Piotr Glinicki ^1,2* Nadia Sawicka-Gutaj ³ Marta Araujo-Castro ^4,5*

• ¹ EndoLab Laboratory, Centre of Postgraduate Medical Education, Warsaw, Poland
• ² Department of Endocrinology, Centre of Postgraduate Medical Education, Warsaw, Masovian, Poland
• ³ Department of Endocrinology, Metabolism and Internal Medicine, Poznan University of Medical Sciences, Poznań, Greater Poland, Poland
• ⁴ Endocrinology & Nutrition Department. Hospital Universitario Ramón y Cajal., Madrid, Asturias, Spain
• ⁵ University of Alcalá, Alcalá de Henares, Madrid, Spain

biochemical research in metabolomics and proteomics, supported by bioinformatics techniques based on artificial intelligence (AI). New biochemical methods for the diagnosis and monitoring of patients with adrenal tumors are currently being developed in two parallel directions. The first direction of research involves classical immunological (immunochemical) methods based on the antigen-antibody reaction. In particular, automated immunochemical methods based on the chemiluminescence reaction (CLIA, ECLIA) are being evaluated, primarily as widely available, inexpensive, and easy-to-use screening tools [1]. These studies include the use of these methods as screening tests in the diagnosis of patients with suspected primary aldosteronism (PA), the feasibility and clinical value of assaying various hormones, e.g. aldosterone, direct renin concentration (DRC), plasma renin activity (PRA), cortisol in tests aimed at differentiating between different subtyping of PA, and, thirdly, the feasibility of assaying free hormone fractions in various biological materials (e.g., free cortisol in saliva) [2][3][4]. The other direction involves chromatographic methods. New possibilities for biochemical diagnostics are enabled by metabolomics research using modern analytical techniques: Liquid chromatography-mass spectrometry (LC-MS/MS), Gas chromatography/mass spectrometry (GC/MS) and others. The development of various chromatographic techniques, especially the LC-MS/MS technique, makes it possible to determine multiple hormones during a single analysis (e.g., steroid hormone profile in plasma or daily urine collection, free metanephrines profile in plasma, etc.) in routine diagnostics [5,6]. The introduction of the ability to determine hormone panels using various chromatographic techniques (mainly LC-MS/MS and GC/MS) into biochemical diagnostics enables new studies both in screening diagnosis of adrenal tumor lesions and in other diagnostic and therapeutic areas. An additional unique feature of hormonal profile studies in biological material from patients with adrenal tumors can be their personalized nature, which can be used in the individual diagnostic-therapeutic process in a given group of patients [7,8]. The advances in the field of molecular studies in adrenal tumors have been made in the molecular characterization of the pheochromocytoma, adrenal Cushing syndrome, PA, and adrenocortical carcinoma (ACC). As for pheochromocytomas, although some older studies reported a prevalence of germline genetic variants in 10% -15% of the patients, more recent studies performed with NGS technology (Next-generation sequencing technology) describe genetic variants in up to 35% -40% of the patients [9][10][11]. The prevalence of genetic variants in these patients has been increasing over the years since new genes such as CSDE1, H3F3A, MET, MERTK and IRP1, have been discovered [10].Furthermore, recent advances in the studies on pheochromocytomas have revealed new molecular events in these tumors. Events that are linked to their genetic landscape and, therefore, provide new therapeutic platforms. In relation to adrenal Cushing syndrome, one of the most recent discoveries has taken place in patients with primary bilateral macronodular adrenal hyperplasia (PBMAH) with the discovery of the role of variants in KDM1A in GIP-mediated cases of Cushing syndrome. It has been reported that 100% of the patients with PBMAH and GIP-responsive Cushing syndrome had a germline variant in KDM1A, compared with 0% of the patients of the control group [11]. The discovery of genetic alterations such as ARMC5 and KDM1A in PBMAH allows early detection of PBMAH in patients' relatives [12]. Regarding PA, several genetic defects in the germline or somatic state have been identified. Although only 5% of the PA are familial [13], currently it is known that about 90% of aldosterone producing adenomas (APAs) are due to somatic variants in genes encoding ion channels or transporters including KCNJ5, CACNA1D, ATP1A1, and ATP2B3 [14]. In the last years, new somatic variants have been identified, including a new one in CACNA1H [15]. In addition, more recently, co-existence of CTNNB1 with G Protein Subunit Alpha Q (GNAQ)/G Protein Subunit Alpha 11 (GNA11) variants has been documented in 59% of APAs [16]. Finally, advances in genetics of ACC have also been reported. Although there are not many current therapy options directly targeting reported ACC alterations, some studies have detected variants in TP53, BRD9, TERT, CTNNB1, CDK4, FLT4 and MDM2 as potentially