Pheochromocytomas (PCCs) and paragangliomas (PGLs), together known as PCCs and PGLs (PPGLs), are catecholamine-producing neuroendocrine tumors arising from the enterochromaffin cells, embryologically originating from the neural crest of the fetus. Historically they were classified as two discrete entities by the International Agency for Research on Cancer (IARC), an agency of the World Health Organization (WHO). However, the recently published fifth WHO classification of endocrine and neuroendocrine tumors described PCCs as intra-adrenal PGLs originating from the chromaffin cells of the adrenal medulla [1]. As the pathogenesis of nearly 80% of all PGLs can have a genetic basis, genetic testing using next-generation sequencing (NGS) should be performed in all patients diagnosed with PGL [2]. Nearly 35–40% of the PGLs have germline mutations in susceptibility genes, hence achieving the highest heritability of all human tumors. Additionally, nearly 30% of the PGLs have somatic mutations [2]. Taken together, there could be more than 20 PPGLs gene mutations which can be divided into 3 main molecular clusters: pseudohypoxia cluster 1 (1A and 1B), kinase-signaling cluster 2, and wingless-type (Wnt) signaling cluster 3 [3]. These clusters have distinct biochemical, clinical, and imaging features, and varying prognosis indicating that personalized investigation and treatment is a possibility [3].

The distinct characteristics in the cellular metabolic pathways of PPGLs have important connotations in their pathobiological behavior, metastatic potential, clinical presentation, and therapeutic response. Several of the aforementioned genetic abnormalities also directly or indirectly contribute to structural and/or functional alterations in the cellular enzyme machinery which results in abnormal metabolic products and finally tumorigenesis. This review explores the metabolic pathways, their interlink to the genetic defects, and the related alterations in tumor behavior in PPGLs, with special emphasis on molecular, clinical, and therapeutic implications.

Metabolomics is the science dealing with the characteristics of the cell’s metabolic pathways, their genetic features, transcriptional functions, and protein byproducts [4]. It is important to understand these features for predicting the clinical, biochemical, and biological aspects of tumors especially neuroendocrine tumors which possess specific metabolic characteristics. Pollard et al. [5], through proton nuclear magnetic resonance (¹H-NMR) spectroscopy, illustrated crucial findings such as the elevation of succinate in SDHB-related PGLs. Understanding these unique metabolic characteristics illustrates the pathophysiological processes inherent in PPGLs and provides a framework for exploring targeted therapeutic strategies and diagnostic markers [6]. D-2-hydroxyglutarate (D-2HG) oncometabolite is generated by the neomorphic activity of the mutated isocitrate dehydrogenase (IDH) [7]. Correlations, such as its association with a distinct cytosine-phosphate-guanine (CpG) island methylator phenotype (CIMP) and its regulatory impacts on α-ketoglutarate-dependent dioxygenases, spotlight its multifaceted role within oncogenic processes [8]. Analysing the downstream effects and biological interactions of D-2HG provides a platform for exploring oncogenic metabolites and their subsequent influences on tumorigenesis and tumor progression [7].

Technological advancements, specifically in the realm of mass spectrometry (MS), have facilitated a deeper understanding of the metabolomic study of biological samples. Transitioning from gas chromatography to liquid chromatography (LC) has enabled the analysis of biologically active polar molecules paving the way for a more nuanced understanding of metabolites and their roles within biological systems [9]. Especially within the context of PPGLs, methodologies like LC-tandem MS (LC-MS/MS) provide an invaluable tool for investigating tumor metabolites, guiding research toward more targeted diagnostic and therapeutic applications [10]. The largest metabolomic study to date was done by Richter et al, analyzing 395 fresh frozen PPGL samples and measuring various metabolites, including citrate, isocitrate, cis-aconitate, α-ketoglutarate, the D-2HG, the L-2-hydroxyglutarate (L-2HG), succinate, fumarate, malate, pyruvate, lactate, glutamate, glutamine, aspartate, and asparagine [11]. The clustering analysis discerned two primary clusters: the inaugural cluster, characterized by heightened succinate and predominantly involving succinate dehydrogenase complex (SDHx)-related tumors, and a second cluster which is further subdivided into two sub-clusters, each differentiated primarily by their respective concentrations of citrate, isocitrate, and cis-aconitate. The first sub-cluster comprising tumors linked to hypoxic signaling including von Hippel-Lindau (VHL), endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1), fumarate hydratase (FH), and IDH-related tumors presented diminished levels of citrate, isocitrate, and cis-aconitate. Conversely, the second sub-cluster, mainly involving tumors related to kinase signaling such as those related to rearranged during transfection (RET), neurofibromatosis type 1 (NF1), and transmembrane protein 127 (TMEM127), displayed elevated concentrations of the aforementioned metabolites [11].

