Molecular and Clinical Spectrum of Primary Hyperparathyroidism

Abstract

Recent data suggest an increase in the overall incidence of parathyroid disorders, with primary hyperparathyroidism (PHPT) being the most prevalent parathyroid disorder. PHPT is associated with morbidities (fractures, kidney stones, chronic kidney disease) and increased risk of death. The symptoms of PHPT can be nonspecific, potentially delaying the diagnosis. Approximately 15% of patients with PHPT have an underlying heritable form of PHPT that may be associated with extraparathyroidal manifestations, requiring active surveillance for these manifestations as seen in multiple endocrine neoplasia type 1 and 2A. Genetic testing for heritable forms should be offered to patients with multiglandular disease, recurrent PHPT, young onset PHPT (age ≤40 years), and those with a family history of parathyroid tumors. However, the underlying genetic cause for the majority of patients with heritable forms of PHPT remains unknown. Distinction between sporadic and heritable forms of PHPT is useful in surgical planning for parathyroidectomy and has implications for the family. The genes currently known to be associated with heritable forms of PHPT account for approximately half of sporadic parathyroid tumors. But the genetic cause in approximately half of the sporadic parathyroid tumors remains unknown. Furthermore, there is no systemic therapy for parathyroid carcinoma, a rare but potentially fatal cause of PHPT. Improved understanding of the molecular characteristics of parathyroid tumors will allow us to identify biomarkers for diagnosis and novel targets for therapy.

Graphical Abstract

Graphical Abstract.

Essential Points.

• Although rare, the heritable syndromes of primary hyperparathyroidism have been critical for the discovery of consequential genes that regulate parathyroid growth and function and whose mutation, in either a sporadic or a familial context, causes parathyroid neoplasia

• Analysis of kindreds with familial isolated hyperparathyroidism, after exclusion of MEN1, CASR, CDC73/HRPT2, and GCM2 mutation, suggests that the majority harbor yet to be discovered genetic risk factors for their primary hyperparathyroidism.

• In MEN1, the most common form of syndromic primary hyperparathyroidism, the parathyroid disease is almost always benign despite its high penetrance and frequency of recurrence, and the greatest negative impact on life expectancy results from the presence and spread of functional or nonfunctional gastroenteropancreatic neuroendocrine tumors

• Clinically impactful parathyroid tumors can be small and difficult to localize, even with the best existing imaging techniques, justifying future investment in the discovery, development, and optimization of superior parathyroid-specific imaging modalities for initial and reoperative cases of primary hyperparathyroidism

 

History

The parathyroid glands were serendipitously noted by Richard Owen, an English anatomist while dissecting an Indian rhinoceros in 1850. Ivar Sandstrom, a Swedish anatomist and histologist, is credited for its discovery in humans in 1877. Some 50 years later parathyroid hormone (PTH) was extracted from the gland (1), and another 50 years later when the complete primary amino acid sequence of PTH became known (2, 3).

Embryology

Embryologically, the parathyroid glands are derived from the endoderm of the third and fourth branchial pouches. The superior glands develop from the fourth branchial pouch and are commonly located near the posterolateral aspects of the thyroid. Notably, the inferior glands (typically located near the inferior poles of the thyroid) develop from the third branchial pouch along with the thymus. The inferior glands are in this typical location in ∼80% (4-6). The variable location of the inferior glands is likely explained by their longer descent (Fig. 1A). Most individuals have 4 glands, although deviations may be seen, most commonly 3 or 5 glands. Supernumerary parathyroid glands are seen in 2.5% to 15% patients while <3% patients may have only 3 glands (7, 8).

Figure 1.

(A) Parathyroid glands are derived from the endoderm of the third and fourth pharyngeal pouches. The third pharyngeal pouch gives rise to the inferior parathyroid glands and thymus while the fourth pharyngeal pouch gives rise to the superior parathyroid gland. Note the longer path of descent of inferior parathyroid glands and increased likelihood of its ectopic location. The inferior parathyroid glands may descend lower into the thymus or chest or leave residual tissue along the path of descent. (B) Microscopically, the parathyroid gland is enveloped by a thin fibrous capsule that extends into the parenchyma dividing the gland into multiple lobules. The gland consists of the following parenchymal cells: chief, oxyphil, clear, and transitional. Both chief and oxyphil cells are eosinophilic, express calcium-sensing receptor, and secrete parathyroid hormone. Clear cells decrease with age while oxyphil and transitional oxyphil cells increase with age. The stromal fat content of the gland (∼50%) varies with nutritional status and weight of the patient.

Evolution

Parathyroid glands are found in all land vertebrates but have not been identified in aquatic vertebrates (9). This may be indicative of the need for greater control of calcium homeostasis in terrestrial animals as marine aquatic animals have a constant supply of calcium from seawater. Humans and chickens have 4 parathyroid glands while mice have 2.

Gross Anatomy and Histology

Overall, normal parathyroid glands measure 2 to 7 mm in length, 2 to 4 mm in width, 0.5 to 2 mm in height and weigh 35 to 55 mg. On histology, the gland is enveloped by a thin fibrous capsule that extends into the parenchyma delineating it into multiple lobules (Fig. 1B). It consists of parenchymal cells (chief, oxyphil, clear, and transitional oxyphil cells arranged in nests and cords with a rich capillary network), fat cells, and fibrovascular stroma (10, 11). In addition to these components, tumor-infiltrating lymphocytes are seen in parathyroid tumors although their functional relevance remains unclear (12). Stromal fat content of a parathyroid gland in an adult is approximately 50% of the mass of the gland although it can vary with nutritional status and weight of the individual (13). It is believed that all other parenchymal cell types, including oxyphil cells, are derived from chief cells (14). Both chief and oxyphil cells contain an eosinophilic cytoplasm, but the oxyphil cells are larger and stain lighter. Oxyphil cells contain many large mitochondria which is thought to account for localization of a hyperfunctioning gland using ^99mTc-sestamibi (15). Both chief and oxyphil cells express comparable amount of calcium sensing receptor (CASR) but there may be a difference in their calcium responsiveness (12). The functional relevance of oxyphil cells remains unclear, although they have been shown to secrete PTH in secondary hyperparathyroidism and express the parathyroid-relevant genes to produce PTH-related peptide (PTHrP) and calcitriol (16). Of note, oxyphil cells are not present in the parathyroid glands of rats or other lower animals.

Clear cells are a form of chief cells with abundant cytoplasmic glycogen seen primarily in embryos and fetuses that decline in number with age (17). Cells with cytologic features intermediate between chief and oxyphil cells are termed transitional oxyphil cells. Both oxyphil and transitional oxyphil cells are few at birth but increase with age (18, 19). Immunostaining with GATA-3 and glial cell missing transcription factor 2 (GCM2), transcription factors of parathyroid development, PTH, and CASR can be used to confirm parathyroid tissue on histology (20-22). Parathyroid glands have a rich blood supply from inferior thyroid arteries or from anastomosis between superior and inferior vessels. Parathyroid veins drain into the thyroid vein plexus whereas the lymph vessels drain into the deep cervical and paratracheal lymph nodes. The nerves to the parathyroid glands are vasomotor but not secretomotor. The nerve supply is sympathetic, derived from the thyroid branches of the cervical ganglia.

PTH and PTH-Related Peptide

The chief cells of the parathyroid glands secrete pre-pro-PTH, a 115 amino acid precursor peptide which undergoes processing in the endoplasmic reticulum and Golgi apparatus to mature into PTH—an 84 amino acid peptide (Fig. 2A). PTHrP was first described in 1987 as the cause of humoral hypercalcemia of malignancy (23). However, it has since been noted to have several other physiological functions such as regulation of chondrocyte maturation (24), promotion of bone formation and resorption (25), promotion of branching morphogenesis in mammary glands (26), regulation of keratinocyte differentiation (27), facilitation of tooth eruption (28), regulation of vascular smooth muscle, regulation of beta cell proliferation, and insulin production in the pancreas (29) and promotion of calcium transport in the placenta (30). Thus, unlike PTH, which is produced only in the parathyroid gland, PTHrP is produced locally by many tissues and has autocrine (effects on same cell), paracrine (effects on adjacent cells), and intracrine functions (travels directly following translation into the nucleus or nucleolus) both during development and in the adult. Despite low sequence homology in the receptor-binding domain, PTHrP and PTH bind to the common PTH receptor (PTHR1) with comparable affinity (31) (Fig. 2A). Clinically, abaloparatide, a synthetic PTHrP analog, is FDA approved as an antiosteoporosis therapy to reduce the risk of both vertebral and nonvertebral fractures in postmenopausal women. Recombinant human PTH(1-34) is also FDA approved for postmenopausal osteoporosis, osteoporosis secondary to hypogonadism or glucocorticoid-induced osteoporosis. PTH(1-84) produced by recombinant DNA technology is available commercially for use in patients with hypoparathyroidism.

Figure 2.

(A) PTH is an 84 amino acid peptide with its biologic activity attributed to the first 7 amino acids in its amino terminal. The first 34 amino acids of the peptide are as potent as the intact native hormone, with signaling and receptor binding domains residing within N-terminal and C-terminal portions of PTH(1-34), respectively. PTH(7-34) peptide analogs function as potent antagonists as these fragments are devoid of signaling activity but can bind to the PTH receptor. The PTH(15-34) fragment is the shortest length peptide of native sequence that exhibits detectable binding to the PTH receptor while PTH(1-14) is the shortest length N-terminal peptide that exhibits cyclic adenosine monophosphate signaling activity (although its potency is 5 times weaker, likely from the absence of receptor binding domain). PTH-related peptide is a 141 amino acid peptide with variants that are 139 or 173 amino acids long. Structurally, the region of homology with PTH runs around the N-terminal sequence with the first 13 residues being identical (36). (B) The evolution of PTH assays can be divided into 3 stages: competitive immunoassays (first generation), “sandwich” immunometric assays (second and third generation). In the first-generation assays, a single polyclonal antibody competed for labeled PTH and the serum forms. In comparison, second-generation assays are based on recognition of 2 distinct antibodies (typically monoclonal): 1 carboxyl terminal and the other amino terminal specific (targeting antigenic site located around amino acids 20-25). However, all these assays also measure forms other than intact PTH(1-84); for instance, PTH(7-84), predominantly an issue in patients with chronic kidney disease.

Measurement of PTH Hormone

Various molecular forms of the hormone exist in circulation either due to direct secretion by the parathyroid gland or as byproducts of peripheral metabolism of PTH(1-84). The various circulating forms of PTH, particularly the small and large carboxy-terminal PTH fragments which accumulate in patients with renal failure, present a challenge for measuring PTH(1-84). Roger Guillemin, Andrew Schally, and Rosalyn Yalow developed the first immunoassay for measuring PTH in 1977. To have an assay that only recognizes intact PTH(1-84), the amino terminal antibody used should recognize the very first amino acids (Fig. 2B). The current second- or third-generation PTH assays have equivalent clinical utility in diagnosing primary hyperparathyroidism (PHPT) (32). The use of mass spectrometry is currently being explored for accurate measurement of the intact PTH(1-84) hormone. Nontruncated amino-terminal PTH is a post-transcriptionally modified form of PTH(1-84) that can be picked up on an elevated whole PTH (third-generation)/total (second-generation) PTH assay ratio (n > 0.8). It has been proposed that a progressive rise in the third/second–generation PTH assay ratio or measurement of nontruncated amino-terminal PTH may have clinical utility in monitoring parathyroid cancer recurrence (33).

PTH, Calcium, and Vitamin D Signaling

PTH signaling

All nucleated cells in human body contain the PTH gene but it is expressed only in chief cells of the parathyroid glands. Variants in PTH gene cause heritable forms of hypoparathyroidism (34). PTHrP is encoded by a single copy PTHLH gene. PTH and PTHrP gene share similar exon/intron boundaries and are believed to have arisen through gene duplication on chromosome 11 and 12 respectively (35). PTH/PTHrP receptor (PTHR1) is a family B G-protein–coupled receptor (GPCR), such as receptors for calcitonin, secretin, glucagon, glucagon-like peptide-1, corticotrophin-releasing factor, and others, with its characteristic 7 transmembrane domain protein structure (Fig. 3). There is low amino acid sequence homology between the different family B GPCRs at about 35%; however, these conserved residues likely define the overall protein fold used by these receptors (36). The cDNA encoding the receptor was cloned in 1991 leading to a host of molecular developments in the field (37). Since then, its 3D cryo-electron microscopic structure has also been solved (38). The gene is located on chromosome 3p and consists of 14 coding exons. Evolutionarily, PTHR1 is highly conserved (39) with orthologs detected in fish (40), birds (41), and sea squirt Ciona intestinalis (42). Mutations in PTHR1 cause Jansen's metaphyseal chondrodysplasia and Bloomstrand's chondrodysplasia. The PTH-2 receptor (PTHR2) is found in vertebrates but is absent in birds (43). It can bind to PTH but not to PTHrP. It is heavily expressed in the central nervous system, cardiovascular, and gastrointestinal systems as well as in lung and testes, although the role of PTHR2 in these systems and tissues is unknown.

Figure 3.

Upon ligand (PTH and PTH-related peptide) activation, the PTH receptor undergoes a series of conformational changes resulting in coupling to the stimulatory G-protein located on the inner, cytoplasmic surface of the cell membrane. Gαs activates adenylate cyclase, which results in synthesis of cyclic adenosine monophosphate (cAMP). cAMP binds to the regulatory 1A subunits (R) of protein kinase A (PKA), the primary effector of cAMP. On activation, the catalytic subunits (C) dissociate from the R subunits and phosphorylate several transcription factors, including cAMP-responsive element-binding (CREB) protein which interacts with cAMP-responsive element (CRE) in the nucleus and regulates transcription of target genes. Created with Biorender.com.

