NF1 (Else, 2012)	NF1	Disregulated RAS activation	1:3000	Rare, case reports	Gliomas, malignant nerve sheath tumour, benign neural tumours
Birth-Hogg-Dube (Raymond et al., 2014)	FLNC	mTOR pathway iperactivation	1:100,000	Rare, case reports	Skin hamartomas, pulmonary cysts and pneumothoraces, renal oncocytomas and chromophobe renal cell cancers

3. Clinical presentation, diagnosis, staging

3.1. Clinical presentation

ACC can present with diverse clinical features or remain asymp- tomatic (Fassnacht et al., 2020a, 2018; Tella et al., 2018; Kim et al., 2016; Luton et al., 1990). When symptoms occur, they are most often related to hormonal excess, particularly hypercortisolism, leading to Cushing syndrome. This typically manifests as a dorsal fat hump, dia- betes, muscle weakness, osteoporosis, hypokalaemia, hypertension, mood changes, insomnia, skin atrophy, and increased infection risk (Berruti et al., 2014). Mass effect symptoms include nausea, vomiting, abdominal fullness, and back pain. Tumour compression of the inferior vena cava (IVC) or renal vein may cause leg oedema, abdominal col- laterals, or hydrocele. Although less common, systemic malignancy symptoms like weight loss, night sweats, fatigue, and fever can also occur (Fassnacht et al., 2020a, 2018; Tella et al., 2018; Kim et al., 2016; Luton et al., 1990). As the disease advances, metastasis to lymph nodes, lungs, liver, or bones are common, with bone involvement contributing significantly to morbidity. Brain metastases are rare but should be considered in patients with unexplained neurological symptoms. When present, they may cause hemiparesis, seizures, postural instability, headaches, diplopia, ptosis, lethargy, or aphasia (Turla et al., 2024).

3.2. Diagnosis

Establishing whether an adrenal mass is malignant is critical, as ACC diagnosis is often not obvious. All patients should undergo a compre- hensive hormonal work-up to assess for autonomous hormone excess (Fassnacht et al., 2020a, 2018). This evaluation includes tests for glu- cocorticoids, measuring basal ACTH and cortisol, performing a 1 mg dexamethasone suppression test in cases of suspected hypercortisolism, or quantifying free cortisol in 24-hour urine when overt Cushing syn- drome is present. Hormone excess assessment also includes sex hor- mones, their precursors (17-ß-oestradiol, testosterone, DHEA-S, 17-OH progesterone, androstenedione) and mineralocorticoids, by evaluating kalium, aldosterone, and the aldosterone/renin ratio in patients with hypertension and/or hypokalaemia. In addition, ruling out pheochro- mocytoma is mandatory through serum and 24-hour urinary meta- nephrines, considering that mildly elevated levels (<2 folds) may be non-specific and occasionally observed in ACC (Fassnacht et al.,

2020a, 2018).

Imaging studies play a central role in distinguishing benign from malignant lesions. CT, MRI, and 18F-FDG positron emission tomography (FDG PET) are commonly used, but among these, unenhanced CT is the most reliable for ruling out ACC (Fassnacht et al., 2020a, 2018). Benign ACAs typically appear as homogeneous lesions with low CT density (<10-20 Hounsfield Units [HU]), whereas ACCs are usually large, het- erogeneous, low in fat content (resulting in higher HU), with irregular margins and contrast enhancement of solid components (Fassnacht et al., 2020a, 2018). The prospective multicentre EURINE-ACT study, evaluating 2169 ACCs patients, showed that an unenhanced CT tumour attenuation cutoff of 20 HU is more accurate for the exclusion of ACC (Bancos et al., 2020). No single imaging modality definitively diagnoses ACC. Malignancy is conclusively determined only by the presence of metastatic lesions in sites normally lacking chromaffin cells (Fassnacht et al., 2020a, 2018). Thus, in the absence of such evidence, an adrenal lesion remains only potentially malignant. FDG PET, for instance, has excellent sensitivity and negative predictive value but limited specificity and positive predictive value, so it is reserved for cases with high met- astatic risk, such as local invasion, IVC extension, lymph node involve- ment, tumour size over 5 cm, or known succinate dehydrogenase-B subunit (SDHB) germline mutations.

Additional functional imaging techniques include 68Ga-PET labelled somatostatin analogues (e.g., DOTATATE, DOTATOC, DOTANOC), particularly useful in SDHB mutated disease, and CXCR4 directed PET/ CT with 68Ga-pentixafor (Dreher et al., 2024). The latter has demon- strated uniform CXCR4 expression in both primary tumours and me- tastases (except in lung lesions), suggesting that CXCR4 directed radioligand therapy could have a broad anti-tumour effect and may help identify patients eligible for such targeted treatment.

Despite advances in imaging and hormonal evaluation, the gold standard for ACC diagnosis remains the anatomic pathological exami- nation of the surgical specimen, utilizing the Weiss score (Fassnacht et al., 2020a, 2018; Weiss et al., 1989; Mete et al., 2022). Biopsy is contraindicated due to the risk of tumour spillage and its negative impact on achieving an R0 resection, except when metastatic disease precludes surgery and histopathologic confirmation is necessary for oncological management. The Weiss score, ranging from 0 to 9, is the established tool for differentiating benign from malignant adrenocor- tical lesions. It comprises nine histopathologic criteria, each assigned 1 point:

1. High Fuhrman nuclear grade (III or IV);

2. Mitotic count > 5 per 50 high-power fields (10mm2);

3. Atypical mitosis;

4. Necrosis;

5. Diffuse architecture > 30 % of tumour volume;

6. Clear cells ≤ 25 % of the tumour volume;

7. Capsular invasion;

8. Venous invasion;

9. Sinusoidal (lymphatic) invasion;

A modified Weiss score focuses on five criteria: mitotic count, clear cells, atypical mitoses, necrosis, and capsular invasion, with the first two weighted double (Fassnacht et al., 2020a, 2018; Mete et al., 2022). A score ≥ 3 indicates ACC, while scores between 0 and 2 suggest an ade- noma. The 2022 WHO classification of adrenal cortical tumours further refines the diagnosis by recognizing four subtypes of ACC based on cytomorphological features: conventional, oncocytic, myxoid, and sar- comatoid (Mete et al., 2022). In some subtypes, such as myxoid ACC, assessing certain Weiss parameters (like diffuse growth, nuclear atypia, or lymphatic invasion) can be challenging (Giordano et al., 2021). Therefore, supplementary multiparameter diagnostic algorithms have been developed to aid the workup of adrenocortical neoplasms (Mete et al., 2022; Giordano et al., 2021; Hodgson et al., 2019). Several diagnostic systems are used for adrenocortical neoplasms, including (a) the reticulin algorithm, which is applicable to conventional, oncocytic, and myxoid adrenocortical neoplasms, (b) the Lin-Weiss-Bisceglia sys- tem, specifically for oncocytic adrenocortical neoplasms, and (c) the Helsinki scoring system, which can be applied to conventional, onco- cytic, and myxoid adrenocortical neoplasms (Mete et al., 2022). The reticulin algorithm, known for its reproducibility, diagnoses ACC when an altered reticulin network is identified using the Gordon-Sweet silver stain, along with at least one of the following: a mitotic count greater than 5 per 50 high-power fields, tumour necrosis, or vascular invasion (Mete et al., 2022). The Lin-Weiss-Bisceglia system is designed for oncocytic adrenocortical neoplasms, which often present with a Weiss score of 3 and can be challenging to diagnose. This system requires more than 90 % of the tumour be oncocytic to qualify as a pure oncocytic neoplasm (Mete et al., 2022; Giordano et al., 2021; Hodgson et al., 2019; Bisceglia et al., 2004). It categorizes findings into major criteria (high mitotic rate, atypical mitoses, vascular invasion) and minor criteria (large tumour size or weight, necrosis, capsular invasion, sinusoidal invasion). Malignancy is diagnosed if at least one major criterion is present, while a minor criterion suggests uncertain malignant potential (Mete et al., 2022). The Helsinki score integrates the Ki 67 proliferation index from the most active area with necrosis and mitotic count, yielding a quantitative score: values from 0 to 8.5 suggest an adenoma, scores above 8.5 indicate carcinoma (Mete et al., 2022; Giordano et al., 2021; Hodgson et al., 2019). A panel of immunohistochemical markers is also recommended to support the diagnosis. Markers such as SF1, inhibin alpha, calretinin, melan A help confirm ACC, while chromog- ranin A is useful for differentiating pheochromocytoma (Mete et al., 2022). In paediatric patients, the use of the Weiss criteria, which are effective in adults, tends to over-diagnose tumours that behave benignly (Grisanti et al., 2021; Mete et al., 2022). Therefore, the Wieneke criteria are currently preferred for diagnosing paediatric ACC, ensuring more accurate classification and management in this population (Mete et al., 2022; Wieneke et al., 2003).