Thus, the use of both LC-MS/MS and immunohistochemistry plays a pivotal role in deciphering the pathogenicity of gene variants, especially within the context of uncertain significance identified by multipanel or exome sequencing. Harmonizing metabolic profiles with immunohistochemistry results provides a layered understanding, offering insights into the functional impacts of certain gene variants. This dual-faceted approach not only aids in discerning the pathogenicity of variants but also offers a detailed view of the molecular underpinnings and potential biomarkers related to PPGLs.

The metabolic pathways involved in hormone synthesis and processing within the adrenal medulla and sympathetic ganglia are complex and finely regulated to ensure a proper physiological response to stressors [12]. The synthesis of catecholamines begins with the amino acid tyrosine, which enters the adrenal medulla and sympathetic neurons through the L-type amino acid transporter (LAT) 1 and LAT2 [13]. Through a series of enzymatic reactions, tyrosine is converted to dihydroxyphenylalanine (DOPA) by the enzyme TH, which is the rate-limiting step in catecholamine synthesis [14]. DOPA is then converted to dopamine by DOPA decarboxylase (DDC). In the cytoplasm of adrenal medullary cells and sympathetic neurons, dopamine is transported into storage vesicles, where it is further hydroxylated to norepinephrine by dopamine β-hydroxylase (DBH) [13]. In the adrenal medulla, norepinephrine can be methylated to form epinephrine through the action of phenylethanolamine N-methyltransferase (PNMT) [15]. Once synthesized, catecholamines are stored in vesicles until they are released into the bloodstream in response to various stimuli. A sizeable fraction does leak into the cytoplasm where it is metabolized into metanephrines. The release process is triggered by the arrival of a nerve impulse which causes the vesicles to fuse with the cell membrane and release their contents into the bloodstream. Most epinephrine (around 90%) of the body’s requirement comes from the adrenal medulla and norepinephrine comes from the sympathetic ganglia. Less than 10% of the body’s norepinephrine comes from the adrenal medulla [15].

The synthesis and secretion of catecholamines are tightly regulated by various factors. The activity of TH, for instance, is modulated by the level of catecholamines through a negative feedback mechanism, ensuring the balance in catecholamine levels is maintained. Additionally, other hormonal and neural mechanisms also play a significant role in the regulation of catecholamine synthesis and secretion. In basal conditions the catecholamines function as metabolic hormones and upon stressful stimulus, they exhibit a fight and flight response triggering the cascade of events for release from the stored vesicle [12]. The main rate-limiting enzyme is the TH. TH gene, harboring 13 exons, is situated at 11p15.5 on chromosome 11 and gives rise to four isoforms through alternative messenger RNA (mRNA) splicing [14]. Through its pivotal role in catecholamine synthesis and multifaceted regulation, TH has attracted significant attention across various cancer research areas. Recent investigations have revealed multiple TH polymorphisms in the general population are associated with heightened norepinephrine levels and elevated blood pressure [16]. TH, with an approximate molecular weight of 240 kDa, is largely relegated to adrenal, extra-adrenal chromaffin cells, and postganglionic sympathetic nerve endings. TH relies on cofactors like ferric (Fe³⁺), tetrahydrobiopterin, and molecular oxygen for the hydroxylation step. Originating from guanosine triphosphate (GTP), tetrahydrobiopterin donates hydrogen atoms to keep TH reduced and active [14]. The Figure 1 shows the graphical representation of the synthesis, release, and metabolism of adrenal medullary hormones.

The complexity of controlling catecholamine synthesis involves a nuanced regulation of TH activity, combining short-term post-transcriptional mechanisms with long-term transcriptional ones. The former encompasses enzyme phosphorylation, dephosphorylation, feedback inhibition by catecholamines, and ubiquitination, while the latter primarily engages transcriptional mechanisms [16]. TH degradation is postulated to be mediated by the ubiquitin–proteasome pathway [14]. Catecholamines mitigate TH formation in its reduced, active state through the oxidation of tetrahydrobiopterin to pteridine and also serve as antagonists of tetrahydrobiopterin, obstructing the TH catalytic domain [17]. Short-term TH activity regulation is further realized via the phosphorylation and dephosphorylation of up to four serine residues at its regulatory site, with phosphorylation facilitated by numerous kinases [like protein kinase A (PKA), PKC, Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2), extracellular signal-regulated kinase 1 (ERK1), ERK2, mitogen- and stress-activated kinase 1 (MSK1), p38-regulated/activated protein kinase (PRAK)] freeing it from the catecholamine-induced feedback inhibition and thereby spurring enzyme activity [17].