CASR signaling

CASR is an evolutionarily conserved family C GPCR (along with GABA type B and metabotropic glutamate receptors) expressed predominantly in the parathyroid glands (maximal) and kidneys (Fig. 4). In the parathyroid glands, it interacts with Klotho, a transmembrane protein to regulate PTH secretion (44). In the kidney tubules, CASR regulates calcium reabsorption independent of PTH. CASR expressed on chondrocyte is important for growth plate chondrogenesis, longitudinal bone growth, and skeletal mineralization, probably by counteracting PTHrP–PTH1R signaling. Osteoblast expression of CASR also has a role in skeletal development, mineralization, and remodeling (44). CASR is also expressed in other tissues such as the brain, lungs, vasculature, breast (promotes lactation), gastrointestinal tract, pancreatic islets, and skin where it is involved in diverse physiological roles (44). Inactivating mutations of CASR cause familial hypocalciuric hypercalcemia type 1 (FHH-1) while activating CASR mutations cause autosomal dominant hypocalcemia type 1.

Figure 4.

CASR binds to many physiological ligands including cations such as magnesium, l-amino acids, polyamines, and γ-glutamyl peptides like glutathione. CASR has a large extracellular domain that consists of a bilobed Venus flytrap module and a cysteine-rich domain. The Venus flytrap has 3 distinct extracellular calcium (Ca²⁺_e) binding sites. CASR exists as a dimer through interactions at the amino terminal of the Venus flytrap lobe. The transmembrane domain of the protein is typical of other GPCRs and consists of 7 hydrophobic helical domains connected by 3 extracellular and intracellular loops each. Signal transduction on activation of CASR propagates through G_q/11 family proteins that activate phospholipase C-beta, resulting in the release of 2 second messengers, diacylglycerol (DAG) and inositol 14,5-triphosphate (IP₃). DAG activates protein kinase C, which activates extracellular signal–regulated kinases 1 and 2 (mitogen activated protein kinases [MAPKs] signaling). IP₃ binds to its receptors on the endoplasmic reticulum and raises intracellular calcium by releasing calcium from the reticulum. CASR activates the G_i/o proteins, which results in suppression of adenylate cyclase–mediated cAMP production eventually resulting in decreased PTH secretion and increased urinary calcium excretion. CASR can also activate MAPK signaling via a G-protein–independent mechanism involving β-arrestins. CASR demonstrates biased signaling in showing preferential activation of distinct intracellular signaling responses in different tissues. The mechanism for this functional selectivity is unclear. Ligand-induced insertional signaling drives cell surface expression of CASR by anterograde trafficking. The σ subunit of adaptor protein 2 (AP2σ) mediates the endocytosis and trafficking of CASR in combination with clathrin and β-arrestin. Mutations in CASR, G_q/11 family (GNA11) and AP2σ (AP2S1) can cause familial hypercalcemic hypocalciuria and autosomal dominant hypocalcemia. Created with Biorender.com.

Vitamin D receptor signaling

Vitamin D receptor (VDR) is a ubiquitously expressed, evolutionarily conserved steroid receptor and transcription factor, which is regulated by 1, 25(OH)₂ vitamin D binding (Fig. 5). Inactivating mutations in VDR cause hereditary vitamin D resistant rickets (HVDRR). Alopecia seen in HVDRR and VDR knockout models is not seen in vitamin D deficiency, indicating that VDR may have ligands other than 1,25(OH)₂ vitamin D at least in hair follicles (45). Intravenous or oral calcium reverses the mineral and skeletal phenotype of HVDRR, indicating the critical role of 1,25(OH)₂ vitamin D and VDR on intestinal calcium absorption. Physiologically important VDRs are also present in osteoblasts which mediate 1,25(OH)₂ vitamin D effects on bone homeostasis. Osteoblast VDR signaling modulates transcription of RANKL, which increases osteoclast formation and action.

Figure 5.

Upon binding to 1,25(OH)₂ vitamin D, VDR heterodimerizes with other nuclear hormone receptors, particularly the family of retinoid x receptors (RXRs), and this complex then binds to vitamin D response elements (VDREs) within the promoters of a large number of genes it regulates, modulating their transcription and subsequent effects in a ligand-dependent manner. Coactivators and corepressors are additional proteins that complex with VDR to regulate transcription. Created with Biorender.com.

Vitamin D₃ (cholecalciferol) is the natural form of vitamin D produced in skin from 7-dehydrocholesterol, but it is not biologically active. Adequate vitamin D levels can be difficult to maintain through diet alone, as it is found in only few foods (fortified dairy and fish oils). Inactivating mutations in CYP27B1 (CYP2R1), encoding 25(OH)D 1-α-hydroxylase cause vitamin D–dependent rickets type 1 (VDDR1), also known as pseudovitamin D deficiency rickets. CYP27B1 is only expressed in kidney and placenta (during pregnancy) in states of health and, additionally, in macrophages in sarcoidosis or Crohn disease. Unlike CYP27B1 expressed in kidneys, CYP27B1 expressed in macrophages is not suppressed by elevated 1,25(OH)₂D_3. The parathyroid glands also express CYP27B1; however, the functional relevance of this expression remains unknown. Rare cases of vitamin D–dependent rickets type 1B (VDDR1B), caused by inactivating mutations in CYP27A1, expressing 25-hydroxylase have been described (46, 47). Inactivating mutations in CYP24A1, encoding 24,25-α-hydroxylase, cause idiopathic infantile hypercalcemia characterized by hypercalcemia, hypercalciuria, and recurrent nephrolithiasis. VDR signaling during negative calcium balance decreases matrix mineralization to preserve serum calcium. However, the role of VDR during normal calcium balance remains unclear. Growing evidence suggests a role of fibroblast growth factor 23 (FGF23) in calcium homeostasis by increasing calcium reabsorption in distal tubules (48). During pregnancy and lactation, the level of both calcitonin and prolactin are increased, and these hormones can stimulate CYP27B1, expressed in fetal trophoblasts and maternal decidua of the placenta, to increase intestinal calcium absorption for the growing fetus.

Calcium Homeostasis

The total body content of calcium is ∼1000 to 1200 g. Ninety-nine percent of this body calcium resides in the skeleton while the remaining 1% is freely exchangeable (∼0.9% intracellular and ∼0.1% in extracellular fluid). Total serum calcium is composed of 3 distinct compartments: ionized (48%)—physiologically active form; protein bound (46%)—largely bound to albumin; and calcium complexed with inorganic compounds such as citrate or phosphate (7%). Calcium absorption in the intestine occurs by an active, carrier-dependent process (vitamin D dependent) and a passive, paracellular process (Fig. 6A). Since the passive absorption is independent of vitamin D, high calcium intake can reduce the effect of vitamin D insufficiency and consequent poor calcium absorption efficiency. Calcium homeostasis is maintained through a complex network of cellular interactions involving the kidney (Figs. 6B and 7), skeleton, gastrointestinal tract, parathyroid gland, and mammary glands (during pregnancy and lactation) (Fig. 7).

Figure 6.

(A) Course of calcium absorption and contribution of active and passive calcium absorption over the course of jejunum, ileum, and colon. Most of the total calcium absorption (65%) takes place in ileum because transit time through the ileum is almost ten times longer than through duodenum. Created with Biorender.com. (B) The adult kidney has a glomerular filtration rate (GFR) of ∼100 mL/min and produces >8000 mg of calcium in the GFR/24 hours; ∼98% of the filtered calcium is reabsorbed, with only about 200 mg/24 hours of calcium appearing in the urine. Sixty to 70% of the filtered calcium is reabsorbed at the proximal convoluted tubule mainly by passive diffusion. However, 10% to 15% of total proximal tubule calcium reabsorption occurs via active transport and is mainly regulated by parathyroid hormone and calcitonin. No calcium reabsorption occurs within the thin segment of the loop of Henle. Twenty percent of the filtered calcium is reabsorbed in the cortical thick ascending limb, 10% in the distal convoluted tubule, and another 3% to 10% in the connecting tubule. PHP, pseudohypoparathyroidism. Created with Biorender.com.

Figure 7.

Vitamin D₃ is transported via vitamin D binding protein (DBP) to the liver for synthesis of 25-hydroxyvitamin D3 [25(OH)D₃], the major circulating form of vitamin D. Subsequently, 25(OH)D₃ is then transported via DBP to the kidney where it is taken up by the tubular epithelial cells. Low calcium sensed through the calcium sensing receptor (CASR) in parathyroid cells stimulates release of parathyroid hormone (PTH). PTH hormone acts via PTHR1 expressed predominantly in osteoblasts and osteocytes (induces release of calcium from bone) and renal proximal (decreases rate of phosphorus reabsorption, stimulates the rate of transcription of CYP27B1, encoding 25(OH)D 1-α-hydroxylase) and distal (increases rate of calcium reabsorption) tubule cells. 25(OH)D 1-α-hydroxylase converts 25(OH)D₃ to 1,25(OH)₂D₃, the functionally active form of vitamin D. The primary function of 1,25(OH)₂D₃ and VDR is intestinal calcium absorption, which although most rapid in the duodenum occurs primarily in the distal segments of the intestine (only ∼10% in duodenum). 1,25(OH)₂D₃ in turn suppresses PTH synthesis directly at the level of transcription of PTH gene (through vitamin D response element (VDRE) within PTH) and indirectly by increasing serum calcium and upregulating the expression and transcription of CASR (by binding to VDRE within the CASR promoter). 1,25(OH)₂D₃ regulates its own synthesis by inhibiting CYP27B1. 25(OH)D₃ (and 1,25(OH)₂D₃ acting as the preferred substrate) can also be converted to 24,25(OH)₂D₃, products targeted for excretion by the enzyme 24,25-α-hydroxylase (encoded by CYP24A1, present in all cells containing VDR). This process helps in regulation of levels of circulating and, possibly, intracellular 1,25(OH)₂D₃. The regulation of CYP24A1 is opposite to CYP27B1—stimulated by 1,25(OH)₂D₃ and inhibited by low calcium and PTH. 1,25(OH)₂D₃ and elevations in serum phosphate stimulate production of fibroblast growth factor-23 (FGF23), a “phosphate-wasting” glycoprotein produced by osteoblasts and osteocytes. FGF23 and its coreceptor, α-klotho, suppress 1-α-hydroxylase and induce 24,25-α-hydroxylase. In addition to the parathyroid gland, CASR is expressed at a low level in the proximal convoluted tubule (regulates expression of 25(OH)D 1-α-hydroxylase and inhibits PTH-mediated phosphate excretion); distal convoluted tubule (increases calcium reabsorption via transient receptor potential cation channel subfamily V member 5 (TRPV5) channel when tubular fluid calcium concentration is high); and is highly expressed in the thick ascending limb, where it has a PTH-independent key role in maintaining calcium homeostasis (senses increases in calcium and promotes calcium excretion via Claudin 14 tight junction protein), and in renal collecting ducts (prevents development of hypercalciuria-mediated nephrocalcinosis by increasing urinary acidification and water excretion).

Epidemiology of Parathyroid Disorders

Parathyroid disorders are a heterogenous group that encompasses disorders of PTH excess, deficit, or signaling defect. The majority of parathyroid disorders are sporadic. Recent data suggest an increase in the overall incidence of parathyroid disorders, with PHPT being the most prevalent (49-51). Parathyroid disorders can be broadly divided into (1) PTH excess disorders, (2) PTH deficiency disorders, and (3) PTH signaling defects. This review will focus on the molecular and clinical spectrum of PHPT—disorders of PTH excess.

PHPT (Disorders of PTH Excess)

PHPT has an estimated prevalence of 23 cases per 10 000 women and 8.5 per 10 000 men, with an incidence of 66 cases per 100 000 person years in women and 25 per 100 000 person-years in men (52). The reason for higher prevalence of PHPT in women remains uncertain but may be explained by estrogen signaling through ERβ1 and ERβ2, which have been demonstrated to be widely expressed in parathyroid tumors or through epigenetic mechanisms (53, 54). PHPT is associated with morbidities (fractures, kidney stones, chronic kidney disease [CKD]) and increased risk of death (55-57). Understanding the pathophysiology of parathyroid tumors is critical for improving the management of patients affected with this disorder.

The disease is characterized by inappropriate excess of PTH in circulation relative to the serum calcium level due to a primary parathyroid pathology. It is typically caused by tumors of the parathyroid glands which may be (1) single gland adenomas, (2) due to multiple diseased glands (multi-gland disease), or (3) parathyroid carcinomas (PCs). Multigland disease is typically seen in heritable forms of PHPT (10-15%) and include both hyperplasia affecting multiple glands and double adenomas (58). There are no specific diagnostic criteria to differentiate parathyroid hyperplasia from adenoma. The diagnosis of adenoma vs hyperplasia is a clinical one based on history of involvement of single or multiple glands. The 2022 WHO classification of parathyroid tumors supports the use of the term “primary hyperparathyroidism related multiglandular parathyroid disease” as a germline susceptibility-driven multiglandular parathyroid neoplasia (59). The term “parathyroid hyperplasia” is reserved for secondary hyperplasia in the context of CKD. Most parathyroid tumors are solid; however, ∼4% can be cystic (typically >50% of tumor volume) or partly cystic based on imaging or pathology (60). Tumors arising from chief or oxyphilic cells have partly overlapping but distinct molecular profiles (61). Lipoadenomas of the parathyroid gland are rare sporadic tumors manifesting as enlarged glands with >50% fat on histology in the setting of PHPT (62). Most of the tumors of the parathyroid gland (approximately 75%) were initially believed to be monoclonal in origin (12, 63). However, current evidence supports heterogeneity in their clonal origin with affected glands being typically composed of multiple “clonal” neoplastic proliferations (12, 59).

Tumors of parathyroid gland result from 2 events: (1) proliferation of the cells of the parathyroid gland, or (2) altered calcium sensing mechanism leading to inappropriate excess of PTH first followed by secondary proliferation of parathyroid cells (64). The genes identified to cause sporadic parathyroid tumors support the former as the more common mechanism. Although several studies suggest an impairment of the PTH secretory capacity in patients with PHPT, mutations in CASR have not been identified in patients with sporadic parathyroid adenomas (PAs).

PHPT is frequently diagnosed incidentally in an asymptomatic patient (80%) due to ease of diagnostic testing in the Western world. However, in resource-limited countries patients are often symptomatic (neuromuscular weakness, fatigue, impaired memory, obtundation) due to presentation with more advanced disease leading to complications of kidney stones or fractures (65). PHPT can occur either sporadically or due to underlying heritable predisposition discussed below.