3.3. Staging

The ENSAT/TNM classification is recommended for assessing ACC stage and is endorsed by the UICC and WHO (Fassnacht et al., 2020a, 2018). According to ENSAT, ACC is categorized into four stages (Table 3). Stage I (≤5 cm) and Stage II (>5 cm) tumours remain confined to the adrenal gland. Stage III tumours extend into adjacent tissues, such as para-adrenal fat or nearby organs or involve locoregional lymph

Table 3 ENSAT staging system. T1: tumour ≤ 5 cm; T2: tumour > 5 cm; T3: tumour infiltration into surrounding tissue; T4: tumour invasion into adjacent organs or venous tumour thrombus in vena cava or renal vein. N0: no positive lymph nodes; N1: presence of positive lymph nodes. M0: no distant metastases; M1: presence of distant metastases.

ENSAT Stage	Definition
I	T1, N0, M0
II	T2, N0, M0
III IV	T1-T2, N1, M0
	T3-T4, N0-N1, M0
	T1-T4, N0-N1, M1

nodes, while Stage IV is defined by the presence of distant metastases.

To improve prognostication in advanced cases, a modified ENSAT (mENSAT) system has been proposed. In this system, Stage III includes tumours invading surrounding tissues/organs or the renal/vena cava, and Stage IV is further subdivided into IVa, IVb, and IVc based on the number of metastatic sites (>2, 3,>3 organs, including nodal metasta- ses) (Fassnacht et al., 2020a, 2018; Libé et al., 2015). Therefore, a CT scan covering the chest, abdomen, and bones is indicated for staging, with abdomen MRI added if hepatic or IVC infiltration is suspected (Fassnacht et al., 2020a, 2018). Although the ESE guidelines (Fassnacht et al., 2018) in 2018 did not recommend routine [18 F]-FDG PET/CT, recent data challenge this view (Fassnacht et al., 2023, 2020b). In a multicentre prospective study, a tumour SUVmax to liver SUVmax ratio > 1.5 demonstrated high accuracy, suggesting that FDG PET/CT is effective in evaluating adrenal masses (Guerin et al., 2017). These findings are further supported by data from a recent meta-analysis (Kim et al., 2018).

4. Therapeutic options

4.1. Surgery

Surgical resection remains the primary treatment for localized ACC, particularly in stages I, II, and select stage III cases, offering the only potentially curative option (Fassnacht et al., 2020a, 2018, 2023, 2020b). Preoperative hormonal evaluation is essential to assess tumour func- tionality, especially cortisol secretion, and to anticipate postoperative adrenal insufficiency, which may require hormone replacement therapy. Guidelines from the NCCN (Shah et al., 2021), AACE (Kiseljak-Vassiliades et al., 2020), and AAES (Yip et al., 2022) recom- mend open adrenalectomy with en-bloc lymph node dissection to reduce peritoneal spread and enable removal of adjacent structures when necessary. Meticulous surgical technique is critical to prevent capsule rupture and spillage. Achieving R0 resection with negative margins is key to minimizing recurrence and may involve removal of surrounding organs such as the kidney, pancreas, spleen, liver, or diaphragm (Fassnacht et al., 2023, 2020b; Shah et al., 2021; Kiseljak-Vassiliades et al., 2020; Yip et al., 2022). Laparoscopic adrenalectomy yields good outcomes in experienced centres but is less widely adopted. Although both open and laparoscopic methods can achieve R0 resection, con- version to open surgery is associated with poorer survival, underscoring the importance of careful patient selection. Robotic adrenalectomy is an emerging option, while retroperitoneoscopic approaches are less com- mon, especially for tumours > 4-6 cm. Routine nephrectomy is discouraged unless there is evidence of renal invasion, favouring nephron-sparing techniques. Splenectomy may be required for left-sided tumours involving splenic vessels, and partial IVC resection may be necessary for right-sided tumours invading the IVC.

Locoregional lymphadenectomy, including periadrenal and renal hilum nodes, is recommended for suspected or confirmed ACC, with extended dissection for suspicious nodes. Nodal involvement signifi- cantly impacts prognosis: median OS is 88 months for node-negative

patients, 35 months with 1-3 positive nodes, and 16 months with ≥ 4 positive nodes. Comprehensive lymphadenectomy lowers recurrence risk and improves survival (Fassnacht et al., 2023, 2020b; Shah et al., 2021; Kiseljak-Vassiliades et al., 2020; Yip et al., 2022).

Recurrence affects up to 75 % of patients and requires individualized management (de Ponthaud et al., 2024). In patients with a disease-free interval ≥ 12 months, repeat surgery or ablation is recommended if complete removal is feasible. A meta-analysis of 11 studies showed improved outcomes with reoperation versus non-surgical treatment, though benefit declines with multiple recurrences or shorter disease-free survival (DFS) (Zhang et al., 2022). In select centres, cytoreductive surgery with hyperthermic intraperitoneal chemotherapy (HIPEC) is used for peritoneal involvement. Preliminary studies report median DFS of 12-19 months, but larger trials are needed to define HIPEC’s role (Hughes et al., 2018).

4.2. Neoadjuvant therapy

Upfront surgery is effective for early-stage ACC but may be less optimal for locally advanced or metastatic cases due to high recurrence risk. In patients with resectable or borderline resectable (BR) ACC, neoadjuvant chemotherapy followed by surgery may be considered (Fassnacht et al., 2023, 2020b; Shah et al., 2021). About 50 % of patients respond to the standard EDP-M regimen, which can shrink tumours, increase R0 resection rates, and preserve adjacent organs. It also allows nephrotoxic agents to be used before nephrectomy and helps identify those most likely to benefit from surgery (Bednarski et al., 2014). BR disease includes tumours that are challenging due to anatomical, bio- logical, or patient-related factors. Anatomically, these may require multi-organ resection or carry a high risk of positive margins. Biologi- cally, it includes resectable oligometastatic or potentially metastatic tumours. Patient-related factors such as comorbidities may contraindi- cate immediate surgery. A retrospective study of neoadjuvant EDP-M in BR ACC showed similar margin-positive resection rates compared to resectable cases, with median DFS of 28 months in BR patients versus 13 months in resectable ones, with similar OS (Bednarski et al., 2014). While randomized data are lacking, multidisciplinary evaluation is essential, as aggressive multimodal strategies can lead to favourable long-term outcomes.

4.3. Adjuvant therapy

Mitotane, a derivative of DDT, is used as adjuvant therapy in high- risk resected ACC and as treatment for inoperable or metastatic dis- ease, either alone or in combination with chemotherapy (Fassnacht et al., 2020a, 2018, 2023, 2020b). Mitotane selectively destroys adrenal cortical cells, impairing steroidogenesis through sterol-O-acyltransferase 1 (SOAT1) inhibition, inducing mitochondrial lipid accumulation and endoplasmic reticulum stress, promoting apoptosis and regulating hormone hypersecretion (Flauto et al., 2024).