targetable genetic alterations in patients with ACC [17]. Utilizing blood-based NGS to characterize genomic alterations in advanced ACC is feasible in over 80% of patients the patients, being 50% of them potentially targetable [18]. Thus, in conclusion, advances in the knowledge of the genetic context of functioning adrenal tumors have allowed a better characterization of these tumors, with important implications in the management of these patients, including the personalization of follow-up and treatment, and its importance in the face of genetic counseling for patients and their relatives. The aim of the first article [19] was to compare the clinical presentation and laboratory hormonal diagnostics in patients with two forms of PA: familial hyperaldosteronism (FH) and primary hyperaldosteronism (PA). The study was a meta-analysis conducted on a systematic review of the literature to identify patients with FH. A total of 360 FH cases (246 FH type I, 73 type II, 29 type III and 12 type IV) and 830 sporadic PA patients (from the SPAIN-ALDO registry) were included in the study. Analysis of the results showed a different clinical presentation of patients with FH-I and III compared to sporadic forms of PA. In this regard, FH-I is characterized by a low prevalence of hypokalemia, while FH-III is characterized by severe aldosterone over-secretion causing hypokalemia in more than 85% of patients. The clinical and hormonal phenotype of types II and IV is similar to patients with sporadic PA.Another original article [20] focused on the evaluation of post-operative management of patients with pheochromocytoma assessing hemodynamic stability as one of the main causes of serious complications after surgical treatment, and in extreme cases leading to patient death.The aim of this retrospective study by Canu et al. [21] was to evaluate changes in Luteinizing hormone (LH), sex hormone binding globulin (SHBG), total testosterone (TT) and calculated free testosterone (cFT), the prevalence and type of hypogonadism and sexual function, the latter before and after androgen replacement therapy (ART) in adjuvant mitotane therapy (AMT) treated patients with ACC.Authors suggest to evaluate LH, SHBG, TT and cFT and sexual function during AMT, and start ART in the hypogonadal patients with ACC with sexual dysfunction.The research group of Araujo-Castro et al. [22] investigated to evaluate the prevalence of recurrence among patients with pheochromocytomas and sympathetic paragangliomas (PGLs; together PPGLs) and to identify predictors of recurrence (local recurrence and/or metastatic disease). This retrospective multicenter study included information of 303 patients with PPGLs in follow-up in 19 Spanish tertiary hospitals. The conclusions of this study are since PPGL recurrence can occur at any time after the initial diagnosis of PPGL, it is recommended close follow-up of all patients with PPGL, especially in patients at higher risk of recurrence.In the following article, the authors [23] performed a transcriptomic analysis of adrenal signaling pathways in different forms of endogenous Cushing syndrome to define areas of dysregulation and targets that can be treated. NGS analysis was performed on adrenal samples from patients with PBMAH (n = 10) and control adrenal samples (n = 8). Validation groups included cortisol-producing adenoma (CPA, n=9) and samples from patients undergoing bilateral adrenalectomy for Cushing disease (BADX-CD, n=8). The conclusion of this project was: therapeutic effect was independent of the actions of ACTH, postulating a promising application of PPARG activation in endogenous hypercortisolism.The other article [24], by Díaz-López et al. concerns severe hypokalemia causing rhabdomyolysis (RML) in PA is a rare occurrence, with only a few cases reported over the last four decades. This systematic review and case report aims to gather all published data regarding hypokalemic RML as a presentation of PA, in order to contribute to the early diagnosis of this extremely rare condition. Early detection and management are essential to reduce the frequency of complications such as acute kidney injury.Mansour et al. [25] studied to investigate an integrated diagnostics approach for prediction of the source of aldosterone overproduction in PA in 269 patients from the prospective German Conn Registry with PA were included in this study. Integration of clinical parameters into a radiomics machine learning model improves prediction of the source of aldosterone overproduction and subtyping in patients with PA.Szatko et al. [26] have prepared a mini-review summarizing current data on pathophysiological pathways of cardiac damage caused by catecholamines, the clinical presentation of PPGL-induced cardiomyopathies, and discusses treatment options.The aim of the next study [27] was to assess the diagnostic value of salivary cortisol and cortisone in patients with suspected hypercortisolism including 155 patients with adrenal incidentaloma, and 54 patients with suspected Cushing syndrome. The authors stated that late night salivary cortisol is not sufficiently