Sporadic PHPT

Sporadic PHPT is characterized by inappropriate excess of PTH in circulation relative to the serum calcium level in the absence of a family history of parathyroid tumors. Sporadic PHPT can result from an adenoma or carcinoma of the parathyroid and is caused by the accumulation of somatic mutation(s) in parathyroid cells over time. Additionally, parathyromatosis, autoimmune hypercalcemia, or lithium-associated PHPT may result in disease phenotype (Fig. 8).

Figure 8.

Spectrum of disorders leading to primary hyperparathyroidism (PHPT). The heritable forms of the disease are followed by the gene causing the disease enclosed in brackets. Similar to sporadic PHPT, heritable forms of PHPT can also be classified based on histological findings as parathyroid adenoma, carcinoma, or parathyromatosis.

Sporadic PA

While only multiple endocrine neoplasia type 1 (MEN1) and cyclin D1 (CCND1) are established drivers of parathyroid tumorigenesis based on in vivo clinical and preclinical studies, several candidate driver genes have emerged in the recent years with the advent of Next Generation Sequencing. These findings are summarized below.

Multiple endocrine neoplasia type 1

Somatic biallelic inactivation of MEN1 accounts for 30% to 40% of sporadic PAs (66-68). Parathyroid-specific Men1 knockout mice (Men1^f/f; PTH-Cre) develop parathyroid hyperplasia and hypercalcemia establishing MEN1 as the driver of tumorigenesis in sporadic adenomas (69). Germline inactivating mutations in MEN1 cause multiple endocrine neoplasia type 1 (MEN1) discussed below under “Heritable Hyperparathyroid Disorders” subsection “Multiple endocrine neoplasia type 1”. The gene was initially identified in the context of familial MEN1 through linkage analysis (70, 71) with a precise locus on 11q13 as the underlying cause of MEN1 and later confirmed by positional cloning (72, 73). Both somatic intragenic mutations (indels, frameshift, nonsense, and missense mutations) and larger deletion or mitotic recombination events detected as loss of heterozygosity (LOH) at MEN1 locus occur in sporadic PAs, the latter being more common, occurring in nearly 50% of cases (74).

MEN1 is ubiquitously expressed and encodes for menin, a tumor suppressor protein. It is a multifunctional protein with a role in transcription and epigenetic regulation; cell adhesion, division, motility and signaling; cytoskeletal structure, DNA repair and genomic stability (75). Menin is required for epithelial to mesenchymal transition and loss of menin may be the mechanism by which a progenitor cell differentiates towards a more neuroendocrine cell-like state (76). The 3D crystal structure of menin has been successfully solved and resembles a “curved left hand” with a pocket formed by the “thumb” and the “palm” (77, 78). The 3D structure of menin shows a central cavity that forms a binding pocket for protein interaction but no obvious DNA binding domain, indicating that menin is dependent on its interactions with components of the transcription regulatory machinery and does not directly bind to DNA to control transcription (78). Studies investigating the function of menin have been challenging due to the lack of (1) similarity between menin and other known proteins, (2) recognizable functional motifs/domains in menin, (3) normal or menin-null endocrine cell lines, and (4) successful ex vivo models of MEN1 tumors and corresponding normal tissues (organoids or patient-derived xenograft) (75).

The interaction of menin with over 50 different proteins of known function provides some insight about its role. It interacts with the mixed lineage leukemia (MLL) fusion protein as a transcription cofactor. This fusion protein drives about 10% of acute leukemias, called the MLL-rearranged (MLLr) leukemias. The interaction of menin with MLL fusion protein is critical for the maintenance of MLL fusion–driven gene expression program (79). Small molecules that inhibit the interaction of MLL with menin are currently in clinical trials for the treatment of MLLr leukemias (NCT04067336, NCT04065399) (75). MLL1 fusion genes are gain-of-function mutations in contrast to MEN1-associated tumor cells where menin acts as a tumor suppressor, limiting the utility of menin–MLL interaction inhibitors in MEN1.

Cyclin D1 (CCND1)

Pericentromeric inversions of chromosome 11 involving the PTH promoter and a gene at the 11q13 locus was initially described in sporadic PAs and named parathyroid adenoma 1 (PRAD1) gene (80). The gene was later renamed as cyclin D1 (CCND1) after the encoded protein was identified as a novel member of the cyclin family (81) that drives the transitions through the cell cycle. The name reflects their fluctuating levels through the cell cycle. The chromosomal rearrangement identified in adenomas places the PTH promoter upstream of CCND1 which drives the overexpression of CCND1 in parathyroid glands. Cyclins provide the binding interface for their cognate cyclin-dependent kinases (CDKs). D cyclins coordinate cell cycle transitions with extracellular stimulation like specific physical and chemical cues (82). Cyclin D1 binds to CDK4 and CDK6 at the G1–S phase transition of the cell cycle. Although cyclin D1 protein overexpression has been described in 18% to 40% PAs (83-86), rearrangements involving CCND1 occur in only about 8% of PAs indicating presence of unidentified mechanism(s) of cyclin D1 overexpression (87, 88).

Transgenic mouse model of the PTH-CCND1 rearrangement with parathyroid-specific overexpression of cyclin D1 result in parathyroid cell proliferation and subsequent biochemical PHPT, indicating proliferation of parathyroid tissue as the primary process for CCND1-driven parathyroid tumorigenesis (89, 90).

Cyclin-dependent kinase inhibitor

Cyclin-dependent kinases (CDKs) require the binding of their cognate cyclins for phosphorylation of target proteins. CDK inhibitors (CDKIs) bind to CDK/cyclin complexes and stop or delay the progression of cell cycle (91, 92). Two CDKI families have been identified: Cip/Kip family (CDKN1) and INK4a/ARF (CDKN2). CDKN1 proteins inhibit CDKs while CDKN2 proteins can activate or inhibit both cyclin and CDKs. CDKN1B encoding P27KIP1 (p27) appears most relevant of the CDKN1 family in parathyroid tumorigenesis. Germline mutations of CDKN1B cause multiple endocrine neoplasia type 4 (MEN4), discussed below in “Heritable Hyperparathyroid Disorders” subsection “Multiple Endocrine Neoplasia Type 4”. Somatic CDKN1B mutations have been reported in adult T-cell leukemia/lymphoma (93), breast cancer (94), myeloproliferative disorder (95), hairy-cell leukemia (96), and small intestinal neuroendocrine tumor (NET) (97, 98). Somatic CDKN1B mutations are rare however in sporadic PA or neoplasia (99, 100). Both somatic and germline mutation in CDKN2C, encoding P18INK4C (p18) have been described (101, 102).

When associated with germline MEN1 mutation, CDKNIB can act as a disease modifier suggesting a common tumorigenesis pathway between the 2 (103, 104). Alternatively, it is plausible that mutations in CDKIs alone are insufficient for parathyroid tumorigenesis as somatic mutations in MEN1 have been described in a large proportion of tumors with CDKN1B mutation or underexpression (100, 105). Nevertheless, low mRNA expression of CDKN1A, encoding P21CIP1 (p21) in 53% and of CDKN2C, encoding P18INK6 (p18) in 42% of parathyroid tumors suggests a functional association of CDKIs with parathyroid tumorigenesis (106). Furthermore, methylation defects and low mRNA or protein expression of CDKN2A, encoding P16INK4A and CDKN2B encoding P15INK4B have been described in parathyroid tumors (107, 108). Thus, CDKIs are rare predisposition alleles for sporadic PAs.

β-Catenin (CTNNB1) and WNT pathway

The WNT family is a group of proteins implicated in cell proliferation, differentiation, survival, angiogenesis, and cell renewal. WNT proteins can signal via a canonical β-catenin dependent pathway or a noncanonical β-catenin–independent pathway. Mutation in the genes involved in the WNT signaling pathway have been identified in several cancers. CTNNB1 encodes β-catenin, the primary signal propagator of the canonical pathway. Somatic mutations in CTNNB1, previously thought to be common, are now established to be rare in PAs with an incidence of <1% (74, 109-112). Furthermore, the CTNNB1 mutations reported in sporadic parathyroid tumors are yet to be demonstrated to drive parathyroid tumorigenesis in in vivo models. Aberrant nuclear localization and overexpression of β-catenin appears to be rare in PAs (84, 109, 110, 113, 114).

Among the other genes of the WNT pathway implicated in parathyroid tumorigenesis, LRP5 was noted to be expressed as an alternatively spliced transcript in 86% of sporadic PAs (115). There has been a rare report of biallelic loss of LRP5 from a germline heterozygous mutation and an acquired LOH in the tumor in a patient with apparently sporadic PA (68). However, the LRP5 mutation co-occurred with a frame-shift deletion in MEN1 limiting the ability to evaluate its functional significance. Transcriptional repression of negative regulators of WNT signaling like APC, RASSF1A or members of the secreted frizzled-related proteins, resulting in the accumulation of β-catenin in the nucleus has been described in sporadic PAs (107, 108, 116, 117). Germline inactivating mutations in the tumor suppressor gene APC cause familial adenomatous polyposis syndrome defined by numerous adenomatous polyps which can progress to colorectal carcinoma. An increased prevalence of colon cancer in patients with PHPT has been suggested (118, 119). While absence of APC expression and promoter hypermethylation have been demonstrated in a significant proportion of parathyroid tumors, including ∼75% of PCs (116, 120-124), a mutation or gene amplification in APC has not been identified. Thus, the role of β-catenin and WNT pathway in parathyroid tumorigenesis remains to be established.

Enhancer of zeste homolog 1 and 2

The chromatin modifier, enhancer of zeste homolog 2 (EZH2) is the enzymatic subunit of the complex that methylates lysine 27 of histone H3 (H3K27) to promote epigenetic silencing. EZH2 overexpression was first identified in prostate cancer and subsequently in several other cancers including breast, esophageal, gastric, anaplastic thyroid, nasopharyngeal, and endometrial cancer (125). Somatic mutations in EZH2 have been reported in hematologic malignancies and associated with poor clinical outcomes (126). Apparent mutational activation of EZH2 is rare in sporadic PAs (67, 68, 127, 128). It seems likely that perturbations in EZH2 are a downstream effect of mutations in another driver gene of parathyroid tumorigenesis as it has been implicated in pathways involving other parathyroid tumor genes like MEN1 (129, 130). Activating mutations in EZH2 could theoretically phenocopy inactivating mutations in MEN1. In addition, EZH2 may be involved in WNT signaling (131, 132).

Somatic mutations in enhancer of zeste homolog 1 (EZH1) are rare in sporadic PAs (67). EZH1 mediates the methylation of H3K27 like EZH2. Mutations in EZH1 have been implicated in several hematological malignancies and thyroid tumors (133, 134). Thus, both EZH1 and EZH2 mutations are rare in sporadic PAs.

Zinc finger X-linked

Zinc finger X-linked (ZFX) located on the X chromosome encodes a nearly identical protein to the 1 encoded by ZFY, located on the Y chromosome. It is a transcription factor that regulates embryonic stem cell renewal (135, 136). It has been implicated in the initiation or progression of several different types of human cancers including prostate, breast, colorectal, renal, gastric, nonsmall cell lung, laryngeal squamous cell, gallbladder cancer, and gliomas (137). ZFX mutations have been described in 5% of sporadic PAs with the identified variants located in the DNA-binding domain which are likely to have resulted in alterations in ZFX's DNA-binding affinity or specificity (67, 138, 139). In the context of PAs, the reported variants in ZFX in the DNA-binding domain appear to function as a direct-acting oncogene (140). Overexpression of ZFX has been associated with unfavorable prognosis in other tumor types (141). However, functional studies to establish ZFX as a driver of parathyroid tumorigenesis are yet to be performed. An evaluation for mutations in ZFY in 117 sporadic PAs did not reveal any (142).

Glial cell missing transcription factor 2

Somatic glial cell missing transcription factor 2 (GCM2) mutations are rare in PAs (143, 144). Germline activating mutations in GCM2 cause familial isolated hyperparathyroidism (FIHP) discussed in “Heritable Hyperparathyroid Disorders” subsection “GCM2-mediated PHPT” below. GCM2 is a homolog of the Drosophila glial cells missing (Gcm) gene that was identified for its role in gliagenesis (145, 146). Subsequently, 2 analogous genes were discovered in mouse, rat and human—Gcm1 and Gcm2, with the DNA-binding domain being the only common well-conserved region (147-151). Gcm2 is expressed in kidney, fetal brain, gliomas, medulloblastomas and 4 of 8 nonneuroepithelial tumor cell lines, namely pancreatic adenocarcinomas, leukemias, renal carcinomas, and teratocarcinomas (151). It is the master regulator of parathyroid gland development as evidenced by congenital hypoparathyroidism in Gcm2-deficient mice (152). Intrathymic PAs express GCM2 although normal thymus does not, further supporting the parathyroid-specific nature of the gene. The expression of GCM2 in human hyperplastic and neoplastic parathyroid glands has been shown to be higher in comparison to normal parathyroid tissue (153, 154).

Calcium sensing receptor

Mice with parathyroid-specific CASR ablation develop severe hyperparathyroidism and hypercalcemia. Reduced expression of CASR has been found in majority of sporadic PHPT tumors (155-160). However, mutations in CASR or VDR have not been identified (161, 162). Epigenetic deregulation of CASR via hypermethylation and histone modification has been proposed (163). In addition, regulator of G protein signaling 5 (RGS5), orphan adhesion GPCR, GPR64/ADGRG2, filamin A, and Yes-associated protein 1 (YAP1) have been proposed as physiologic regulator(s) of calcium signaling through CASR in tumor tissue (164-167).

Other candidate genes

Two reported cases of AIP mutations in sporadic PAs were both germline in nature despite the lack of a family history of PHPT (168). Similarly, somatic mutations in REarranged during Transfection gene (RET) are rarely associated with PAs (169, 170). Germline mutations in RET cause MEN2 discussed below in “Heritable Hyperparathyroid Disorders” subsection “Multiple Endocrine Neoplasia Type 2”. Somatic mutations in CDC73 are rare in sporadic PAs but frequent in a PC as discussed below. Germline inactivating CDC73 mutations cause hyperparathyroidism jaw-tumor (HPT-JT) syndrome discussed in “Heritable Hyperparathyroid Disorders” subsection “Novel MEN syndrome—MEN5” (171, 172). While mutations in PIK3CA have been reported in parathyroid malignancy (see Sporadic Primary Hyperparathyroidism subsection “Sporadic Parathyroid Carcinoma”), these are rare in PAs (127, 173).