Despite its efficacy, mitotane therapy is challenging. In advanced ACC, plasma levels above 14 mg/L correlate with better outcomes, but optimal exposure in the adjuvant setting is still debated (Fassnacht et al., 2020a, 2018).

The value of systemic adjuvant therapy remains controversial. While mitotane is often used in high-risk patients (e.g., Ki67 >10 %, Stage III, R1 resection), evidence is mixed (Fassnacht et al., 2020a, 2018). A 2007 retrospective study by Terzolo et al. showed improved recurrence-free survival (RFS) with adjuvant mitotane (Terzolo et al., 2007), whereas a 2016 study by Postelwait et al. found no significant improvement in RFS or OS in high-risk patients (Postlewait et al., 2016). A meta-analysis of five retrospective studies (n = 1249) did report improved RFS and OS with adjuvant mitotane (Tang et al., 2018).

More recently, the randomized phase III ADIUVO trial, which enrolled low- to intermediate-risk post-surgical patients (target plasma levels 14-20 mg/L), found no significant increase in 5-year RFS, 79 %

(95 %CI 67-94) in the mitotane group, compared to surveillance group, 75 % (63-90) (HR 0.74, 95 %CI 0.30-1.85); the trial was discontinued prematurely due to slow enrolment (Terzolo et al., 2023). These findings suggest that low- to intermediate-risk patients (Stage I-III, Ki67 <10 %) are unlikely to benefit from adjuvant mitotane.

Ongoing trials, such as the ADIUVO-2 phase III study (NCT03583710), aim to compare adjuvant mitotane alone versus mitotane combined with chemotherapy in high-risk post-surgical ACC patients (A Randomized Registry Trial of Adjuvant Mitotane vs, 2018). ACACIA trial (NCT03723941) is evaluating the efficacy of cisplatin/e- toposide (EP-M) as compared to observation/mitotane after primary resection of localized ACC. These studies will help clarify the optimal adjuvant approach in ACC management (Calabrese et al., 2019).

The role of adjuvant radiotherapy (RT) in localized ACC remains under debate (Fassnacht et al., 2020a, 2018). A recent study analyzed outcomes in 46 patients who received adjuvant RT following surgery, with a median dose of 45.0 Gy, compared to 59 matched patients with a median follow-up of 36.5 months. The findings indicated that adjuvant RT was associated with a higher 3-year OS rate (87.9 % vs. 79.5 %, P = 0.039), with a particularly significant benefit observed in patients with ENSAT stage I/II disease (P = 0.004). Additionally, adjuvant RT improved median DFS, extending it from 16.5 months (95 %CI, 12.0-20.9) to 34.6 months (95 %CI, 16.1-53.0). The treatment was generally well tolerated, with most adverse effects being mild to mod- erate, though six cases of grade 3 toxicity were reported (Wu et al., 2024).

Another retrospective analysis of 16 patients found no significant benefit in local RFS, distant RFS, or OS compared to 32 matched patients without RT (Habra et al., 2013). Conversely, a study involving 20 matched patients found that while adjuvant RT significantly improved local tumour control (HR 12.59; 95 %CI, 1.62-97.88), it did not impact OS (HR 1.97; 95 %CI, 0.57-6.77) (Sabolch et al., 2015).

A larger study of 171 patients found RT more often administered in patients with positive margins. Although no OS difference was observed overall, RT reduced the yearly risk of death by 40 % in this subgroup (HR 0.60; 95 %CI 0.40-0.92) (Nelson et al., 2018).

A meta-analysis confirmed that adjuvant RT significantly reduces local recurrence (HR 0.24; 95 %CI 0.12-0.49) but does not improve OS (Tsuboi et al., 2024). More recently, a single-institution analysis re- ported improvements in local RFS, overall RFS, and OS with RT (Gharzai et al., 2019).

The 2018 ESE/ENSAT guidelines did not establish a consensus on the routine use of adjuvant RT in ACC (Fassnacht et al., 2020a, 2018). They discourage its use in Stage I-II disease with R0 resection but suggest RT (50-70 Gy over four weeks) may be considered for Stage III cases or those with R1/Rx margins, often in combination with mitotane (Fassnacht et al., 2020a, 2018).

4.4. Systemic therapy

In 2012, Fassnacht et al. showed that combining chemotherapy with mitotane (EDP-M) led to higher overall response rates (ORR) and longer PFS than streptozocin plus mitotane (S-M). The FIRM-ACT trial, the first randomized study in ACC, enrolled 304 patients and evaluated EDP-M: etoposide (100 mg/m2 IV on days 2-4), doxorubicin (40 mg/m2 IV on day 1), cisplatin (40 mg/m2 IV on days 3-4), and daily oral mitotane, targeting plasma levels of 14-20 mg/L. Although OS did not differ significantly between arms (14.8 vs. 12.0 months; HR 0.79; P = 0.07), EDP-M showed superior ORR (23.2 % vs. 9.2 %; P < 0.001) and PFS (5.0 vs. 2.1 months; HR 0.55; P < 0.001) (Fassnacht et al., 2012). EDP-M is now the first-line standard for ACC, despite increased toxicity, mainly bone marrow suppression and infections. Mitotane monotherapy may be appropriate for patients with low tumour burden or poor performance status (Megerle et al., 2018).

Mitotane can be initiated with either a low-dose (starting at 1 g/day, increasing to 3 g/day over two weeks) or a high-dose regimen (starting

at 1.5 g/day, increasing to 6 g/day over 4-6 days). No significant dif- ferences in therapeutic levels or side effects have been reported between the two approaches (Kerkhofs et al., 2013).

Several alternative regimens have been explored. A phase II study of docetaxel and cisplatin administered every three weeks in metastatic ACC yielded a 21 % ORR, with neutropenia as the most common grade 3-4 toxicity; median PFS and OS were 3 months (95 %CI, 0.7-5.3) and 12.5 months (95 %CI, 6-19), respectively (Urup et al., 2013).

In a multicentre phase II study of 28 patients, second/third-line therapy with gemcitabine and 5FU/capecitabine (maintaining mito- tane) produced one complete response (CR) and one partial response (PR), with median PFS of 5.3 months (1-43) and OS of 9.8 months (3-73), accompanied by manageable toxicity (Sperone et al., 2010).

Capecitabine plus bevacizumab, used as salvage therapy in 10 pa- tients, demonstrated no objective responses and a median survival of 4.1 months (Wortmann et al., 2010). In 2016, Kroiss et al. evaluated tro- fosfamide in 27 refractory ACC patients; most experienced only grade 1-2 adverse events, with 23 % achieving stable disease (SD), a median PFS of 84 days (95 %CI, 74-95) and median OS 198 days (95 %CI, 89-307) (Kroiss et al., 2016).

Another study by Kroiss on thalidomide in 27 refractory ACC patients showed no responses, with median PFS of 2.8 months and OS of 9.1 months, and only mild side effects (Kroiss et al., 2019a).

Retrospective evaluation of temozolomide in 28 pretreated ACC patients, revealed a 21.5 % (95 %CI, 6.5-27.5) ORR, a 35.8 % (95 %CI, 17.8-53.8) disease control rate (DCR) with median PFS of 3.5 months (1.2-24.2) and OS of 7.5 months (2-38.8) (Cosentini et al., 2019).

A phase II trial with cabazitaxel in 25 patients as second/third-line therapy reported no objective responses, though 36 % had SD; median PFS and OS were 1.5 months (0.3-7) and 6.0 months (1-22.2 months), respectively (Laganà et al., 2022).

Ferrero et al. investigated low-dose metronomic chemotherapy in 5 patients with refractory ACC progressed on gemcitabine/capecitabine. Treatment with daily oral etoposide (50 mg) or cyclophosphamide (50 mg) plus mitotane yielded PRs or SD in two patients with good tolerability (Ferrero et al., 2013).

Turla et al. investigated the efficacy of Megestrol Acetate in addition to EDP-M as first line therapy in 24 patients with metastatic or unre- sectable ACCs with low performance status (Turla et al., 2023). The association was well tolerated and non-inferior to EDP-M administered in patients will good performance status.