Moreover, protein phosphatase 2A (PP2A) and, to a lesser degree, PP2C facilitate dephosphorylation, reinstating catecholaminergic suppression of TH [18]. This inhibition is mediated through alpha2-adrenergic or D2-dopaminergic receptors, activating cyclic adenosine monophosphate (cAMP) or Ca²⁺/calmodulin-dependent protein phosphatases [17]. Persistent stimulation of catecholamine synthesis catalyzes TH protein synthesis induction via several cAMP-dependent pathways that trigger TH gene transcription. An overview of the regulation of synthesis and secretion of adrenal medullary hormones is given in Table 1.

In adrenal medullary chromaffin cell vesicles, the norepinephrine undergoes conversion to epinephrine via the enzymatic action of PNMT [15]. PNMT utilizes S-adenosylmethionine as a co-substrate and methyl donor. Not exclusive to a single substrate, PNMT also participates in the production of additional N-methylated trace amines [19]. The regulatory control of PNMT expression is orchestrated through mechanisms mediated by the glucocorticoid receptor, in synergy with an array of transcription factors like early growth response-1 (Egr-1), activator protein 2 (AP2), specificity protein 1 (Sp1), and Myc-associated zinc-finger protein (MAZ) [20]. Ensuring elevated circulating glucocorticoid levels, the closeness of adrenocortical cells to the adrenal medulla allows glucocorticoids to diffuse passively through the chromaffin cell membrane [20]. Upon binding to the intracellular glucocorticoid receptor, the hormone-receptor complex transitions into the cell nucleus, associating with the glucocorticoid response element of the promoter region of the PNMT gene, situated at chromosome 17q12, thereby initiating gene transcription [21]. This mechanism elucidates why the adrenal gland is paramount as the principal source of epinephrine in the body, while extra-adrenal PNMT expression remains confined to a select group of neurons in the clinical nurse specialist (CNS) and a subset of cardiomyocytes.

Kimura et al. [22] demonstrated that while TH mRNA was identifiable in all patients with PCC samples, the presence of PNMT mRNA was exclusively observed in epinephrine-type tumors. Immunohistochemical analysis involving 70 PPGLs-wherein every PCC possessed cells showcasing immunoreactivity to TH, with PNMT expression being isolated to epinephrine-producing PCC type alone [22]. Hence PNMT still retains its capacity not only in physiological conditions but in tumor states when the production of hormone from the lesion is increased by multiple folds. In a study involving array comparative genomic hybridization (CGH) and gene expression profiling of 12 fresh frozen PPGLs samples, it was observed that while most cases had limited copy number aberrations irrespective of their malignancy status, 390 genes were differentially expressed between benign and malignant tumors [23]. Notably, the expression of PNMT, was significantly reduced in malignant PPGLs. This key finding positions PNMT downregulation as a potential hallmark of malignancy in PPGLs and underscores its prominence as a differentially expressed gene between the two tumor types [23].

Metyrosine, a recognized TH blocker, has been validated for its efficacy and safety, particularly in pre-surgical or pre-intervention contexts [24]. Metyrosine not only demonstrates a propensity to enhance intraoperative hemodynamics but also substantively mitigates catecholamine-mediated symptoms in a subset of patients [25, 26]. The strategic integration of metyrosine into treatment regimens may be particularly beneficial for specific patient cohorts. These cohorts include individuals who persistently exhibit hypertension despite the initiation of alpha-adrenergic blockade; those who exhibit intolerance towards alpha-adrenergic blockade; patients are identified as high-risk due to tumor size or location, which predisposes them to substantial catecholamine release; and patients scheduled for interventions such as chemotherapy or ablative therapy, where a significant catecholamine release is plausible [27]. Consequently, metyrosine provides a robust option to safeguard against potential clinical complications associated with catecholamine surges in these patient profiles. Hence a thorough knowledge of the metabolism of the PPGL can be used to prognosticate patients in difficult circumstances and offer additional therapeutic advantages.