The telomerase reverse transcriptase (TERT) gene is responsible for maintaining the telomere length. Mutations in TERT have been implicated in several endocrine tumors. Nuclear staining for TERT was noted to be positive in PC but absent in PA (174, 175). However, mutations in TERT were noted to be rare in a study investigating 88 PAs, 22 PCs and 10 atypical tumors (176). Similarly, Protection of telomeres 1 (POT1) gene binds and protects telomeres. Germline variants in POT1 have been associated with several familial cancers including glioma, melanoma, and colorectal cancer while germline and somatic variants in POT1 have been associated with chronic lymphocytic leukemia. Somatic POT1 variant was observed in 1 study in a single sporadic PA with a high mutation rate and with LOH involving multiple chromosomes indicating genomic instability (66). Hypermethylated in cancer 1 (HIC1) is a tumor suppressor gene that is frequently deleted or epigenetically silenced in most cancers and associated with poor clinical outcomes (177). Homozygous deletion of Hic1 is embryonically lethal (178) while heterozygous mutants develop several spontaneous tumors in an age-dependent manner (179). HIC1 was found to be underexpressed in parathyroid tumors regardless of functional status of the tumor (180). In addition, overexpression of HIC1 lead to a decreased survival of parathyroid tumor cells, supporting its growth-regulatory role in the parathyroid glands (180).

Recurrent somatic mutations in Additional sex combs like 3 (ASXL3) were identified in 2 patients with sporadic PA (127). ASXL3 gene has an important role in deubiquitination and is expressed in several organ systems. Germline mutations in ASXL3 are associated with a neurodevelopment disorder, Bainbridge—Ropers syndrome. Somatic ASXL3 variants have been identified in myeloid leukemias and in solid tumors like breast, prostate, and pancreatic cancer (181). Mutation in Ras-related protein, RAP1B, a GTP-binding protein associated with the Ras family has been reported to be involved in several different malignancies with its abnormal expression associated with poor prognosis (182). Somatic variant in RAP1B was reported in PA from 1 patient with co-occurring somatic MEN1 mutation making it difficult to interpret the functional relevance of this gene in parathyroid tumorigenesis (68). Somatic variants in SCL25A3 were observed in 3 PAs in a study (67). SCL25A3 is a phosphate carrier protein located in the mitochondrial inner membrane with a key role in oxidative phosphorylation. Germline mutations in this gene cause mitochondrial phosphate carrier deficiency which is fatal in the first year of life. A role for early stem core genes SOX2, POU5F1/OCT4, and NANOG has been suggested in parathyroid tumorigenesis (183). In addition to GCM2, aberrant expression of embryonic transcription factors like HOX (184), T-box transcription factor 1 (TBX1), and the microRNA cluster C19MC has been described in parathyroid tumors (185, 186). Epigenetic changes of DNA methylation, regulation of gene expression by noncoding RNAs and post-translational histone methylation have been described in parathyroid tumors and are particularly attractive because of the prospect of reversibility (54, 187-191). To summarize, the molecular signature in a large majority of sporadic PAs remains unknown. Somatic mutations in MEN1 are the most frequent genetic change seen in sporadic PAs.

Sporadic PC

The first case of PC was reported by Fritz de Quervain in 1909—a 68-year-old man who presented with a large neck mass and died from lung metastases (192). The incidence of PC has increased by 60% from 1988 to 2003 with a 10-year all-cause mortality rate of 33.2% and the 10-year cancer-related mortality rate of 12.4% (193). Thus, overall, PC is an indolent disease (194, 195). Nevertheless, it has a high recurrence rate at 50% to 100% with majority of the recurrences occurring in the locoregional field which increases the risks of reoperations (196-199). Survival rates for patients who undergo complete en bloc resection with negative margins is up to 90% at 5 years and 67% at 10 years (200).

An unequivocal diagnosis of parathyroid malignancy can only be established in the presence of invasion into adjacent structures, capsular, lymphovascular, or perineural invasion and/or documented distant metastases (201, 202). There is an increasing emphasis on standardizing the pathological reporting of PC and atypical parathyroid tumors (APTs) (203). APTs are parathyroid tumors that lack unequivocal histological signs of malignancy but share some histological features of PC, such as solid growth pattern, fibrous bands, cellular atypia, etc. The distinction between PC and APTs can be difficult and it is not unusual for an initial diagnosis of APT to be modified to PC on follow-up and occurrence of unequivocal features of malignancy particularly distant metastases (204). It is unclear if APTs represent an early stage of PC. However, most patients with APT have a good prognosis without any recurrence (205, 206).

One of the biggest challenges in the management of PC is the lack of a biomarker to establish the diagnosis preoperatively. PC should be suspected in the presence of abnormal high serum calcium concentration (>12 mg/dL) and iPTH levels >3 times upper limit of normal, parathyroid lesions >3 cm, or in patients with PHPT who present with a palpable neck mass associated with hoarseness or dysphagia (207-209). Preoperative suspicion for PC is important in planning the extent and radical nature of initial surgery. Cytology is not useful in differentiating between adenoma vs malignancy in parathyroid tumors. In addition, fine needle aspiration predisposes patients to the risk of tumor seeding and is, hence, avoided. En bloc resection to achieve negative margins is the preferred surgical procedure. Intraoperatively, PC may be suspected by features of local invasion, adhesion to surrounding structures or an enlarged firm to hard gland with greyish white cut surface. Differentiation of benign vs malignant parathyroid tumors cannot be reliably established on frozen section immediate-read pathology although frozen section pathology can help identify metastases to regional lymph nodes. A revision surgery with en bloc resection may be needed if gland excision was initially performed given higher rate of recurrence with the latter (51% vs 8%) (210). Several putative markers for diagnosis of PC have been suggested (Table 1). Most PCs arise de novo and do not represent progression from APTs or PAs as evidenced by their distinct molecular distribution profile (248-250).

Table 1.

Markers of potential diagnostic utility in patients with parathyroid carcinoma

Biomarker	Description	Current data
Gene
CDC73	Chromosomal location 1q31.2; encodes parafibromin; germline mutation associated with HPT-JT	Somatic inactivating mutations can be seen in parathyroid carcinoma and adenoma (particularly, atypical parathyroid tumors).
Copy number loss at 1p and 13	Chromosomal locations harbor putative tumor suppressor genes?	Seen in CDC73-mutated PCs but not CDC73-mutated Pas (211)
CD24, HMOX1, VCAM1, and KCNA3	Overexpression of transcript and/or protein in PC relative to PA	Potential marker for PC (212)
RNA
miR-296	Small, noncoding RNAa (micro-RNA)	Downregulated in PCs compared to normal parathyroid tissue (213)
miR-126	Small, noncoding RNAa (micro-RNA)	Most accurate differentiator between PC and PAs (71% sensitivity, 82% specificity) (214)
C19MC (miR-371-3)	micro-RNA cluster	Aberrations characteristic of PC (186)
Combined analysis of miR-139 and miR-30b		Can distinguish PC from PAs (215)
hsa_circ_0075005 and MYC mRNA	Circular RNA (noncoding RNAs)	Differentially expressed in PCs (216)
LINC00959, Inc-FLT3-2:2, lnc-FEZF2-9:2, and lnc-RP11-1035H13.3.1-2:1	Long noncoding RNA	differentially expressed in PCs (217)
lncRNAPVT1 and GLIS2-AS1	Long noncoding RNA	Increased in PCs (218)
exosome has-miR-27a-5p	exosomal miRNA	upregulated in serum exosomes of patients with PC (219)
Protein
Parafibromin	Tumor suppressor encoded by CDC73—contains nuclear and nucleolar localization signals; predominantly nuclear protein	Potentially useful in diagnosis of APTs (220, 221) Loss of staining shows ∼70% sensitivity for diagnosis of PC (222, 223-231) High specificity for PC (∼95%) (224, 226, 227, 232) Loss of staining may predict lower overall survival in PC (233)
Galactin-3	Member of lectin family; overexpression related to ability of tumor to evade apoptosis	Strong staining in ∼95% PCs but low sensitivity (223, 229, 230, 234) Can serve as an adjunct to histology for PC diagnosis particularly in combination with parafibromin (231)
Protein gene product 9.5 (PGP9.5)	Encodes ubiquitin carboxy-terminal hydrolase L1 (UCHL1); initially thought to have an expression specific to NETs but has since been shown to be expressed in large number of non-NETs.	Strong staining for PC (sensitivity: 78%; specificity: 100%) (224, 230, 231) Stains 22-35% APTs and <22% PAs thus unlikely to be useful as diagnostic marker of PC Predictor for recurrent hypercalcemia in PC and APTs (220)
Ki-67	Monoclonal antibody Ki-67 identifies a nuclear protein antigen (encoded by gene MKI67) that is associated with cellular proliferation; marker in cells that increases as they prepare to divide into new cells	Higher proliferation index suspicious for malignancy (223, 228, 229, 234) Poor sensitivity for PC diagnosis (230) Potentially useful in combination with other immunohistochemical markers (235)
Cyclin D1	Cyclin encoded by the gene CCND1; required for progression through the G1 phase of the cell cycle	No difference in expression in PCs, APTs, and PAs (221, 230, 235-237)
Adenomatous polyposis coli (APC)	Encoded by APC gene; tumor suppressor that is negative regulator of WNT signaling; germline mutations associated with familial adenomatous polyposis (FAP)	Not informative (231, 238)
Calcium-sensing receptor (CASR)	Calcium-sensing GPCR expressed in parathyroid cells and kidney tubules; negative regulator of PTH secretion	Downregulation of CASR strongly associated with ↑ malignant potential in PC (239)
Retinoblastoma (RB)	Tumor suppressor encoded by RB1 associated with childhood intraocular cancer	88% of PC had absence of nuclear staining which was not seen in any of the 19 adenomas (240) However, subsequent studies showed lower sensitivity with loss of expression in 30% PCs only (223)
B-cell lymphoma-2 (BCL-2)	Encoded by the gene BCL2; regulates programmed cell death (apoptosis); positive expression in PAs	Positive expression can potentially distinguish PAs from APTs and PCs (234, 235) Staining can be positive in ∼40% PCs (234) Potentially useful in combination with other immunohistochemical markers (235)
APOLLON	Encoded by gene BIRC6; inhibits apoptosis by facilitating the degradation of apoptotic proteins by ubiquitination	Increased expression in PC (241)
E-cadherin	Component of adherens junction integral in cell adhesion Loss promotes tumor metastasis	Expression detected in normal parathyroid and PAs (242) Findings in APTs not consistent (243) Loss of membranous staining in PC (242)
CDKN1B	P27Kip1, a.k.a. cyclin-dependent kinase inhibitor 1B, encoded by the CDKN1B gene; cell cycle inhibitor and tumor suppressor: loss of staining anticipated in PC; germline mutations associated with MEN4	Not useful as a diagnostic marker between different tumor types (223) Poor sensitivity and specificity (234) Potentially useful in combination with other immunohistochemical markers (235)
Mouse Double Minute 2 (MDM2)	Encoded by MDM2 gene; negative regulator of p53 tumor suppressor; overexpression associated with several cancers	Not useful as a diagnostic marker between different tumor types (223) Potentially useful in combination with other immunohistochemical markers (235)
Programmed death-ligand 1 (PD-L1)	Encoded by CD274; transmembrane protein that suppresses the adaptive arm of immune response, involved in interaction of tumor cells with host immune response	Expression not increased in normal parathyroid (244) Deficient PD-L1 expression in PAs, PCs, and APTs suggesting limited effectiveness of immunotherapy although studies not consistent (245, 246) No difference in expression between APTs and PCs (247)
Hector Battifora mesothelial cell-1 (HBME-1)	marker of mesothelial cells, positive in various thyroid carcinomas	Not informative as a diagnostic marker for PC (229)

Abbreviations: APT, atypical parathyroid tumor; GPCR, G-protein–coupled receptor; HPT-JT, hyperparathyroidism jaw tumor; PA, parathyroid adenoma; PC, parathyroid carcinoma; PTH, parathyroid hormone; NET, neuroendocrine tumor.

About 6% to 30% patients with PC can present with lymph node metastases (194, 197). The utility of TNM cancer staging in PC and its correlation to clinical outcomes remains controversial (193, 251). However, recent studies demonstrate that the presence of distant metastasis predicts overall survival (193, 252). The most common sites of distant metastasis are lung, bone, and liver occurring in approximately 25% patients (193, 194, 202). While mostly functional, PCs may rarely present as nonfunctional (253). Nonfunctional PCs tend to be larger at presentation and patients may present with locally compressive symptoms such as palpable neck mass or hoarseness from recurrent laryngeal nerve invasion (254, 255).

The efficacy of adjuvant systemic therapy has not been evaluated in clinical trials. Case reports of remission with immunotherapy have been described (256, 257). Sorafenib was associated with decrease in the size of lung metastases in 1 patient with metastatic PC (258). The role of radiotherapy in PC remains controversial (210). The morbidity and mortality from PC are primarily the sequelae of hypercalcemia and its complications. Cinacalcet, bisphosphonate, denosumab and intravenous fluids are all treatment options to be considered for management of hypercalcemia in patients who are not surgical candidates. We discuss the genes that have implicated in the pathogenesis of PC below.