ACC develops resistance to mitotane and chemotherapy through pharmacokinetic, cellular, and microenvironmental mechanisms. Mito- tane is rapidly metabolized by hepatic CYP3A4, overexpressed in ACC, reducing drug levels and efficacy (Seidel et al., 2020). Although mito- tane disrupts mitochondria to induce apoptosis, resistant cells upregu- late anti-apoptotic proteins such as BCL-2, BCL-XL, and MCL-1. TP53 mutations further impair apoptosis. Compensatory steroidogenesis via STAR and CYP11A1 offsets mitotane’s inhibition of cholesterol meta- bolism (Ronchi et al., 2009), while ABC transporters (ABCB1, ABCG2) lower intracellular drug concentrations (Morrison et al., 2021). Che- moresistance is also driven by enhanced DNA repair, with over- expression of ERCC1, BRCA1, and RAD51 enabling survival against DNA-damaging agents like cisplatin and etoposide (Greb and Reikes, 2021). Survival pathways including NF-KB and AKT promote expression of additional anti-apoptotic factors. The tumour microenvironment contributes via HIF-1a-mediated MDR1/ABCB1 upregulation and im- mune suppression under hypoxia. Moreover, stromal cells release IL-6 and TGF-6, activating JAK2/STAT3 signaling and further supporting tumour survival and immune evasion (Georgantzoglou et al., 2021). Table 4

4.4.1. Immune checkpoint inhibitors

Immunotherapy has emerged as a potential treatment for advanced ACC, though clinical results remain mixed. While ACC exhibits a high rate of single germline mutations, its low somatic mutation burden

generally limits responsiveness to immune checkpoint inhibitors (ICIs) (Assié et al., 2014; Zheng et al., 2016). However, a subset of patients, particularly those MMR deficiencies, present in up to 14 % of cases, may benefit from ICIs (Assié et al., 2014; Zheng et al., 2016; Remde et al., 2023) (Table 5).

Several phase II trials have investigated pembrolizumab, an anti-PD- 1 antibody, in advanced ACC. In the NCT02721732 trial, 14 patients were treated with an ORR of 14 % (95 %CI, 2-43 %), including two PRs, seven SD, and five progressions. Six patients experienced disease stabi- lization for over four months, suggesting benefit in patients with indo- lent disease (Habra et al., 2019).

Another trial (NCT02673333) involving 39 patients showed an ORR of 23 % and DCR of 52 %. Median PFS was 2.1 months (95 %CI, 2.0-10.7), and OS was 24.9 months (95 %CI, 4.2-NR), with 13 % of patients experiencing grade 3 or 4 treatment-related adverse events (TRAEs). Neither PD-L1 expression nor MSI-H/MMR-D status was pre- dictive of response (Raj et al., 2020).

Naing et al. evaluated pembrolizumab in a phase II basket trial with 127 rare cancer patients, reporting an ORR of 15 % and clinical benefit rate of 54 % in the ACC subgroup, supporting pembrolizumab’s inclu- sion in NCCN guidelines, particularly for patients unfit for chemo- therapy or with slowly progressing tumours (Shah et al., 2021; Naing et al., 2020).

Nivolumab, another PD-1 inhibitor, was studied in a small phase II trial (NCT02720484) involving 10 patients who had failed platinum- based therapies. Only one unconfirmed PR and two DS were observed, leading to early trial termination due to limited efficacy (Carneiro et al., 2019).

The JAVELIN phase Ib trial (NCT01772004) assessed avelumab, a PD-L1 inhibitor, in 50 ACC patients, half of whom continued mitotane. The ORR was 6.0 %, with three PRs. SD was observed in 42 %, yielding a DCR of 48 %. Median PFS and OS were 2.6 months (95 %CI, 1.4-4.0) and 10.6 months (95 %CI, 7.4-15.0), respectively. While ORR was higher in PD-L1 + tumours (16.7 % vs. 3.3 %), the difference was not statistically significant. Most common TRAEs were nausea, fatigue, and hypothyroidism (Le Tourneau et al., 2018).

Atezolizumab, anti-PD-L1, combined with cabozantinib, was evalu- ated in a phase II basket trial including 24 ACC patients. ORR was 8.3 %, and median PFS was 2.9 months (Hernando et al., 2024).

No dedicated trials have assessed dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) in ACC, though small cohorts exist. The CA209-538 study enrolled six ACC patients treated with nivolumab and ipilimumab, reporting a 33 % ORR and 66 % DCR (Klein et al., 2021).

The SWOG S1609 basket trial included 21 ACC patients, most of whom were previously treated. It showed an ORR of 14 %, with 6-month PFS and OS rates of 24 % and 76 %, respectively (Patel et al., 2024). In another study evaluating durvalumab, anti-PD-L1, with tremelimumab, anti-CTLA-4, two ACC patients were included, but ACC-specific results were not reported (Edenfield et al., 2021).

MMR/MSI status is the most reliable biomarker for predicting ICI response in ACC, with MSI tumours showing higher responsiveness, consistent with other cancers (Araujo-Castro et al., 2021). PD-L1 expression has limited value, while low tumour mutational burden ap- pears more influential in determining resistance. Cortisol-producing tumours may suppress systemic immunity, further reducing efficacy. The tumour microenvironment, characterized by low T-cell infiltration and regulatory immune cells, also impairs response. Other markers, like interferon-gamma signatures and HLA genotypes, remain inconclusive (Georgantzoglou et al., 2021; Araujo-Castro et al., 2021). Currently, immunotherapy is mainly considered for MSI-H or, less commonly, high-tumour molecular burden (TMB) ACCs (Shah et al., 2021). Pem- brolizumab, approved for MMR-deficient tumours, has shown the most encouraging results. Ongoing research aims to refine biomarker-driven patient selection and address resistance mechanisms.

Table 4 List of trials exploring the efficacy of chemotherapy and/or Mitotane.