sensitive or specific to be used for screening patients with suspected hypercortisolism.Instead, late-night salivary cortisone seems to be a promising alternative in patients with adrenal The metanalysis by Li et al. [30] focused on comparing the advantages of robotic posterior retroperitoneal adrenalectomy (RPRA) over laparoscopic posterior retroperitoneal adrenalectomy (LPRA), a total of 675 patients were included. It was found that RPRA is associated with a significantly shorter hospital stay compared to LPRA, while demonstrating comparable operative time, blood loss, conversion rate, and complication rate.The next article [31] describes machine learning models for predicting the difficulty of retroperitoneal laparoscopic adrenalectomy by combining clinical and radiomic characteristics. The models can help surgeons evaluate surgical difficulty, reduce risks, and improve patient benefits.In the following study [32], the authors presented a universal mathematical model for the differential diagnosis of all morphological types of ACC in adults is introduced. The method involves determining eight diagnostically significant indicators that enable the calculation of ACC development probability using specified formulas.Zhanghuang et al. [33] presented an interesting case report of a bilateral adrenal giant medullary lipoma and perform a review of the literature. Patients with adrenal myelolipoma complicated by disorders of sex development can be monitored after resection of the myelolipoma, prior to oculoplastic surgery. In some cases, patients with disorders of sex development may experience spontaneous relief of abnormal manifestations of the external genitalia.In the next article [34] authors presented a case of ACC with liver metastases treated with systemic antitumor therapy combined with local therapy for liver lesion (mitotane combined with TACE+MWA) is reported. The treatment outcome was partial response, and the progression free survival of the patient has been extended to about 28 months so far she was still alive up to now (September 2022) after so far.The Research Group of Enguita et al. [35] found significant differences in the miRNA expression profiles of paragangliomas and pancreatic neuroendocrine tumors, leading to the identification of 6 key miRNAs (miR-10b-3p, miR-10b-5p, and the miRNA families miR-200c/141 and miR-194/192) that can effectively differentiate between the two types of tumors. The next article [36] was presented by authors from Italy. A total of 24 patients with ACC were included in this study. Testosterone deficiency was reported in 10 patients (41.7%) at baseline. In addition, mitotane therapy exposes these patients to further elevated risk of hypogonadism that should be promptly detected and counteracted, since it might have a negative impact on quality of life.Kimura et al. presented a prospective study [37] on mixed corticomedullary tumor of the adrenal gland is an extremely rare tumor characterized by an admixture of steroidogenic cells and chromaffin cells in a single tumor mass simultaneously producing adrenocortical hormones and catecholamines; it is associated with ectopic adrenocorticotropic hormone in some cases.The review [38] described clinically "silent" PPGLs which are characterized by the absence of signs and symptoms associated with catecholamine excess. "Non-secretory" tumors are those with absence of clear catecholamine secretory activity, "biochemically negative" PPGLs are those characterized by plasma or urinary metanephrines below the upper cut-offs of reference intervals and "non-functional" tumors are those with absent catecholamine synthesis as determined from measurements of catecholamines in the tumor tissue.The next case report [39] highlight the remarkable response of a patient with an ACC microsatellite instability-high tumor, MLH1 spice variant, and high tumor mutational burden to treatment with a novel combination of mitotane, etoposide, paraplatin and sintilimab.The research group of Zhang et al. [40] investigated steroid profiling by LC-MS/MS led us to select DHEA as a candidate reference hormone for cortisol secretion. Lateralization and different steroid ratios showed that each steroid and all three steroidogenic pathways may be affected in patients with PBMAH. In patients with germline ARMC5 variants, the androgen pathway was particularly dysregulated.In the last study [41] it was found that amongst 23 patients carrying germline NF1 variants, targeted sequencing revealed additional pathogenic germline variants in DLST (n=1) and MDH2 (n=2), and two somatic variants in H3-3A and PRKAR1A. Thus, the authors concluded that variants affecting genes involved in different pathways (pseudohypoxic and receptor tyrosine kinase signaling) co-occurring in the same patient could provide a selective advantage for the development of PPGL and explain the variable expressivity and incomplete penetrance observed in some patients.In summary, this Research Topic illustrates the challenges in diagnosis and treatment of patients with adrenal tumors as well as new diagnostic and therapeutic options.