CDC73

While somatic mutations in CDC73 are rare in PAs, these are seen in 40% to 75% of patients with sporadic parathyroid cancer (171, 199, 259-261). Germline mutations in CDC73 cause HPT-JT discussed in “Heritable Hyperparathyroid Disorders” subsection “Hyperparathyroidism Jaw Tumor Syndrome” . Parathyroid-targeted deletion of Cdc73 by crossing floxed-Cdc73 mice with PTH-Cre mice, resulted in parathyroid gland features consistent with APTs and PCs in both homozygous and heterozygous mice (262). All patients with seemingly sporadic PC should be screened for germline variants in CDC73, even in the absence of a family history, given incomplete penetrance seen with CDC73 mutations (259). CDC73 encodes a ubiquitously expressed, evolutionarily conserved 531 amino acid protein called parafibromin, a predominantly nuclear protein (263, 264). Cytoplasmic parafibromin may be distinct from nuclear parafibromin. In vitro studies suggest that parafibromin needs to interact with targets in the nucleolus to execute its tumor suppressor functions as indicated by simultaneous loss of nucleolar localization and acquisition of a growth stimulatory phenotype in mutant cells (265). Parafibromin is a component of the Polymerase Associated Factor 1 (PAF1) transcriptional–regulatory protein complex in human, fly, and yeast. The PAF1 complex binds to the C-terminal domain of RNA polymerase II and is involved in transcriptional initiation, elongation, and post-translational events (266, 267). The clinically relevant mutations reported in CDC73 occur throughout the coding region and splice sites of the gene (268). Exons 1, 2, and 7 account for the maximum frequency of mutations but this is not explained by their larger size as these exons remain overrepresented even when mutation rate is calculated accounting for exon size (268). There is no reported difference between the types of germline and somatic CDC73 mutations with majority being frameshift (54% in germline and 42% in somatic) (268). The crystal structure of the N-terminal 111 amino acids of human parafibromin has been solved (269-271). The precise mechanism by which inactivation of CDC73 results in parathyroid neoplasia remains elusive. It is possible that parafibromin may act as both a tumor suppressor and an oncogene, depending on the tissue micro-environment (272). Some studies suggest a role of parafibromin in WNT signaling (273, 274). Upon tyrosine dephosphorylation, parafibromin acquires the ability to stably bind β-catenin, thereby activating promitogenic WNT signaling (275).

Multiple endocrine neoplasia type 1

While PC associated with germline mutations in MEN1 is rare and may occur in up to 2% of patients, somatic mutations in MEN1 have been reported to occur in ∼15% PCs (276-281). However, overall, mutations (germline or somatic) in MEN1 appear to be rare in PC.

Rearranged during Transfection

There are rare case reports of PC in MEN2A (discussed in “Heritable Hyperparathyroid Disorders” subsection “Multiple Endocrine Neoplasia Type 2”); however, the germline or somatic status of RET mutations is not always clarified (282). Overall, mutations in RET appear to be rare in PC.

PI3K/mTOR

The PI3K/AKT/mTOR pathway has emerged as a major oncogenic pathway and may serve as a diagnostic marker for PC as it appears to be rarely involved in PAs. The identification of mutations in PIK3CA, PTEN, AKT1, mTOR, and TSC1 (20-30%) in the presence of the wild-type CDC73 allele suggests their potential role in driving parathyroid neoplasia (124, 283, 284). However, functional characterization of these mutations has not been performed. An activating PIK3CA mutation found in primary PC sample was surprisingly noted to be lost in the evolution of the tumor from primary to recurrence (285). Variants in TSC1 and NF1 with allele frequency >0.5 indicating LOH were observed among 29 samples of PC (286). More recently, gene alterations in PI3K/AKT/mTOR pathway (somatic or germline) were found in ∼80% of tumors on whole genome sequencing of 23 PC samples (261).

WNT pathway

Increased nuclear accumulation of active, nonphosphorylated β-catenin in PC tumor samples relative to adjacent normal control samples implicate the canonical WNT/β-catenin pathway as an important oncogenic pathway in PC (123). Activation of the WNT pathway due to epigenetic regulation such as hypermethylation of APC has been proposed as the underlying mechanism of increased β-catenin accumulation. Inactivating somatic mutation(s) in APC and RNF43 were observed in 1 of 24 PC sample each, although it is unclear if this was a biallelic event (124). Furthermore, functional characterization of these variants was not performed.

Cyclin D1/CCND1

Higher CCND1 mRNA levels and protein expression are frequent (∼70%) in PC in contrast to PAs (∼20%) (287). Of the 5 PC samples with CCND1 gene amplifications, only 1 also harbored a somatic CDC73 mutation (124). However, the mechanism linking CCND1, and malignancy remains unknown, and it is possible that gene amplifications in CCDND1 synergize with mutations with other candidate drivers of carcinogenesis.

Other candidate genes:

• Somatic activating GCM2 mutations have been described in patients with sporadic PC (154, 288).

• Tumor protein 53 (TP53) encodes p53, a transcription factor and tumor suppressor. Somatic mutations in TP53 have been reported in a subset of PC and PAs although their contribution to tumorigenesis remains unknown (284, 286, 289, 290). Low expression of p53 is seen in both PC and PAs.

• Recurrent germline and somatic mutations in prune homolog 2 (Drosophila) (PRUNE2) have been described in 1 study of exome sequencing on 8 paired PCs (291). The reported PRUNE2 mutations were a single germline and 2 somatic missense mutations in a CDC73 wild type sample. PRUNE2 is a tumor suppressor reported to be mutated in prostate cancer.

• Glycogen synthase kinase 3 beta (GSK3B) protein expression was found to be lost in ∼35% PCs (121). However, loss of GSK3B in parathyroid tumors was not associated with an increased β-catenin or CCND1 expression indicating an unrelated mechanism. Furthermore, mutations in GSK3B have not been described in PC. GSK3B regulates glycogen synthesis, energy homeostasis and apoptotic pathways and is implicated in the pathogenesis of Alzheimer and Parkinson disease.

• While decreased expression of ten-eleven translocation-1 (TET1) and ten-eleven translocation-2 (TET2) in PC was seen in 1 study, mutations in TET proteins have not been described (292). The TET family of proteins regulate DNA methylation and play an important role in various cancers.

• EZH2 and ZFX, which are rarely mutated in PAs, appear to be even more rarely mutated in PCs (293, 294). Gene amplification of EZH2 has been reported in ∼60% PCs (295). Furthermore, overexpression of EZH2 mRNA has been observed in samples without gene amplification suggesting indirect mechanism possibly through its interaction with WNT-β-catenin pathway or histone modification via HIC1 (180).

• PC samples demonstrated a strong presence of the mutation signature of apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like, particularly in samples with higher mutational burden (124, 291).

• Allelic loss of RB1 has been observed in 30% to 100% of PCs and reported to be associated with recurrence (236). In contrast, allelic loss of RB1 is only seen in <5% of PAs (240-297). Decreased RB1 expression has been described in >85% PCs. However, no mutations in RB1 have been identified (298).

• FAT3 encodes a large protein with multiple cadherin and EGF-like motifs which is predicted to enable calcium ion binding activity and have a role in cell adhesion (299, 300). It has been implicated in PCs (124) and multiple other cancers including lung (301).

• Extracellular matrix–related genes have been implicated in the early phase of parathyroid carcinogenesis while upregulation of MMP9, ANGPTL4, BMP7, FGFR1, and SOX2, and downregulation of ERBB3, TBX1, FBP1, and RAB25 contribute to metastatic phenotype of PC (219, 248, 302).

Thus, the genomic landscape of PC remains largely unknown and is further limited by the rarity of the disease and availability of tissue samples being limited to archived formalin-fixed paraffin embedded samples.

Parathyromatosis

Parathyromatosis is a rare cause of recurrent PHPT due to presence of nodules of benign hyperfunctioning parathyroid tissue scattered outside the gland usually in the neck and mediastinum, that was first described in 1975. It affects women more than men. It can be of 2 forms (303):

1. Primary or type 1 parathyromatosis results from proliferation of parathyroid rests during its descent in embryologic development.

2. Secondary or type 2 parathyromatosis is the more common form and results from seeding during parathyroidectomy or percutaneous ablation from rupture of capsule, typically in patients with CKD (304-306). The metabolic derangements in CKD likely provide a persistent stimulation for PTH secretion.

Parathyromatosis may be considered a low-grade locally invasive malignancy. Preoperative diagnosis of parathyromatosis is rare and the diagnosis is typically made based on clinical presentation, imaging findings and histology. These nodules can be localized on ^99mTc-sestamibi, 4-dimensional computed tomography (CT) or ¹⁸F-fluorocholine positron emission tomography/CT, although available data on sensitivity in disease is limited (307, 308). Parathyromatosis tissue shows positivity for chromogranin and synaptophysin and is positive for PTH and cytokeratin (CAM 5.2). While the distinction of parathyromatosis from APTs and PCs can be challenging, this distinction is unlikely to change management as surgery is first line for all 3 (309). Management of parathyromatosis is challenging with a high failure rate with both surgical and medical management. The possibility of unidentified miliary parathyroid tissue dissemination should be considered in the medical decision-making for these patients. Cinacalcet and bisphosphonates, alone or in combination are the main treatment options for medical therapy. Recent reports of denosumab use for long-term therapy have also emerged (310-314).

Other Acquired Disorders With Primary Hyperparathyroidism

Autoimmune Hypocalciuric Hypercalcemia

These patients have clinical features of FHH (biochemical phenotype of PHPT and hypocalciuria—see “Heritable Hyperparathyroid Disorders” subsection “FHH and Neonatal Severe Hyperparathyroidism”) but negative genetic testing and presence of autoantibodies against CASR with lymphocytic infiltration of the parathyroid gland (315-317). Typically, this is seen in a patient with other autoimmune conditions. Mechanistically, these antibodies can induce biased signaling of CASR. These patients may respond to glucocorticoid or calcimimetic therapy (318).

Lithium-associated hypercalcemia

Lithium-associated hypercalcemia has a prevalence of 26% among all patients on lithium for bipolar disease (319) and presents as PHPT phenotype with PTH-mediated hypercalcemia. The speculated mechanisms for this association include (1) the inhibition of inositol monophosphatase by lithium in chief cells results in alterations in intracellular calcium levels and subsequent PTH secretion, (2) interaction of lithium with CASR resulting in an increased calcium-PTH secretory “set point” (extracellular calcium concentration at which PTH secretion is half maximally suppressed) and increased PTH secretion through the development of hyperplasia or adenoma, (3) inhibition of glycogen synthase kinase 3 by lithium may contribute to irregular WNT/β-catenin signaling, which is implicated in parathyroid tumorigenesis, and (4) unmasking of an adenoma. Multigland disease with persistent/recurrent disease seems more common in lithium-associated hypercalcemia indicating that bilateral neck exploration should be considered in these patients (319).

Heritable Hyperparathyroid Disorders

Approximately 10% to 15% of PHPT consists of heritable forms with MEN1 being the most common heritable form, affecting 2% to 4% of patients with PHPT (320). Hyperparathyroidism is often the first manifestation of the syndromic forms of heritable PHPT (MEN1, MEN4, and HPT-JT). Early diagnosis of these disorders is useful in planning the extent of surgery and establishing an optimal surveillance for recurrence of PHPT and the associated extraparathyroidal manifestations of these disorders (Table 2). The distinction between sporadic vs heritable form of PHPT can often be difficult due to (1) the incomplete penetrance of some heritable syndromic forms, (2) presence of de novo mutations in patients with no family history, and (3) occult PHPT in family members limiting the utility of family history in such cases. Thus, consideration of heritable causes of PHPT should be done in patients of any age, particularly those with additional risk factors.

Table 2.

Heritable forms of hyperparathyroidism

Disorder	Genetic cause	Gene location	Functional effect	Associated clinical features
Multiple endocrine neoplasia type 1	MEN1	11q13	Inactivating	Pituitary tumors, GEP-NETs
Multiple endocrine neoplasia type 2	RET	10q11.21	Activating	Medullary thyroid cancer, pheochromocytoma
Multiple endocrine neoplasia type 4	CDKN1B	12p13.1	Inactivating	“MEN1-like”; pituitary tumors, GEP-NETs, milder disease phenotype
Multiple endocrine neoplasia type 5	MYC-associated factor X (MAX)	14q23.3	Inactivating	Pheochromocytoma, pituitary tumors, (evolving phenotype)
Hyperparathyroid jaw tumor syndrome	CDC73	1q31.2	Inactivating	Jaw, uterine, kidney tumors; 20-25% risk of parathyroid carcinoma; incomplete penetrance
Familial hypocalciuric hypercalcemia type 1	CASR	3q13.3-q21.1	Inactivating	Mild elevation in serum calcium and PTH, minimal target organ effect, Asymptomatic, parathyroidectomy does not resolve hypercalcemia, present since birth
Familial hypocalciuric hypercalcemia type 2	GNA11	19p13.3	Inactivating
Familial hypocalciuric hypercalcemia type 3	AP2S1	19q13.32	Inactivating	Most severe form of FHH; may be associated with target organ effects (for example, kidney stones or fractures)
Neonatal severe hyperparathyroidism	CASR (homozygous/compound heterozygous)	3q13.3-q21.1	Inactivating	Seen in first week of life; life-threatening
Familial isolated hyperparathyroidism	GCM2	6p24.2	Activating	Poor penetrance; parathyroid carcinoma reported; enriched in Ashkenazi Jewish populations

Abbreviations: FHH, familial hypocalciuric hypercalcemia; GEP-NET, gastroenteropancreatic neuroendocrine tumor; PTH, parathyroid hormone.

Syndromic Forms of Primary Hyperparathyroidism

Syndromic forms of PHPT are conditions that are characterized by a set of associated signs, symptoms, and/or extraparathyroidal manifestations like the multiple endocrine neoplasias and HPT-JT discussed below. Multiple endocrine neoplasias are characterized by tumors in at least 2 endocrine tissue.

Multiple endocrine neoplasia type 1

History

MEN1 was first recognized as a distinct clinical and familial syndrome by Wermer accounting for why the original name of MEN1 was Wermer syndrome (321). It has an estimated prevalence of 3-20/100 000 and 1 % to 18% among patients with primary hyperparathyroidism (75, 322), 16% to 38% in patients with gastrinomas (323) and less than 3% in patients with pituitary tumors (324-326). It is an autosomal dominant disease; however, its prevalence seems to be higher in females.

Clinical manifestations

Patients with MEN1 (Fig. 9A) are at an increased risk of premature death (327) justifying the need for genetic screening (328) and periodic prospective clinical screening of carriers of MEN1 mutation (329-331). While the management of MEN1-related neuroendocrine tumors (NETs) is no different to the management of the respective NETs in sporadic cases, the treatment outcomes are not as successful (325). This is likely explained by (1) tumor multiplicity reducing the probability of surgical cure, (2) higher frequency of occult metastatic disease in MEN1, and (3) tendency for MEN1-related NETs to be larger, more aggressive, and therefore more resistant to treatment (325).

Figure 9.