Trial Name	Trial Design	Authors and Year of Publication	Population	Investigated Schemes	Outcomes	References
ADIUVO	Phase III RCT Open Label	Terzolo M,	91 pts with R0, stage I-III, Ki67 ≤ 10% ACC	Adjuvant Mitotane for 2 years vs Surveillance	OS (primary);	(Terzolo et al.,
		Fassnacht M et al. 2023			RFS (secondary)	2023)
ADIUVO-2	Phase III RCT	Mouhammed A	240 pts with High Risk ACC (Stage I-III, R0 relapsed within 90 days, R1, Rx, Ki67 >10%)	Adjuvant EDP x 4 cycles plus Mitotane for 2 years vs Adjuvant Mitotane for 2 years	RFS (primary); OS, Safety, QoL (secondary)	(A Randomized
		Habra Last Update October 2024, ongoing				Registry Trial of Adjuvant Mitotane vs, 2018)
ACACIA	Phase III RCT Open Label		pts with ACC and Ki67 ≥ 10 % after resection (Stage I-III)	Adjuvant EP x 4 cycles plus Mitotane for 2 years vs adjuvant mitotane for 2 years or observation	RFS (primary); OS (secondary)	NA
FIRM-ACT	Phase III RCT Open Label	Fassnacht M, Terzolo M et al. 2012	304 pts with Advanced ACC	EDP-Mitotane vs STZ- Mitotane	OS (primary); PFS, Tumour Response, QoL	(Fassnacht et al., 2012)
NCT00453674	Multicentre cohort study	Megerle F, Herrmann W et al.	127 pts with advanced ACC	Mitotane monotherapy	(secondary) PFS, OS	(Megerle et al., 2018)
		2018
Comparison of two mitotane starting dose regimens in patients with advanced adrenocortical carcinoma	Prospective, open-label, multicentre trial	Kerkhofs TM, Baudin E, Terzolo M et al. 2013	40 mitotane-naïve pts with metastatic ACC	Low vs High Mitotane dose regimen	The difference in median mitotane plasma levels between both treatment groups	(Kerkhofs et al., 2013)
NCT00324012		TUrup, W Z Pawlak et al. 1994	19 pts with metastatic ACC	Docetaxel plus Cisplatin	ORR (primary); Safety, PFS, OS	(Urup et al., 2013)
					(secondary)
Gemcitabine plus metronomic 5- fluorouracil or	Multicenter Phase II Study	Sperone P, Ferrero A et al. 2010	28 pts whit II/III-line advanced ACC	Mitotane plus Gemcitabine plus metronomic 5-FU or capecitabine	PFS (primary); Tumour Response Rate, TTP, OS (secondary)	(Sperone et al., 2010)
capecitabine as a second-/third-line chemotherapy in advanced ACC: a multicenter phase II study
Activity and safety of temozolomide in advanced adrenocortical carcinoma patients	Retrospective study	Deborah Cosentini, Giuseppe Badalamenti et al. 2019	28 pts with pretreated advanced ACC	Temozolomide 200 mg/ m2/die given for 5 consecutive days every 28 days	DCR (primary); OS, PFS, Safety (secondary)	(Cosentini et al., 2019)
NCT03257891	Single-arm Phase II trial	Laganà M, Grisanti S et al. 2022	25 pts with II/III-line advanced ACC	Cabazitaxel plus Mitotane (only in pts with secretory ACC)	PFS (primary); ORR, OS, hormone response in secretory ACC	(Laganà et al., 2022)
					(secondary) ORR (primary) TTP, OS (secondary)
Etoposide, doxorubicin and cisplatin plus mitotane in the treatment of advanced adrenocortical carcinoma: a	Multicentre Phase II trial	Berruti A, Terzolo M et al. 2005	72 pts with non resectable advanced ACC	EDP for maximum 6 cycles plus Mitotane		(Berruti et al., 2005)
large prospective phase II trial
Mitotane associated with etoposide, doxorubicin, and cisplatin in the treatment of advanced adrenocortical carcinoma. Italian Group for the Study of Adrenal Cancer	Multicentre Phase II trial	Berruti A, Terzolo M et al. 1998	28 pts with non resectable advanced ACC	EDP plus Mitotane	ORR, TTP; hormone response	(Berruti et al., 1998)
Phase II trial of mitotane and cisplatin in patients with adrenal carcinoma: a Southwest Oncology Group study	Phase II trial	Bukowski RM, Wolfe M et al. 1993	37 pts with residual or metastatic ACC stratified by risk	CDPP 100 mg/m2 or 75 mg/m2 plus Mitotane	ORR, DOR	(Bukowski et al., 1993)
Streptozocin and o,p'DDD in the treatment of adrenocortical cancer patients: long-term survival in its adjuvant use	Phase II trial	Khan TS, Imam H et al. 2000	40 pts with ACC	Mitotane plus STZ	DFS, OS	(Khan et al., 2000)
Gemcitabine-Based Chemotherapy in Adrenocortical Carcinoma: A Multicentre Study of Efficacy and Predictive Factors	Phase II trial	Henning JEK, Deutschbein T et al. 2017	145 pts with advanced ACC	Mitotane plus Gemcitabine plus capecitabine	ORR, PFS	(Henning et al., 2017)

Table 5 Trials evaluating IO, MKIs and combination therapies in ACCs. PFS-4: Progression Free Survival at 4 months; PI: Principal Investigator; QoL: Quality of Life; MTD: Maximum Tolerated Dose; DLT: Dose-Limiting Toxicity; NPR: Non-Progression rate; DOR: Duration of Response; NA: Not Applicable. Case reports and observational studies were excluded.

Trial Name/ID	Trial Design	Authors, Year of Publication	Population	Drugs	Outcomes	Status	References
727P-abstract	Retrospective	Li et al. 2023	37 pts with pretreated advanced ACC	Anlotinib + Tislelizumab	ORR (primary); OS, DCR, PFS, safety (secondary)	Terminated	(LI et al., 2023)
NCT04318730	Phase II open label	Zhu et al. 2024	21 pts with pretreated advanced ACC	Apatinib + Camrelizumab	ORR (primary); OS, DCR, PFS, safety (secondary)	Recruiting Estimated study completation 2025-04-30	(Zhu et al., 2024)
						2025-04-01
NCT01255137	Phase II open label	O'Sullivan et al. 2014	13 pts with pretreated advanced ACC	Axitinib	ORR (primary); OS, DCR, PFS, safety (secondary)	Terminated	(O'Sullivan et al., 2014)
NCT03612232	Phase II open label	PI: Kroiss	Estimated enrollment of 37 pts	Cabozantinib	PFS4 (primary); ORR, PFS, OS, Safety (secondary)	Active, not recruiting. Estimated study completation 2025-04-30	(Kroiss et al., 2019b)
(CaboACC)
NCT03370718	Phase II open label	Campbell et al. 2024	18 pts with pretreated advanced ACC	Cabozantinib	PFS4 (primary); ORR, PFS, OS, Safety (secondary)	Terminated	(Campbell et al., 2024)
NCT02867592	Phase II		Estimated enrollment of 109 pts with sarcomas, Wilms or other rare tumors	Cabozantinib	ORR (primary); Safety, Tissue- banking (secondary)	Active, not recruiting. Estimated study completation 2025-06-25	NA
SAT-175 Trial	Phase II open label	Bedrose et al. 2020	10 pts with pretreated advanced ACC	Cabozantinib	PFS4 (primary); ORR, PFS, OS, Safety (secondary)	Terminated	(Bedrose et al., 2020b)
NA	Retrospective	Kroiss et al. 2020	16 pts with pretreated advanced ACC	Cabozantinib	PFS, OS (primary)	NA	(Kroiss et al., 2020)
NCT04400474, (CABATEN/ GETNE-T1914)	Phase II open label	Hernando et al. 2024	24 pts with pretreated advanced ACC	Cabozantinib + Atezolizumab	ORR (primary); PFS, OS, Safety (secondary)	Terminated	(Hernando et al., 2024)
NCT06006013	Phase II	PI: Bassel Nazha	Estimated enrollment of 21 pts	Cabozantinib + Pembrolizumab	ORR (primary); PFS, OS, Safety (secondary)	Active, not recruiting. Estimated study completation	NA
						2025-12-16
NCT05036434 (ACCOMPLISH)	Phase II single- arm	PI: Tak Yun	Estimated enrollment of 30 pts	Lenvatinib + Pembrolizumab	ORR, DCR (primary)	Enrolling by invitation. Estimated study completation 2026-08-31	NA
NA	Retrospective case series	Bedrose et al. 2020	8 pts with pretreated advanced ACC	Lenvatinib + Pembrolizumab	ORR (primary); PFS, Safety (secondary)	NA	(Bedrose et al., 2020a)
7959-abstract	Single-centre, retrospective	Yaylaci Mert et al. 2024	17 pts with pretreated advanced ACC	Cabozantinib/Lenvatinib + Pembrolizumab	ORR, DCR, PFS, OS, safety (primary)	NA	(Yaylaci Mert et al., 2024)
NA	Single-centre study	Miller et al. 2020	8 pts with unresectable/ advanced ACC	Cabozantinib/Lenvatinib + Pembrolizumab	ORR, DCR, OS, PFS (primary)	Teminated	(Miller et al., 2020)
NCT00453895 (SIRAC)	Phase II open label	Kroiss et al. 2012	39 pts with pretreated advanced ACC	Sunitinib	ORR, PFS (primary);	Terminated	(Kroiss et al., 2012)
NA	Phase I	Rini et al. 2009	5 pts with pretreated advanced ACC	Sunitinib + Bevacizumab	MTD, DLT	Terminated	(Rini et al., 2009)
NA	Phase I	Brell et al. 2012	1 pt with pretreated advanced ACC	Sunitinib + Gemcitabine	MTD	Terminated	(Brell et al., 2012)
NCT00786110	Phase II open label	Berruti et al. 2012	10 pts with pretreated advanced ACC	Sorafenib + Paclitaxel	ORR (primary)	Terminated	(Berruti et al., 2012)
NCT01514526	Phase II open label	Garcia-Donas et al. 2013	17 pts with unresectable/ advanced ACC	Dovitinib	ORR (primary); PFS, OS, Safety, QoL (secondary)	Terminated	(Garcia-Donas et al., 2013)
NA	Phase II multi- centre	Gross et al. 2006	4 pts with pretreated advanced ACC	Imatinib	ORR	Terminated	(Gross et al., 2006b)