The adrenal medulla generates and releases not only catecholamines but also a wide array of enzymes, peptides, and proteins that oversee catecholamine synthesis and secretion through autocrine and paracrine actions, some of which are neuropeptide Y (NPY), adrenomedullin (AM), peptides pituitary adenylate cyclase-activating polypeptide (PACAP), and catestatin [20]. NPY, a 36-amino acid neuropeptide distributed broadly within the brain and sympathetic nervous system, stimulates catecholamine secretion by enhancing TH expression and intracellular calcium levels [28]. AM, a peptide initially isolated from a PCC, plays a crucial role in both autocrine and paracrine activities by binding to various receptors, subsequently augmenting the adrenal blood flow and stimulating catecholamine release, along with other systemic effects such as vasodilatation and natriuresis stimulation [29–31]. The concentration of AM is also markedly increased in patients with adrenal PCC and can be a usual biomarker differentiating between PPGL in certain special patients when there is an imaging constraint [31]. PACAP, another neuropeptide, amplifies catecholamine secretion through transcription induction and the activation of biosynthetic enzymes like TH, DBH, and PNMT, also influencing the expression and secretion of additional peptides like brain natriuretic peptide and enkephalins in normal adrenal medullary chromaffin cells [32, 33]. It is very important to note that PNMT expression is mainly influenced by genetic backgrounds rather than tumor location [34]. Lastly, catestatin, derived from the proteolytic cleavage of chromogranin A, predominantly acts as a noncompetitive nicotinic cholinergic antagonist, thereby providing robust negative feedback inhibition of catecholamine secretion [33]. These peptides collectively modulate chromaffin cell functionality through an array of membrane receptors, primarily belonging to the G protein-coupled receptors (GPCRs) family, orchestrating a complex network of hormonal regulation and release within the adrenal medulla [35].

Reports indicate that an annual PPGL incidence ranges from two to eight cases per million people, while in hypertensive patients, PPGL prevalence is estimated to be between 0.2% and 0.6% [36] Notably, 5% of patients with adrenal masses incidentally identified through radiology were found to have PPGLs [36]. Molecular analysis demonstrates that there is an elevated gene expression tied to cellular cycle, tumor evolution, metastasis, hypoxia, angiogenesis, and the Wnt signaling pathway in PPGLs when contrasted with normal adrenal glands [10, 12, 37, 38]. Around 35–40% of PPGLs exhibit a hereditary predisposition due to germline mutations in more than a dozen susceptibility genes [39]. Additionally, large genomic studies reveal approximately 30% of cases possessing tumor-specific somatic mutations, thus identifying tumorigenesis drivers in roughly 60–70% of cases [40, 41]. Identifying pathogenic variants in susceptibility genes is pivotal as it modifies the patients’ and potentially asymptomatic relatives’ required surveillance, screening, and clinical care [42]. These susceptibility genes fall into three main clusters: pseudohypoxia cluster (cluster 1), Kinase signaling cluster (cluster 2), and Wnt signaling cluster (cluster 3) [38, 43–45].

The clinical presentation for the inherited group substantially varies from the sporadic group in the realm of PPGLs. Notably, age stands out as an important factor, with those harboring hereditary PPGLs typically developing the disease roughly a decade earlier than their sporadic counterparts [58]. However, there is a significant variation in the age at initial diagnosis among patients with hereditary syndrome. The hereditary cluster 1 PPGL patients has a very early age at first diagnosis (28.7 years) than hereditary cluster 2 (46.8 years) and sporadic PPGL patients (52.9 years) with the age at initial diagnosis of cluster 2 PPGL being almost like that of sporadic patients [59]. Multiple PGLs are much more common in the hereditary group than the sporadic PPGLs [2, 57]. A considerable 17% to 85% of hereditary PPGL patients manifest with multiple PGLs with only 1.2% prevalence in sporadic occurrences [1, 2, 55, 60].

Furthermore, VHL-related PGLs secrete only norepinephrine, providing a unique biochemical signature to this hereditary variant of the disease [45]. Gender disparities in occurrence also take the forefront in distinguishing between hereditary and sporadic forms. Hereditary PGLs are common in both genders, while sporadic instances have a female predominance, with a notable 71% to 29% female-to-male ratio [60]. Bilateral PCCs, which are characteristic of RET, VHL, and NF1 genes, do not co-exist with multiple PGLs, a phenomenon commonly associated with SDHx syndromes [61]. Hence, patients presenting with multiple PGLs are almost certain to be diagnosed with a hereditary form of the disease. Moreover, among patients with sympathetic PGLs, a substantial 25% have the presence of a hereditary syndrome (Table 3) [62].