Clinical manifestations in syndromic forms of primary hyperparathyroidism with its reported frequency. (A) Multiple endocrine neoplasia type 1 (MEN1). (B) Multiple endocrine neoplasia type 2 (MEN2). (C) Multiple endocrine neoplasia type 4 (MEN4). (D) Multiple endocrine neoplasia type 5 (MEN5); *comprehensive phenotype evolving. (E) Hyperparathyroidism-jaw tumor syndrome (HPT-JT). Frequency reported in parenthesis.

Primary hyperparathyroidism

PHPT is the most penetrant manifestation of MEN1 occurring in ∼100% patients by age 50 (332, 333). Comparison with sporadic PHPT, the PHPT seen in MEN1 has an earlier age of onset (20-25 year vs 55 year), more often involves several glands, has an equal gender predilection, greater reduction in bone mineral density (334), and a higher risk of recurrence (325). The youngest reported case of PHPT in MEN1 is at age 4 years (335). Preoperative localization is of limited value in index operations as bilateral neck exploration is recommended irrespective of the findings on preoperative localization studies. There is no consensus regarding the timing of surgery for index or reoperation in MEN1-related PHPT and a shared decision-making approach considering the surgical experience, access to testing for long-term monitoring of serum calcium, and acceptance of risk for possible postsurgical hypoparathyroidism is recommended. Our practice is to evaluate patients for symptoms and complications and delay the initial parathyroid surgery in the absence of either, in view of likely need for future reoperations.

Preoperative diagnosis of MEN1 helps guide the extent of initial parathyroidectomy and is associated with lower recurrence rates (336). Open bilateral neck exploration is recommended given the preponderance of multigland disease in MEN1. Subtotal parathyroidectomy with removal of ∼3.5 glands with bilateral transcervical thymectomy is the surgery of choice for index operations in MEN1-related PHPT to balance the risks of recurrent or persistent disease vs those from permanent hypoparathyroidism (325, 337-344). The choice of remnant creation (weighing 60 mg, approximately twice the size of a normal gland) is guided by relative macroscopic normality, the accessibility of the preserved gland for subsequent reoperations (inferior glands lie more anteriorly, away from the recurrent laryngeal nerve and are preferred), and vascular viability which should be confirmed before resection of other glands (75). Autotransplantation of fresh or cryopreserved parathyroid tissue has been used when no functioning parathyroid tissue is assumed to remain in the body, predominantly for reoperations; however, its success is largely dependent on storage duration in the case of cryopreserved tissue and its consequent vitality. Cinacalcet, may be useful for the medical management of PHPT in patients who are not surgical candidates (345). Prophylactic transcervical thymectomy is performed to mitigate risk of mediastinal recurrence from ectopic or supernumerary glands seen in 12% MEN1 cases (346).

The multiglandular parathyroid involvement in MEN1 can be asymmetrical and asynchronous. The involved glands are grossly enlarged (>6-8 mm) and increased in weight (>40-60 mg). Multiglandular parathyroid chief cell hyperplasia of nodular type composed of multiple monoclonal proliferations, constituting multiple microadenomas is the major manifestation of MEN1-related PHPT (347-349). MEN1-related parathyroid proliferations lack the characteristic atrophic rim of nonlesional parathyroid tissue. When present, these rim-like areas are cellular rather than atrophic. Cystic parathyroid changes may be seen occasionally in MEN1 (350). APTs or PC is rarely seen in PC (351). Typically, PHPT is the earliest and most penetrant manifestation of MEN1. Fortunately, given the availability of widely available biomarkers like serum calcium and PTH, patients with MEN1 can be easily monitored for the onset or recurrence of PHPT.

Pituitary adenomas

The incidence of pituitary tumors in patients with MEN1 varies from 15% to 50% with a mean age of onset 38.0 ± 15.3 year (325, 335, 352-354). Pituitary disease is the initial presenting manifestation of MEN1 in ∼20% cases (352) and may occur as early as age 5 (355-357). Plurihormonal adenomas and multiple pituitary adenomas are seen at a higher frequency in MEN1 patients (353). In addition, a higher proportion of MEN1-related pituitary tumors are macroadenomas and show invasive features with reduced response to therapy in comparison to tumors in non-MEN1 patients (326, 352, 353, 358). However, prevalence of pituitary carcinoma is not increased in MEN1 (359) and asymptomatic nonfunctional adenomas may be followed safely with serial MRI (354). A recent study showed a lower prevalence of macroadenomas in MEN1-related pituitary adenomas (about 30%) than older reports (85%), perhaps reflecting detection bias from improved and increased imaging and assays (352, 354). There are no histological characteristics distinguishing MEN1-related pituitary adenomas from the corresponding non-MEN1 pituitary tumors (353).

Functional pituitary adenomas are more common in MEN1 with a frequency of 60% for prolactinomas, 25% for growth hormone–secreting tumors, 5% for Cushing disease (360) and remaining being nonfunctioning or glycoprotein subunit secreting, although these numbers may represent detection bias (325). The increased female to male ratio in the frequency of pituitary adenomas in MEN1 patients (352, 354) is similar to the observation in non-MEN1 patients (361). The management of MEN1-related pituitary adenomas is identical to its management in non-MEN1 patients (362-364). There are 2 MEN1 variants in which prolactinomas seem to be predominant: (1) Burin or prolactinoma variant (365, 366), indicating the region of origin from the Burin peninsula of Newfoundland with a high occurrence of prolactinomas and a low occurrence of gastrinomas with nonsense mutations (Tyr312Stop and Arg460Stop) in MEN1, and (2) Tasmania variant (367), with a splice site mutation (c.446-3c→g). In this variant however, the prolactinomas were not evenly distributed, present in >50% of MEN1 affected members in branches of overall genealogy but uncommon in the remaining pedigree (368).

Gastroenteropancreatic neuroendocrine tumors

About 30% to 80% of patients with MEN1 develop gastroenteropancreatic neuroendocrine tumors (GEP-NETs) which may be functional, resulting in distinct clinical syndromes, or nonfunctional (325, 369). Comparison with sporadic GEP-NETs, MEN1-related GEP-NETs have an earlier age of onset and are frequently multiple (332, 335, 370), making it challenging to correlate circulating hormonal status with tumor source. GEP-NETs including nonfunctional GEP-NETs increase the risk of death in MEN1 (371). Unlike patients with sporadic GEP-NETs where patients are often diagnosed after the disease has metastasized, in MEN1 there exists a window of opportunity to prevent metastatic disease. One of the goals of research in MEN1 is to develop biomarkers that can predict aggressive vs indolent GEP-NETs early in the disease course and prevent metastases.

Functional GEP-NETs

Zollinger–Ellison syndrome/gastrinomas

Zollinger–Ellison syndrome (ZES) is characterized by gastric acid hypersecretion and resulting severe peptic ulcer disease and diarrhea caused by secretion of the hormone gastrin by GEP-NETs (gastrinomas). It is important to note that the designation of a tumor as gastrinoma relies on the presence of symptoms of hypergastrinemia and not on histological or immunohistochemical characteristics. Gastrinomas are the most common functional GEP-NETs in MEN1, present in some 30% to 50% of patients (325). Approximately 20% to 30% of all gastrinomas occur in patients with MEN1 (325). Gastrinomas predominantly occur in the submucosa of the first part of the duodenum, with only about 20% to 25% of gastrinomas occurring in the pancreas (372). Nevertheless, pancreatic gastrinomas metastasize to the liver more frequently than duodenal gastrinomas (372, 373). Rarely, gastrinomas may arise in other abdominal (eg, stomach, liver, lymph nodes, bile duct, ovary) or extra-abdominal (lung) sites. MEN1-related gastrinomas have an aggressive disease course with distant metastases and early death however, some inconsistencies in the reported studies regarding prognosis prevail (373-376). Treatment of associated PHPT with restoration of normocalcemia has been reported to ameliorate symptoms of hypergastrinemia in approximately 20% of MEN1 patients with ZES (377). Current evidence only supports surgery for pancreatic gastrinomas greater than 2 cm (373, 374). The risk of hepatic metastases, a prognostic marker of MEN1-related gastrinomas increases with tumor size (374). The presence of lymph node metastases does not appear to adversely affect survival in MEN1-related gastrinomas (373).

Insulinomas

Insulinomas constitute 10% to 30% of all pancreatic neuroendocrine tumors (PNETs) in MEN1 and approximately 4% of patients with insulinomas will have MEN1 (325). MEN1-related insulinomas differ from sporadic insulinomas in presenting at an earlier age and in having a higher rate of recurrence (21% in MEN1 vs 7% for non-MEN1) (378). Of note, a recurrence before 4 years suggests fracture of the insulinoma at original enucleation (379). Insulinoma may be the presenting manifestation of MEN1 in about 10% of MEN1 patients (325). Insulinomas can be associated with other PNETs in 10% MEN1 patients and the tumors may arise at different times (380) necessitating specialized insulinoma localization procedures like selective pancreatic arterial calcium stimulation testing.

Other functional GEP-NETs seen more rarely in MEN1 include glucagonoma, VIPoma, GHRHoma, and somatostatinoma.

Nonfunctioning GEP-NETs

Nonfunctioning PNETs (NF-PNETs) are the most frequent PNET in MEN1 (369, 381) and are a clinically heterogenous group (382). NF-PNETs have been reported to occur in children (370, 383). Thus, imaging assessment for GEP-NET is recommended starting at age 10 (325). Malignant NF-PNET are the commonest cause of death in MEN1, likely due to delayed diagnosis given lack of clinical syndrome (327, 371, 381, 384). Nevertheless, the clinical relevance of detecting small GEP-NETs in an asymptomatic individual remains uncertain. The optimal frequency of surveillance and timing of surgery remains controversial and may be personalized based on tumor size, location, grade, symptoms, overall clinical status, and operative risk (385). Surgical resection is currently recommended for tumors >2 cm in size or those with significant progression (doubling of tumor size within 3-6 month or increase > 1 cm) (325, 386-390).

Carcinoid tumors

The term “carcinoid” means “carcinoma-like” underscoring a benign behavior despite microscopic resemblance to a carcinoma (391). Carcinoids occur in about 3% MEN1 patients arising predominantly from the foregut (thymus, bronchopulmonary system and stomach) (325). Carcinoid syndrome characterizes the constellation of symptoms including diarrhea, episodic flushing, bronchoconstriction, and right sided valvular heart disease mediated by the release of humoral factors like serotonin into systemic circulation. Most MEN1 patients with carcinoid tumors remain asymptomatic and lack a consistent biochemical abnormality (325). Rarely, ectopic hormonal secretion, such as adrenocorticotropin may occur from thymic or bronchopulmonary carcinoid in MEN1 (392-394).

Thymic carcinoids

The thymus contains elements derived from all 3 germinal layers. Thymomas and thymic carcinoids should be considered in the differential diagnosis of an anterior mediastinal mass in MEN1 (395, 396). While both are epithelial in origin, thymomas have a dual population of epithelial cells and lymphocytes, not seen in carcinoids. Additionally, the staining for neuroendocrine markers in thymomas, if present, is more focal (397). Somatic LOH at the MEN1 locus was recently demonstrated as the mechanism for tumor development in both thymic carcinoids and thymomas (398). An association of thymomas with myasthenia gravis and paraneoplastic syndromes, while not uncommon in the non-MEN1 setting, has not been described in MEN1. About 25% of thymic carcinoids arise in patients with MEN1 (399).

Thymic carcinoids may be the initial manifestation of MEN1 (400, 401) and have been reported as early as age 16 (402). These tumors have an aggressive disease course with a 10-year survival of 35% to 45% (402-405). Almost half of MEN1 patients with thymic carcinoids already have distant metastases at the time of diagnosis which may explain their poor prognosis (403). Transcervical thymectomy does not afford prevention from thymic carcinoids raising the question of prophylactic thymectomy in high-risk patients (406, 407). The rarity of the manifestation and lack of definitive evidence have precluded the determination of the best approach to patient care in these high-risk patients. Male gender, a smoking history, and a familial history of thymic carcinoids appear to be risk factors for thymic carcinoids in MEN1 (402, 405, 408, 409).

Bronchopulmonary carcinoids

Multifocal tumorlets and neuroendocrine cell hyperplasia may represent the precursor lesions of bronchopulmonary carcinoids (410). Their natural course seems benign although, poorly differentiated aggressive bronchopulmonary carcinoids can increase mortality (404, 405, 410-413). There is no consensus regarding management (observation vs resection) of bronchopulmonary carcinoids in MEN1. MEN1 mutations have been described in sporadic carcinoid tumors of the lung (414) and are associated with poor prognosis (415).

Gastric carcinoids

Gastric carcinoids (type II) are associated with MEN1 and ZES and may be detected incidentally on endoscopy in 70% MEN1 patients. These are usually multiple, smaller than 1.5 cm and have an indolent clinical course. Endoscopic resection followed by endoscopic surveillance is adequate therapy for most patients.

Adrenal tumors

About 20% to 73% of patients with MEN1 have adrenal tumors detected on imaging, the majority of which are asymptomatic. The spectrum of adrenal lesions includes cortical adenomas, hyperplasia, multiple adenomas, nodular hyperplasia, cysts, or carcinomas (325). About 10% to 15% of these lesions are functional with aldosteronomas and cortisol-secreting adrenocortical tumors being the most frequent (360, 416, 417). Pheochromocytoma and adrenocortical carcinoma (1%) are rare in MEN1 (416, 417). Similarly, somatic MEN1 mutations are rare in sporadic adrenocortical carcinomas (418). Adrenal tumors with radiological characteristics concerning for malignancy, with significant increase in size, or those larger than 4 cm should be considered for surgery (417, 419).

Skin findings

Multiple angiofibromas (22-88%), collagenomas (0-72%), and lipomas (33%) are cutaneous manifestations of MEN1 (420-424). The presence of skin lesions does not correlate with age, disease duration or other MEN1 features (423). As a diagnostic criterion, the combination of multiple angiofibromas (more than 3) and any collagenomas had the highest sensitivity for identifying MEN1 (423, 425). Management is conservative; however, these may need to be removed for cosmetic reasons. Risk of recurrence does not appear to be increased with surgical removal (325).