(continued on next page)

Table 5 (continued)

Trial Name/ID	Trial Design	Authors, Year of Publication	Population	Drugs	Outcomes	Status	References
NCT00354523	Phase I/II	Halperin et al.	5 pts with pretreated advanced ACC	Imatinib + Dacarbazine + Capecitabine	MTD (primary); ORR (secondary)	Terminated	(Halperin et al., 2014)
NCT02721732	Phase II	Habra et al. 2019	157 pts unresectable or advanced rare tumours	Pembrolizumab	NPR (primary); ORR, PFS, Safety (secondary)	Active, not recruiting. Estimated study completation 2025-12-31	(Habra et al., 2019)
NCT05563467 (PEMBR-01)	Phase II multi- centre	NA	Estimated enrollment of 21 pts with pretreated advanced ACC	Pembrolizumab	ORR (primary); Safety, QoL (secondary)	Active,recruiting. Estimated study completation 2027-05-31	NA
NCT02673333	Phase II	Raj et al. 2020	39 pts with pretreated advanced ACC	Pembrolizumab	ORR (primary); PFS, OS (secondary)	Unknown status	(Raj et al., 2020)
NCT06066333	Phase II	PI: Raj Nitya	Estimated enrollment of 12 pts with pretreated advanced ACC	Pembrolizumab + Radiotherapy	Safety	Active, recruiting. Estimated study completation 2026-09-27	NA
NCT05634577	Phase II	PI: Habra Mouhammed	3 pts with advanced ACC	Pembrolizumab + Mitotane	ORR	Terminated	NA
NCT04373265	Phase Ib	Habra et al. 2021	15 pts with cortisol producing advanced ACC	Pembrolizumab + Relacorilant	DLT (primary); NPR, PFS, Relacorilant plasma concentrations,	Terminated	(Habra et al., 2021)
					Safety (secondary) ORR, DCR, PFS, OS, Safety
NA	Retrospective	Remde et al. 2023	54 pts with pretreated advanced ACC	Pembrolizumab:59 % Nivolumab:24 % Avelumab:11 % Atezolizumab:2 % Ipilimumab and nivolumab:4 %		NA	(Remde et al., 2023)
NCT01772004	Phase 1b dose expansion	Le Tourneau et al. 2018	50 pts with pretreated advanced ACC	Avelumab (50 % of the pts received concurrent mitotane)	ORR, DCR, PFS, OS, Safety	Terminated	(Le Tourneau et al., 2018)
NCT02720484	Phase II open label	Carneiro et al. 2019	10 pts with pretreated advanced ACC	Nivolumab	ORR (primary); PFS, OS, Safety (secondary)	Terminated	(Carneiro et al., 2019)
NCT03333616	Phase II	McGregor et al. 2021	55 pts with rare genitourinary malignancies,	Nivolumab + ipilimumab	ORR (primary); DOR, PFS, OS, Safety (secondary)	Terminated	(McGregor et al., 2021)
			16 pts whit ACC 6 pts with pretreated advanced ACC		DCR (primary); Immuno-signature or predictive biomarkers identification (secondary)	Terminated	(Klein et al., 2021) (Patel et al., 2024)
CA209-538	Phase II open label	Klein et al. 2021		Nivolumab + ipilimumab
NCT02834013	Phase II	Patel et al. 2024	21 pts with pretreated advanced ACC	Nivolumab + ipilimumab	ORR (primary); DCR, PFS, OS (secondary)	Active, not recruiting
NCT04187404 (SPENCER)	Phase I/II	PI: Paillarse Jean-Michel	ACC and malignant pheochromocy toma/	Nivolumab + EO2401	Safety (primary); PFS, OS, Immuno- genicity assessment (secondary)	Terminated	(Baudin et al., 2022)
NCT02637531 (MARIO-1)	Phase I	Hong et al.	paraganglioma 219 pts with advanced solid tumours, including 5 ACC pts (Cohort G)	Nivolumab + IPI-549 (Eganelisib)	DLT, ORR, DOR, PFS, OS, IPI-549 plasma concentrations	Active, not recruiting	(Hong et al., 2023)
NA	Phase II	Edenfield et al. 2021	50 pts with rare cancers, including 2 pts with ACC	Durvalumab + tremelimumab	ORR, Safety	Terminated	(Edenfield et al., 2021)

4.4.2. Multi-kinase inhibitors (MKIs)

ACC frequently shows upregulation of growth factor pathways (VEGF, FGFR, IGF, etc.) that promote angiogenesis and tumour prolif- eration, prompting trials of multi-kinase inhibitors (MKIs) targeting these pathways. While not yet standard of care, several MKIs have demonstrated modest activity (Table 5).

A phase II trial of dovitinib, an FGFR inhibitor, in 17 patients with metastatic or locally advanced ACC reported no objective responses, a median PFS of 1.8 months (95 %CI, 1.3-2.25), and SD in 23 % at 6 months (Garcia-Donas et al., 2013). Despite FGFR overexpression in ACC, this outcome suggests inadequate target inhibition or resistance via alternative pathways.

Axitinib, anti-VEGF, showed a median PFS of 5.48 months and OS of 13.7 months in 13 metastatic ACC patients (O’Sullivan et al., 2014).

Cabozantinib, targeting c-MET, VEGF, AXL, and RET, yielded a me- dian PFS of 6 months in 18 patients with progressive ACC following mitotane (Campbell et al., 2024). The ongoing CaboACC phase II trial (NCT03612232) is further assessing cabozantinib in advanced ACC (Kroiss et al., 2019b).

A promising combination of Lenvatinib with pembrolizumab showed PRs s in 25 % and SD in 12.5 % of patients in a retrospective series (Bedrose et al., 2020a), supporting the ACCOMPLISH phase II trial (NCT05036434).

Sunitinib produced a median PFS of 2.8 months and OS of 5.4 months in 35 refractory ACC patients, suggesting limited benefit (Kroiss et al., 2012).

A phase II trial of Apatinib, VEGFR-1/2/3 inhibitor, with Camreli- zumab, anti-PD-1, in 21 pretreated ACC patients showed an ORR of 52 % (95 %CI, 30-74 %) and DCR of 95 % (95 %CI, 76-100 %). For the 11 patients who achieved PR, the median time to response was 2.8 months (0.8-12.6), and median duration of response was not reached. The median PFS was 13.3 months (95 %CI, 8.4-NR) and the median OS was 20.9 months (95 %CI, 11.0-NR). TRAEs occurred in 9 patients (Zhu et al., 2024).

Anlotinib, VEGFR, FGFR, PDGFR, c-Kit inhibitor, combined with Tislelizumab, anti-PD-1, in 37 patients yielded an ORR of 35.1 % and DCR of 78.4 %. Median PFS was 8.2 months (95 %CI, 5.5-15.2) and median OS was 30.6 months (95 %CI 21.1-NR) (LI et al., 2023).

Sorafenib combined with paclitaxel failed to achieve objective re- sponses in a phase II trial of 25 patients who progressed after mitotane and chemotherapy, with all experiencing disease progression (Berruti et al., 2012).

Imatinib, a c-Kit inhibitor, also showed no activity in a phase I/II basket trial (Gross et al., 2006a).