Due to its precision and consistency, LC-MS/MS has emerged as the benchmark. A value three times greater than the upper limit of normal values signifies a positive outcome [52]. Nonetheless, delayed diagnosis may result from the presence of pseudo-silent PPGL and a heavy tumor burden in certain patients. Hence, any findings beyond the standard range should be regarded as indicative of PPGL especially when prospectively screening for hereditary disease. Biochemical profiling is useful for assessing PPGL syndromes, as tumors can be classified based on their profile (Table 4) [52].

The three clusters are aligned in their approach to radionuclide therapy, utilizing ¹³¹I-MIBG radionuclide therapy in cases where a positive MIBG scan has been obtained, and employing PRRT in cases where a positive gallium DOTATAE scan has been obtained [2, 89, 103]. ¹³¹I-MIBG, including the novel high specific activity (HSA) ¹³¹I-MIBG, is the best-studied first-line therapeutic option for patients with PPGLs showing slow to moderate progression [2, 131–137].

Metastatic cluster 1 SDHx-related diseases, particularly those related to SDHB, may show lower positivity rates on ¹²³I-MIBG scans [2, 138]. They are detected in the gallium DOTATAE scans and, therefore, treated with SSTR-based PRRT [139–147]. PRRT is recommended by the National Comprehensive Cancer Network (NCCN) and the European Society of Hypertension for treating PPGL [2]. Generally, patients tolerate PRRT well, and causes minimal side effects [2]. The need for treatment must be balanced with the possibility of severe bone marrow suppression, especially if radionuclide therapy is followed by chemotherapy [2]. TKIs could be an alternative second-line treatment to chemotherapy in patients who experience severe hematological toxicity as a result of their radionuclide therapy [2].

Nausea, myelosuppression, and fatigue are the most common side effects of radionuclide therapy [148]. Whenever a second application is indicated, a delay of at least 3 months should be considered until platelets and neutrophils are within normal ranges [149]. Potential long-term side effects include secondary hematological malignancies, such as myelodysplastic syndrome, acute myeloid leukemia, and acute lymphocytic leukemia [148].

The Averbuch (CVD) scheme is the preferred chemotherapy treatment for rapidly spreading metastatic disease with a high tumor burden and for tumors unsuitable for radionuclide therapy [2, 88, 103]. The CVD regime (cyclophosphamide 750 mg/m² on day 1, vincristine 1.4 mg/m² on day 1, and dacarbazine 600 mg/m² on days 1 and 2) is given at 21-day intervals [89]. This therapy is well-established and effective for aggressive and rapidly progressive PPGLs, especially in patients with SDHB mutations [108, 150, 151]. The most prevalent adverse events linked to the CVD regimen were myelosuppression, neuropathy, nausea, vomiting, and hemorrhagic cystitis. Less prevalent side effects are diarrhoea, alopecia, and oral ulcers [108].

As a form of maintenance therapy, it may be advisable to continue treatment with Temozolomide monotherapy if the patient has successfully completed 6–9 cycles of CVD chemotherapy or has been treated for a prolonged period with 20 cycles of CVD chemotherapy [150–152]. The standard Temozolomide regimen is 150 mg/m² every 28 days on days 1–5 [89]. This is especially relevant for cases with SDHB mutations. In patients with SDHB mutation, who have limited performance status or co-morbidities and a lower disease burden, Temozolomide can be a substitute for CVD chemotherapy, achieving a disease control rate of up to 80% [progression-free survival (PFS) 13.3 months] and has more favourable side effect profile compared to CVD chemotherapy [3, 108, 151].

The understanding about tumor metabolism in PPGLs and its clinical, diagnostic, prognostic and therapeutic implications have evolved significantly in the past few years. Discovery of new culprit genes and metabolic pathways are expected to improve the knowledge about the pathobiology of these uncommon tumors to enhance diagnostic and management strategies in future. Prompt recognition of the genetic abnormalities in patients with PPGLs through NGS would help clinicians to identify and categorize these tumors for appropriate design of management and follow up plan including genetic counseling of family members. More research involving multinational collaboration would empower clinicians to deal with this enigmatic disease in future.