Other associations

Central nervous system tumors including ependymoma, schwannomas, and meningiomas are a part of the MEN1 syndrome (325). LOH at the MEN1 locus was demonstrated in the resected meningioma from a MEN1 patient (426). These typically are asymptomatic and show no growth (426). Thyroid tumors comprising adenomas, colloid goiters, and carcinomas occur in 25% MEN1 patients (325). However, it is assumed that this may represent an incidental finding resulting from the high background prevalence of such thyroid disorders combined with the increased imaging studies performed in MEN1 patients (427). Somatic MEN1 mutations have been reported in 4% of patients with Hurthle cell thyroid carcinoma, a rare thyroid malignancy which often presents at an advanced stage (428). Leiomyomas of the esophagus, uterus and ureter but not lung have been demonstrated to be part of MEN1 syndrome, arising as independent clones (429, 430). Menin loss has been implicated in breast cancer via stimulation of estrogen receptor (431). While an association may exist, current level of evidence does not support heightened surveillance (432, 433). Similarly, somatic MEN1 mutations were identified in 4% of BRAF mutant colorectal neoplasia samples supporting a role for menin as a tumor suppressor in colorectal tissue (434). Nevertheless, existing data does not suggest an increased predisposition to colon cancer in MEN1 patients. A higher than expected frequency (0.5%) of pathogenic or likely pathogenic germline variant in MEN1 was observed in patients with osteosarcoma of European ancestry (435). While osteosarcomas have not been reported in MEN1 patients, these findings suggest a role of menin as a tumor suppressor in bone.

Genetic and molecular characteristics

Sequencing and deletion/duplication analysis can identify a heterozygous MEN1 germline mutation in 70% to 90% of families with MEN1 (325). The mutations in MEN1 cover the entire coding region with no hot spots or phenotype–genotype correlations (436, 437). Large deletions (2.5%) or nonsense (14%), frameshift (42%), and splice-site (10.5%) mutations predict premature truncation of menin while missense mutations (25.5%) and in-frame insertion or deletion of 1 or more amino acids (5.5%) do not predict obvious menin inactivation (75). Genetic testing for MEN1 should be offered to (1) index cases with 2 or more MEN1-associated endocrine tumors, (2) asymptomatic first-degree relatives of a MEN1-mutation carrier, (3) symptomatic first-degree relatives of a MEN1-mutation carrier presenting with at least 1 MEN1-associated tumor, and (4) patients with multigland parathyroid disease, gastrinoma, or multiple pancreatic islet tumors at any age (59, 75). Germline MEN1 mutations are not found in 10% to 30% patients with MEN1 and this group of germline MEN1-mutation–negative MEN1 seems to have a distinct clinical course compared with patients with germline MEN1-mutation–positive MEN1 (438, 439).

The various mice models of MEN1 are summarized below:

• Germline homozygous knockout of Men1 (Men1^−/−) is embryonically lethal at E11.5-E14.5 (440, 441).

• Germline heterozygous knockout of Men1 (Men1^−/+) develop tumors from the 3 primary tissues affected in MEN1 at age >12-15 months: anterior pituitary (mainly prolactinomas), pancreatic islets (mainly insulinomas), and parathyroid glands (mainly hyperplasia) (440, 442, 443). A second hit to the wild type allele of Men1 resulting in LOH and subsequent biallelic inactivation is essential for tumor formation in Men1^+/− mice (444). A pretumor stage of hyperplasia and dysplasia is seen prior to LOH at the Men1 locus in Men1^+/− mice. Furthermore, knockout of the cell cycle genes (Rb1, Tp53, Cdk2, Cdk4, Cdkn2c, and Cdkn1b) in the Men1^−/+ mice suggests that Cdkn2c inactivation and Cdk4 activation may be critical for islet tumor formation after menin loss (445-449).

• Conditional mouse models of MEN1:

  • Mice with conditional loss of menin in the liver (Men1^f/f; ALB-Cre) do not develop tumors in the liver (450).

  • Mice with conditional loss of menin in the whole pancreas (Men1^f/f; PDX1-Cre) develop tumors of β-cells but not from the exocrine pancreas (451). Of note, mice with conditional loss of menin in the β-cells of the pancreatic islets (Men1^f/f; GLU-Cre) primarily develop insulinomas but not glucagonomas (452). It has been speculated that this may be due to (1) transdifferentiation of α-cells into β-cells after menin loss (453) or (2) paracrine signals form menin-null α-cells inducing β-cell proliferation. As expected, β-cell-specific Men1 knockout mice (Men1^f/f; RIP-Cre) develop insulinomas (454). However, in addition to insulinomas these mice also develop prolactinomas due to leaky expression of the RIP-Cre transgene in pituitary lactotroph cells (455).

Although the conditional Men1 knockout mice do not depend on a spontaneous second hit for LOH of Men1, tumor development is nevertheless delayed until about 8-10 months after embryonic menin loss, the reason for which remains unclear (456). The reason for the tissue-restricted pattern of MEN1 syndromic tumors despite the ubiquitous expression of MEN1 remains uncertain. In the pancreatic islets, it is thought to be related to the regulation of tissue-specific target genes by menin such as Hlxb9 and other β-cell differentiation factors (457-460).

Multiple endocrine neoplasia type 2

MEN2 is an autosomal dominant disorder with an estimated prevalence of 1:30 000. It is caused by a germline gain-of-function in the RET proto-oncogene encoding the RET tyrosine kinase receptor, a cell surface molecule involved in signal transduction for cell growth and differentiation. RET is expressed in cells derived from the neural crest, branchial arches, and urogenital system (461). Activation of RET results in activation of several downstream pathways, including the RAS/MAPK and PIK3/AKT pathways. The primary clinical manifestations of the disease are multiple endocrine tumors that typically include medullary thyroid carcinoma (MTC), pheochromocytoma (often bilateral), and PHPT (typically mild, single gland adenoma with hyperplasia). A genotype–phenotype correlation has been recognized in that RET codon mutations predispose to characteristic phenotypic expressions of MEN2 (see below).

MEN2 (previously called MEN2A)

While MTC and pheochromocytoma commonly precede the diagnosis of PHPT in MEN2 (Fig. 9B), rare reports of PHPT as the initial manifestation have been described (462). All parathyroid glands should be examined during surgery and current preference is for resection of only visibly enlarged glands with intraoperative PTH monitoring to document surgical cure (461, 463). Given the low penetrance of PHPT (30%), prophylactic parathyroidectomy at the time of thyroidectomy is not advised (461).

Four disease variants have been identified:

1. Classic MEN2 with manifestations of MTC (90%), pheochromocytoma (10-50%), and PHPT (30%): RET codon 634 mutation is associated with a moderate penetrance of PHPT and mutations in codons 609, 611, 618, and 620 are associated with a penetrance between 2% and 12% (464).

2. MEN2 with cutaneous lichen amyloidosis: These dermatological lesions are most frequent in the scapular region.

3. MEN2 with Hirschsprung disease: Paradoxically, RET mutations associated with Hirschsprung disease are loss-of-function mutations in contrast to the gain-of-function RET mutation causing MEN2. This dual occurrence is explained by constitutive activation of RET being sufficient to trigger neoplastic transformation of C-cells and adrenal chromaffin cells but not in the precursor neurons due to a lack of expression of the RET protein at the cell surface (465).

4. Familial medullary thyroid cancer: These patients develop neither pheochromocytoma nor PHPT.

MEN3 (previously called MEN2B)

This constitutes about 6% of previously classified MEN2 patients (466). Unlike MEN2, PHPT is not seen in MEN3. MTC is the most frequent component of the syndrome and is typically more aggressive and has an earlier onset than MEN2 (461). In addition, these patients can have the specific clinical feature of mucosal neuromas of the lips, tongue and conjunctiva, musculoskeletal abnormalities (467), narrow long face, thickened lips and ganglioneuromas of the gastrointestinal tract resulting in abnormal gastrointestinal motility leading to diarrhea, constipation, or colonic dilatation (megacolon) at a young age. These nonendocrine manifestations can result in an early diagnosis even before the endocrine manifestations (468). Although, 80% patients with MEN3 develop MTC in the first year of life, the median age of thyroidectomy in MEN3 is 14 years (469). The delay in diagnosis may be explained by the preponderance of de novo mutation in MEN3, which typically involves a methionine-to-threonine substitution at RET codon 918 (M918T). Somatic RET mutations have been identified in sporadic MTC (60%), papillary thyroid cancer (10-20%) and nonsmall cell lung cancer (1-2%). A patient with a mixed MEN1/MEN2 phenotype without any mutations in MEN1 or RET has been described (470).

Multiple Endocrine Neoplasia type 4

MEN4 was described initially in rats (MENX) in 2000 and subsequently in humans. The syndrome is caused by germline mutations in Cdkn1b in rats and CDKN1B in humans, encoding P27KIP1 (p27), a gene regulating cell cycle progression (471-473). Rats exhibiting MENX phenotype show development of a wide spectrum of NETs, including pituitary, adrenals, thyroid, parathyroid and gastroduodenal tract. However, the rat models exhibit parathyroid hyperplasia and not tumor development. The human and mouse CDKN1B share >90% sequence homology in cDNA (474). Homozygous knockout mice model of Cdkn2c (p18) show low-penetrance parathyroid neoplasia, which is enhanced in double mutant mice lacking either Cdkn1a (p21) or Cdkn1b (p27) or Men1 in addition to Cdkn2c (p18), indicating redundant or overlapping function (449, 472). CDKN1B is not a classic tumor suppressor gene, and the second allele is rarely mutated or lost by LOH in human cancers (475, 476). The reduced expression of P27KIP1 protein without a somatic mutation in the second CDKN1B allele suggests that haploinsufficiency of CDKN1B is adequate to confer tumorigenicity.

MENX was renamed as MEN4 in 2008 (477). The estimated prevalence of MEN4 among patients with MEN1-like manifestations is 1.5% to 3.7% (478-480). The earliest reported onset of manifestations is at age 15 years (481). While there exists a significant overlap in the clinical features of MEN1 and MEN4 (Fig. 9C), some differences exist: (1) PHPT seems to occur at a later age in MEN4 than in MEN1 (mean age 56 years vs 25 years respectively), (2) PHPT in MEN4 is milder with low likelihood of recurrence, (3) pituitary tumors in MEN4 are not as aggressive as seen in MEN1, and (4) the penetrance of GEP-NET is lower in comparison to MEN1 (482). However, these differences should be considered with caution given the small number of reported cases of MEN4. Recent works suggests that patients with indels (CDKN1B) have higher risk for PHPT vs point mutations and variants in codons 94-96 were associated with higher risk of PHPT and pituitary adenoma (483).

Novel MEN syndrome—MEN5

Rare reports of families with inactivating mutations in MYC-associated factor X (MAX), with paragangliomas, pheochromocytomas (bilateral and/or metastatic), and pituitary adenoma, have been described (484). Additional endocrine tumors reported in patients with germline MAX variant include ganglioneuroma, ganglioneuroblastoma, adrenomedullary hyperplasia, PNETs, and PAs (Fig. 9D). MAX is a transcription factor regulating cell proliferation, differentiation, angiogenesis, and apoptosis. It is a classic tumor suppressor gene, with somatic loss of the second allele. Comprehensive phenotyping of this novel syndrome is evolving.

Hyperparathyroidism Jaw Tumor Syndrome

History

HPT-JT (OMIM #145001) was first characterized in a kindred with familial hyperparathyroidism originally described 3 decades earlier by Jackson (485). Several decades after initial ascertainment, 2 affected members of the third generation of the kindred were shown to have ossifying jaw fibromas similar to 4 of the 5 affected members of the first generation leading to identification of HPT-JT as a clinically and genetically distinct syndrome (486). Linkage analysis mapped a single locus associated with the syndrome to chromosomal region 1q25-q32 (487-489). Using a positional candidate approach through multi-site collaboration, heterozygous germline inactivating mutations in HRPT2 were identified in 13 of 14 families in 2002 (490). After the cloning of the gene, it was found that the homologs were present in several species including Drosophila (54% identical and 67% similar) and yeast (25% identical and 45% similar) (490). The homolog in the yeast called cdc73 (cell division cycle protein 73) had already been well-studied and hence, HRPT2 was renamed as CDC73. The disease has an autosomal dominant pattern of inheritance, but mutations may occur de novo (491). Concomitant demonstration of a somatic mutation in tumor tissue from a patient with germline CDC73 mutation is consistent with biallelic inactivation and a putative tumor suppressor function in accordance with Knudson's “two hit” model of hereditary cancer (492).

In its fullest clinical expression, HPT-JT may manifest with PHPT, jaw tumors involving the maxilla or mandible, kidney cysts or tumors, and uterine tumors (Fig. 9E). HPT-JT is characterized by variable and incomplete penetrance, such that some 20% of obligate or proven CDC73 mutation carriers may exhibit no obvious expressions.

Clinical manifestations

Primary hyperparathyroidism

The most common, earliest, and sometimes sole feature of HPT-JT is PHPT. The earliest reported onset of hyperparathyroidism in a patient with HPT-JT is 7 years (493). Median age of diagnosis of hyperparathyroidism is approximately 28.5 years (range 16-58) (494-497). Penetrance of PHPT in 43 Dutch nonindex CDC73 mutation carriers was shown to increase with age (65% and 83% at ages 50 and 70, respectively) (497). The oldest reported asymptomatic gene carrier is at age 72 years (494) and oldest reported onset of PHPT is at age 60. Approximately 20% of patients with the clinical diagnosis of HPT-JT do not have a detectable pathogenic variant in CDC73 (498). These patients may have CDC73 mutations in the promoter, untranslated regions, or introns or they may have whole exon or gene deletions that can be missed on sequencing analysis (499). Single gland parathyroid involvement is more common than multi-gland disease (86.1 vs 13.9%) in HPT-JT relative to other forms of familial hyperparathyroidism such as MEN1 (500). Thus, a targeted unilateral exploration with selective parathyroidectomy is typically performed in patients with preoperative single-gland localization (501). Bilateral neck exploration is reserved for patients with negative or discordant preoperative localization. Overall, recurrence rate for hyperparathyroidism in patients with HPT-JT is approximately 25% after a median disease-free interval of 8.5 years (500). Recurrence is more frequently seen in patients with discordant preoperative localization (60% vs 9%; P = .06) and subsequent long-term cure is typically achieved after reoperation (500).