Overall, MKIs have shown modest activity in ACC, typically yielding low response rates but occasional disease stabilization. Early agents like sunitinib, sorafenib, and axitinib demonstrated minimal tumour shrinkage, suggesting limited benefit from VEGF/PDGFR inhibition alone, though variable responses reflect tumour heterogeneity. The rarity of ACC has limited randomized trials, with most evidence coming from small, single-arm studies, preventing robust comparisons or meta- analyses.

A key challenge is concurrent mitotane therapy, which induces CYP3A4 and accelerates MKI metabolism, reducing drug levels and ef- ficacy (Fay et al., 2014). Although some studies attempt mitotane washout, this is not always feasible in clinical practice (Wierman, 2024). Additionally, intrinsic tumour resistance and an immunosuppressive microenvironment limit MKI effectiveness. These agents fail to signifi- cantly alter immune infiltration, with persistent suppressive cell pop- ulations impeding antitumor responses (Cerquetti et al., 2021).

This has led to interest in combining MKIs with immune checkpoint inhibitors. Ongoing trials of cabozantinib or lenvatinib with anti-PD-1 agents (e.g., pembrolizumab) aim to enhance immune activation and improve outcomes (Bedrose et al., 2020a). Well-designed trials are ur- gently needed to confirm efficacy, optimize combinations, and inform treatment sequencing.

4.4.3. IGF2/IGF1-R pathway inhibitors

IGF overexpression and activation of IGF-1R and mTOR pathways are common in ACC, prompting trials of targeted therapies. However, clinical results have been limited.

In a phase I study, figitumumab, an anti-IGF-1R antibody, was tested in 14 patients with refractory ACC. While no objective responses were observed, 57 % achieved SD, with mild toxicities such as hyperglycemia, nausea, and fatigue (Haluska et al., 2010).

A 2013 phase II trial combined cixutumumab (IGF-1R antibody) with the mTOR inhibitor temsirolimus in 26 patients. Although no objective responses occurred, over 40 % achieved SD. Side effects included

mucositis, thrombocytopenia, and hyperglycemia (Naing et al., 2013).

Another phase II trial by Lerario et al. evaluated cixutumumab plus mitotane as first-line therapy in 20 patients but was discontinued due to slow enrollment and limited efficacy. Only one PR was seen, with a median PFS of 1.5 months (Lerario et al., 2014b).

Linsitinib, an oral IGF-1R/insulin receptor inhibitor, showed early promise in a phase I trial (Macaulay et al., 2016), but failed in the phase III GALACCTIC trial. Among 139 patients with advanced ACC, no sur- vival benefit was observed over placebo (median OS 323 vs. 356 days; P = 0.77) (Fassnacht et al., 2015).

Despite high IGF2 expression in ACC, targeting IGF-1R has not been effective, likely due to intrinsic resistance and pathway redundancy. Tumours may bypass IGF-1R via insulin receptors or activate alternative pathways such as EGFR or Wnt (Logié et al., 1999). No predictive bio- markers beyond IGF2 have been identified, and since nearly all ACCs overproduce IGF2, it lacks discriminatory value. Only a subset of “IGF2-addicted” tumours may benefit but identifying them remains a challenge.

Currently, IGF-1R inhibitors are not used outside clinical trials due to minimal efficacy. Future strategies may require combination therapies or multi-target approaches.

4.4.4. mTORC1 inhibitors

Hyperactivation of RAS/RAF/ERK and PI3K/AKT/mTOR pathways is common in ACC. Everolimus, an mTOR inhibitor, showed no clinical benefit in a small exploratory study involving 4 advanced ACC patients (Fraenkel et al., 2013). Temsirolimus was used in the combination trial with cixutumumab mentioned above (Naing et al., 2013). As mono- therapy it hasn’t been specifically reported in ACC, but the combo’s lack of success indicates temsirolimus alone likely has minimal activity too.

4.4.5. EGFR inhibitors

EGFR is expressed in ACC and may indicate malignancy but lacks predictive value. Erlotinib showed poor activity in combination with gemcitabine in advanced refractory ACC, in a study enrolling 10 patients with progressive ACC after two to four previous systemic therapies (Quinkler et al., 2008). Despite modest in vitro growth inhibition, EGFR-targeted therapy is not recommended as salvage treatment in advanced ACC.

4.4.6. PLK-1 and PI3Ky inhibitor

Polo-like kinase 1 (PLK-1), involved in cell division, is overexpressed in ACC. In a study of 16 ACC patients treated with the PLK1-targeted siRNA TKM-080301, one showed near-complete tumour necrosis, but most discontinued early due to progression or side effects (Warmington et al., 2023).

Eganelisib (IPI-549), a PI3K-y inhibitor targeting the tumour immune microenvironment, showed a PR in one ACC patient during a phase I trial with nivolumab; however, none of the 5 patients in the expansion cohort responded (Hong et al., 2023). These findings suggest limited and inconsistent activity, highlighting the need for further investigation of these agents in ACC.

4.4.7. Other drugs

Gossypol (AT-101) is a BH3 mimetic derived from cottonseed oil that inhibits BCL-xL, BCL-2, BCL-w, and MCL-1, promoting apoptosis in cancer cells (Flack et al., 1993). Though tested in various malignancies, a phase II trial in 29 advanced ACC patients showed no clinical benefit (Xie et al., 2019).

Progesterone (Pg) has demonstrated anti-tumour activity in ACC. Preclinical studies showed Pg reduced cell viability in ACC cell lines and primary cultures by activating progesterone receptors through genomic and non-genomic mechanisms (Fragni et al., 2019). Pg also inhibited ß-catenin nuclear translocation and enhanced mitotane’s effects.

Nevanimibe, a selective SOAT-1 inhibitor, showed some preclinical activity but no significant responses in a phase I trial involving 48 ACC

patients (Smith et al., 2020).

About 50 % of ACCs produce glucocorticoids, and hypercortisolism is linked to poorer outcomes. Inhibiting the glucocorticoid receptor (GR) may boost immune-related gene expression and anti-tumor immunity. A phase Ib trial (NCT04373265) is evaluating relacorilant, a nonsteroidal GR antagonist, with pembrolizumab in advanced ACC patients with hypercortisolism (Habra et al., 2021).

While these approaches show potential in preclinical or early clinical settings, further investigation is needed to assess their therapeutic role in ACC.

4.4.8. Radiotheranostics

A promising strategy for ACC involves targeted radionuclide therapy, leveraging specific adrenal cortex uptake mechanisms. Radiolabeled metomidate, initially used for imaging, has shown strong uptake in both primary and metastatic ACC (Kreissl et al., 2013). Hahner et al. devel- oped I-131-metomidate to target metastatic lesions (Hahner et al., 2012). In a study of 11 advanced ACC patients ineligible for surgery and showing high [123I]IMTO uptake, treatment with [131I]IMTO (1.6-20 GBq in one to three cycles) resulted in SD in 5 patients, with a median PFS of 14 months. Side effects were mild, primarily bone marrow related (Hahner et al., 2012).

Somatostatin receptors, often expressed in ACC, offer another ther- apeutic target. Grisanti et al. evaluated Ga-68 DOTATOC uptake in 19 patients, finding significant metastatic site uptake in two. One patient treated with Lu-177 DOTATOC had SD for 12 months, another for 4 months with symptom improvement (Hahner et al., 2022, 2024; Grisanti et al., 2020b).

These investigational radioligand therapies illustrate the theranostic approach, using diagnostic imaging (e.g., Metomidate PET or 68Ga- DOTATOC PET) to identify suitable targets, followed by therapeutic radioisotope treatment. Though highly specialized, they offer a novel way to deliver radiation directly to ACC cells and warrant further exploration.

4.4.9. HDAC inhibitors

Histone tails, which contain positively charged amine groups on their lysine and arginine amino acids, interact with the negatively charged phosphate groups of the DNA backbone. Acetylation neutralizes these positive charges, weakening the histone-DNA interaction. This reduced binding allows the chromatin to expand, facilitating genetic transcription. Histone deacetylases (HDACs) remove the acetyl groups, restoring the positive charges on histone tails, which then bind tightly to the DNA backbone. This increased DNA binding condenses the chro- matin structure and inhibits transcription. Hyperacetylated chromatin is transcriptionally active, while hypoacetylated chromatin is transcrip- tionally silent. Demeure et al (Demeure et al., 2013)., Davis et al (Davis et al., 2016)., and Montgomery et al (Montgomery and Hull, 2019). investigated the effects of HDAC inhibitors (HDACi) in ACC. In Demeure’s study, ACC cell lines (NCI-H295R and SW13) were analyzed for gene expression and biomarkers. Dysregulation of the p53 pathway and G2/M transition was identified, particularly overexpression of PTTG1, which encodes securin, an anaphase-promoting complex (APC) substrate that associates with a separin until activation of the APC. PTTG1 overexpression was inversely correlated with survival. To assess its potential as a therapeutic target, researchers treated ACC cells with Vorinostat (SAHA), an HDAC inhibitor known to reduce PTTG1 expression in colorectal cancer. Vorinostat binds the active site of HDACs, chelating zinc ions necessary for their function. It inhibited ACC cell growth, causing a dose-dependent reduction in securin protein levels in both lines. Davis et al. studied HDAC inhibition in SW13 cell subtypes: SW13 + (expressing vimentin) and SW13- (lacking vimentin, BRM, and BRG1, ATPase subunits of the SWI/SNF chromatin remodeling complex). BRM and BRG1 function as tumour suppressors but are often silenced in cancer. Trichostatin-A (TSA), an HDACi, induced a pheno- typic switch from SW13- to SW13 + , restoring BRM expression. This

reinstated BRM’s role in suppressing glucocorticoid receptor-induced transcription, ultimately reducing cell proliferation. Montgomery et al. found FK228 to be the most effective HDACi in this model.

4.4.10. DNA methyltransferase inhibitors

The primary regulators of the methylator phenotype are DNMT1 and DNMT3A. The h-CIMP phenotype is associated with high expression of genes involved in cell proliferation and survival, while genes related to immune response are hypermethylated and silenced, resulting in poor immune cell infiltration and reduced immune response. This contributes to poor prognosis. Based on these findings, a combination of demethy- lating agents (e.g. 5-Azacitidine) and immunotherapy has been pro- posed as a potential treatment strategy for h-CIMP ACC. Suh-I et al (Suh et al., 2010). investigated Decitabine, a DNMT inhibitor that removes methyl groups from silenced promoter sequences. Decitabine has a dose-dependent mechanism: at low doses, it inhibits methylation and reactivates gene expression, at high doses, it induces cytotoxicity by trapping DNMT. The treatment of ACC cells with Decitabine demon- strated antineoplastic effects across key ACC hallmarks, including reduced cortisol secretion, decreased cell proliferation, lower cellular invasion. To confirm its demethylating effects, the study examined gene expression at chromosomal region 11q13, known for LOH in 70-100 % of sporadic ACCs. Six underexpressed genes (DDB1, MRPL48, NDUFS8, PRDX5, SERPING1, TM7SF2) were analyzed. NDUFS8 and PRDX5 showed significant re-expression after Decitabine treatment. Interest- ingly, DDB1 and TM7SF2 expression decreased post-treatment, an un- expected result requiring further investigation. This paradoxical response highlights the complexity of epigenetic regulation and suggests that while demethylation is a promising therapeutic approach, addi- tional studies are needed to refine its application in ACC.

5. Conclusions

Despite substantial progress in understanding the molecular land- scape of ACC, clinical translation of precision oncology remains limited. Tumours often harbour complex, heterogeneous alterations across genomic, epigenetic, and transcriptomic layers, yet reliable predictive biomarkers are lacking. No molecular feature consistently predicts response to mitotane, chemotherapy, or immunotherapy, and markers such as PD-L1, MSI, or TMB have shown inconsistent utility in clinical settings (Araujo-Castro et al., 2021; Garcia-Donas et al., 2013). Most altered genes, including TP53 and components of the Wnt/ß-catenin pathway, remain undruggable (Lippert et al., 2022). Therapeutic resis- tance is further compounded by low immunogenicity, a suppressive tumour microenvironment, and signaling redundancy (Araujo-Castro et al., 2021). Preclinical models fail to fully capture tumour heteroge- neity, underscoring the need for more representative systems such as patient-derived organoids and ex vivo cultures (Grisanti et al., 2022).

From an implementation standpoint, the lack of standardized guidelines for molecular testing, regarding timing, platforms, and interpretation, limits clinical utility. High-yield gene panels and methylation-based classifiers offer feasible diagnostic tools and could be integrated into routine workflows, particularly with the support of specialized multidisciplinary tumour boards. Germline testing should also be systematically considered in selected cases.

Emerging strategies such as liquid biopsy, biomarker-enriched trials, and therapeutic approaches targeting epigenetic or metabolic vulnera- bilities hold promise (Baudin et al., 2022; Lippert et al., 2022). However, their success depends on improving access to testing and fostering clinician education and patient engagement.

To move beyond current treatment limitations, a coordinated effort is needed to implement molecular stratification into clinical decision- making. This includes identifying patients with hypermutated or immunogenic tumours suitable for immunotherapy and refining risk- adapted strategies to avoid overtreatment. Ongoing research and in- ternational collaboration are essential to making precision medicine a

tangible reality in ACC.

Simple summary

Adrenocortical carcinoma (ACC) is a rare and aggressive adrenal malignancy that often presents at an advanced stage and carries a poor prognosis. While surgery is the cornerstone for localized disease, sys- temic treatment options for advanced ACC remain limited and rarely curative. In recent years, advances in genomic and epigenetic profiling have provided new insights into ACC pathogenesis, identifying molec- ular subtypes with prognostic and therapeutic relevance. However, these findings are not yet routinely applied in clinical practice. This review highlights current diagnostic and therapeutic strategies for ACC, emphasizing the need for integrating molecular characterization to improve personalized care and clinical outcomes.

Funding

The author(s) declare that no financial support was received for the research, authorship, and/or publication of this article. Conflicts of In- terest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

CRediT authorship contribution statement

FF: Conceptualization, Supervision, Writing - original draft, Writing - review & editing, Validation. GF: Writing - original draft, Writing - review & editing. MCDM: Supervision, Writing - original draft, Writing - review & editing. RP: Supervision, Writing - original draft, Writing - review & editing. AC: Supervision, Writing - original draft, Writing - review & editing. VD: Supervision, Validation, Writing - review & editing.

Declaration of Competing Interest

All authors declare no conflicts of interest.

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Fabiano Flauto, MD. Medical Oncologist with experience in clinical, laboratory and statistics projects. The main research projects involve all areas involved in genitourinary and adrenal tumours. Medical Oncology Resident at the University of Naples Federico II.

Giulio Ferone, MD. Medical Oncologist with experience in clinical projects, with focus on adrenal tumours. Medical Oncology Resident at the University of Naples Federico II.

Maria Cristina De Martino, MD, Ph.D. Doctor and researcher with experience in clinical and laboratory projects with a lot of publications in renowned journals. The main research projects involve endocrine malignancies and adrenal diseases. Professor at the University of Naples Federico II.

Rosario Pivonello, MD, Ph.D. Doctor and researcher with experience in clinical and laboratory projects with a lot of publications in renowned journals. The main research projects involve endocrine malignancies and adrenal diseases. Professor at the University of Naples Federico II.

Annamaria Colao, MD, Ph.D. Doctor and researcher with experience in clinical and laboratory projects with a lot of publications in renowned journals. The main research projects involve endocrine malignancies and adrenal diseases. Professor at the University of Naples Federico II.

Vincenzo Damiano, MD, Ph.D. Doctor and researcher with experience in clinical and laboratory projects with a lot of publications in renowned journals. The main research projects involve adrenal malignancies, head and neck tumours and thyroid tumors. Medical Oncology and Researcher at the University of Naples Federico II.