On pathology, parafibromin-negative tumors can demonstrate distinctive morphology including extensive sheet-like rather than acinar growth, eosinophilic cytoplasm, nuclear enlargement with distinctive coarse chromatin, perinuclear cytoplasmic clearing, prominent arborizing vasculature and frequently, a thick capsule (502). Loss of immunohistochemical expression of parafibromin can be a marker of biallelic CDC73 inactivation in parathyroid tumors. Parafibromin localizes in the nucleolar compartment (503). Evaluation of not only nuclear but also nucleolar staining for parafibromin increases the sensitivity for detection of malignancy (504). Clinically, loss of parafibromin staining can be particularly useful in predicting the behavior of atypical PAs (220, 222). However, the technique is not widely available, and process can be technically demanding. In addition, the existence of different scoring systems and lack of a standard antiparafibromin antibody or antiserum for immunostaining limit a uniform interpretation.

Patients with HPT-JT have a higher prevalence of atypical adenomas and carcinomas of the parathyroid (20%) (494, 496). Rarely, patients may develop a nonfunctioning parathyroid malignancy although CDC73-mutation status is not reported in most published cases with nonfunctional parathyroid malignancy (253, 255, 505). Patients with high-impact germline CDC73 mutations that predict significant conformational disruption or loss of expression of parafibromin (such as truncation and frameshift mutations and gene deletions) have a higher (6.6-fold) risk of PC in comparison to patients with low-impact mutations despite a similar risk of developing PHPT between the 2 patient groups (506). This may be due to disruption of C-terminal domain of parafibromin by the high-impact mutations. The C-terminal domain is believed to constitute a protein interaction surface that couples PAF1 complex to RNA polymerase II elongation machinery (270).

Jaw tumors

Jaw tumors are rare and seen in approximately 10% to 30% of patients in HPT-JT (495, 496). Pathologically, these tumors are consistent with “ossifying fibromas” (OF) under the class fibro-osseous lesions (507). In HPT-JT, OFs may precede the onset of PHPT (494). Multiple jaw lesions are rare in sporadic OFs but may be seen in about 30% patients with HPT-JT (508). The precise cell of origin from which OF arise remains unclear. It is speculated that OFs are derived from the mesenchymal blast cells of periodontal membrane with the tumor consisting of highly cellular fibrous tissue containing varying amounts of calcified tissue resembling bone, cementum or both (509). OFs are distinct from the “brown tumors” described in osteitis fibrosa cystica or severe PHPT as the former (1) can appear or enlarge even after parathyroidectomy (510), (2) are limited to the maxilla (491) or mandible while brown tumors can appear anywhere in the skeleton, (3) lack the abundant multi-nucleated giant cells seen in “brown tumors” (510), and (4) typically have a sclerotic rim on imaging unlike brown tumors that are purely lytic and lack the sclerotic rim (511). Jaw tumors are the presenting manifestation in about 30% patients with HPT-JT (512). These tumors can be managed conservatively unless excision is warranted due to displacement of teeth or of the inferior alveolar canal. Complete excision of these tumors (vs enucleation or curettage) is recommended considering the possibility of recurrence (511). Somatic CDC73 mutations are rare in sporadic OFs of the jaw (513-515).

Uterine tumors

Several studies have shown a high frequency of uterine tumors in women with HPT-JT (495, 496, 498). The reported uterine pathology includes benign and malignant tumors like adenosarcoma, adenofibroma, leiomyomas, adenomyosis, and endometrial hyperplasia (498). These uterine tumors have a common embryological origin from the mesodermal Mullerian duct system. Affected women typically present with menorrhagia in their second to fourth decade and may need hysterectomy leading to decreased fecundity (494, 498). We reported the successful use of aromatase inhibitor in a young nulligravid woman with HPT-JT and multiple atypical endometrial polyp-like lesions filling the entire uterine cavity subsequently leading to a successful pregnancy (516). Ovarian granulosa cell tumor in a young woman with genotype-positive HPT-JT has been described although LOH in tumor tissue was not demonstrated (517).

Kidney tumors

Kidney tumors including cysts, hamartomas, Wilms’ tumors, mixed epithelial and stromal tumors, and adenocarcinomas have been reported in 25% to 30% of patients with HPT-JT (488, 494, 496, 518). The earliest reported case of kidney tumor is at age 8 (519). While allelic imbalances in CDC73 have been reported in sporadic renal tumors, somatic mutations in the gene are rare (520).

Other manifestations

Thoracic aortic aneurysm was described a young male (age 32) with genotype-positive HPT-JT and his father (CDC73 testing not available in father, mother negative) raising the possibility of it being a feature of this disorder (521). Other manifestations potentially related to HPT-JT are thyroid, renal and colon carcinoma (495). Parafibromin has been shown to be downregulated in other tumors like chromophobe renal cell carcinoma (522), gastric carcinomas (523), and breast cancer (524), although no mutations in CDC73 have been identified in these tumors. The reason for the tissue-restrictive manifestations of CDC73 despite its ubiquitous expression remains unclear.

Genetic and molecular characteristics

Cdc73 null mice are embryonic lethal by 6.5 days postcoitum, the timing of implantation indicating that parafibromin has a key role in embryonic development (525). Increased proliferation rate was noted in the parathyroid glands of the conventional (Cdc73^+/−) mouse models starting from 9 months of age (262). Although the heterozygotes do not develop jaw or renal tumors, female heterozygotes develop uterine neoplasms at approximately 18 months of age (262).

We believe that all patients with seemingly sporadic parathyroid cancer should undergo germline testing for CDC73 because of the potential implications for family members (526). In the absence of published guidelines from a consensus of experts, annual laboratory testing for hypercalcemia with imaging of the jaw and abdomen–pelvis every 4 to 5 years seems to be a reasonable surveillance plan for patients who are germline CDC73 mutation positive.

Nonsyndromic Forms

This subgroup of heritable forms of PHPT is not associated with extraparathyroid signs and symptoms.

FHH and neonatal severe hyperparathyroidism

FHH was first described in 1972 (527). It is a genetically heterogenous group of autosomal dominant disorders (FHH1, FHH2, and FHH3) with a reported prevalence of 1.3 per 100 000 which is likely an underestimation due to the occult nature of the disease (528). It makes up about 2% of patients with PHPT (529). All 3 forms of FHH are caused by mutations in genes involved in calcium signaling. Patients with FHH manifest mild to moderate hypercalcemia, mild hypermagnesemia and “inappropriately” normal or elevated PTH. The underlying pathophysiology is nonneoplastic and results from an increased calcium-PTH secretory “set point” (extracellular calcium concentration at which PTH secretion is half maximally suppressed) due to impaired calcium sensing by, or signal transduction downstream of, the CASR.

Clinically, FHH is characterized by the biochemical phenotype of PHPT and hypocalciuria with a calcium to creatinine clearance ratio <0.01, seen in 80% to 95% of patients with FHH. However, a calcium to creatinine clearance ratio <0.01 can be seen in up to 10% of patients with sporadic PHPT, underscoring the important role of genetic testing (44, 530). In addition, some patients diagnosed as FHH based on the calcium to creatinine clearance ratio do not have an identifiable mutation in any of the genes known to cause the disease. Establishing a correct diagnosis of FHH has implications for management of PHPT in addition to the implications for family. Another tool called “pro-FHH” which factors plasma calcium, PTH, serum osteocalcin and calcium to creatinine clearance ratio has been recently proposed to improve the reliability of diagnosis in comparison to the use of calcium to creatinine clearance ratio alone (531).

The disease typically has a benign course although rare patients may manifest chondrocalcinosis or recurrent pancreatitis. In general, patients with FHH should not undergo parathyroidectomy as it would not correct the hypercalcemia (unless the parathyroidectomy were total). However, rare reports of concurrent PA in FHH patients with improvement in hypercalcemia after parathyroidectomy have been described (532). The use of a calcimimetic may be considered in case of symptoms from hypercalcemia or target organ damage (533). Imaging in FHH can be inaccurate resulting in an inadvertent surgery (534). On pathology, parathyroid glands in FHH demonstrate hyperplasia with multigland involvement. Cardiometabolic phenotyping of patients with FHH has not revealed any alterations of clinical significance in this cohort as might be expected based on the ubiquitous expression and role of CASR (535, 536).

Familial hypocalciuric hypercalcemia type 1

This is the most common and first genetically defined form of FHH (∼65%) caused by germline heterozygous inactivating mutations of CASR described in “PTH, calcium, and vitamin D signaling” (537). Majority (>85%) of these mutations are missense while remaining cases can be attributed to nonsense, deletion, insertion and splice site mutations leading to truncated CASR protein. Almost half of the inactivating mutations causing FHH1 impair the biosynthesis and post-translational processing of CASR (increased proteasomal degradation, decreased anterograde trafficking), while some other mutations located in extracellular and transmembrane domain induce biased signaling (preferential signaling through pERK1/pERK2 or equal signaling via intracellular Ca²⁺ and pERK1/pERK2 instead of preferential coupling with intracellular Ca²⁺ signaling seen in wild-type CASR) (44).

Familial hypocalciuric hypercalcemia type 2

This rare form of FHH is caused by germline heterozygous inactivating mutations of GNA11 encoding the Gα₁₁ protein. The identified mutations in GNA11 impair CASR-mediated signaling and have revealed the key residues critical for Gα₁₁ function.

Familial hypocalciuric hypercalcemia type 3

This is the most severe form of FHH with significant hypercalcemia, hypermagnesemia, and hypocalciuria compared with other forms. It is caused by germline heterozygous inactivating mutations in AP2S1, which encodes AP2σ, a subunit of a multimeric complex involved in clathrin-related endocytosis of GPCRs. Nearly all mutations in AP2σ causing FHH3 identified to date affect the Arg15 residue of the protein. These mutations disrupt an interaction between AP2σ and the CASR intracellular domain, reducing the endocytosis of CASR (538, 539).

Neonatal severe hyperparathyroidism

Children of parents with FHH1 can have homozygous or compound heterozygous CASR mutations manifesting as NSHPT (540). It is a life-threatening disorder with severe hypercalcemia, respiratory distress, rib cage deformities, hypotonia and bone demineralization which can be seen as early as in the first week of life. An urgent parathyroidectomy may be needed (541, 542). Children may respond to cinacalcet initially, but parathyroidectomy is typically needed eventually (543). Occasionally, NSHPT may be caused by sporadic heterozygous CASR mutations that have a dominant negative effect on CASR function despite the presence of a wild-type allele (533).

GCM2-mediated PHPT

In vivo studies in Gcm2 knockout mice suggest that Gcm2 plays an important role in parathyroid cell proliferation and maintenance (544). In humans, germline homozygous deletion in GCM2 has also been shown to cause familial isolated hypoparathyroidism (154, 545, 546). Germline activating mutations of the small C-terminal conserved inhibitory domain that increased the transcriptional activity of GCM2 in in vitro studies have been reported in 18% of patients with FIHP (144). These activating variants appear enriched among PHPT patients of Ashkenazi Jewish descent (547). GCM2 Y282D polymorphism has been observed to be more prevalent in Italian PHPT patients relative to controls with variant GCM2 showing an increased transcriptional activity compared with wild type (548). However, the penetrance of activating variants in the C-terminal conserved inhibitory domain is low in patients with sporadic or heritable PHPT, suggesting that majority of individuals with such variants will not develop a sporadic PA (549-551). The low penetrance raises questions about the utility of including GCM2 in screening for heritable causes of PHPT. GCM2-mediated FIHP has been shown to be associated with a higher preoperative PTH levels, frequency of multigland disease, frequency of persistent hyperparathyroidism and risk of PC (154, 552, 553).

Familial isolated hyperparathyroidism

FIHP is characterized by familial hyperparathyroidism without any apparent extraparathyroidal manifestation. This is a genetically heterogenous group consisting of patients with (1) incomplete expression of MEN1, CDC73 (554-556), or CASR, (2) germline activating GCM2-mutations, and (3) unknown heritable cause of PHPT (557). Patients with FIHP should undergo screening for established heritable causes of PHPT (171, 259). Our preference is to classify the patients who fall in the first 2 categories in their respective group of patients with the same molecular signature. Furthermore, the majority of FIHP patients fall in the third group with an unknown molecular etiology and small kindreds (558). The search for other genetic causes to explain FIHP in majority of kindreds has so far been unrevealing (559) (Fig. 10). Thus, more work to elucidate the genetic cause of FIHP in majority of patients with the disease remains.

Figure 10.

Genes implicated in parathyroid tumorigenesis. The thick outlined boxes represent the genes known to cause parathyroid tumors when mutated in the germline and somatic. PA, parathyroid adenoma; PC, parathyroid carcinoma. Created with Biorender.com.

Perspective: Important Questions for the Future

The broad spectrum of clinical manifestations associated with various disorders of parathyroid function as described above reflects the important role calcium plays in diverse cellular processes as well as the significance of PTH in the regulation of calcium and bone homeostasis. The diverse clinical findings associated with parathyroid disorders also reflect the multiple roles and expressions of a subset of genes vital for parathyroid gland and bone development, function, and growth. Clinical and genetic investigation of the molecular pathogenesis of rare heritable syndromes of PHPT, even though such forms of PHPT represent only a small fraction of total cases, has been critical for our discovery of key genes that regulate parathyroid growth and function and whose mutation can lead to parathyroid neoplasia, whether in a sporadic or familial context. Such investigation first illuminated the importance of genes such as MEN1, CDKN1B, CDC73, CASR, AP2S1, GNA11, and GCM2 in parathyroid neoplasia.

Genetics of Heritable Hyperparathyroidism of Unknown Etiology

Given such historical success, an outstanding challenge for the future is to advance high-yield clinical investigation that delineates additional genes, variants in which predispose to parathyroid tumor development. It seems quite likely that other genes remain to be discovered whose gain- or loss-of-function can also initiate or promote the development of parathyroid tumors. This hypothesis is perhaps best illustrated by the observation that parathyroid disease in the majority of FIHP kindreds results from the germline mutation of genes not presently recognized to play a role in parathyroid tumor formation (see Section “Nonsyndromic Forms” subsection “Familial isolated hyperparathyroidism” above). From FIHP kindreds which are MEN1, CASR, and CDC73/HRPT2 mutation negative, it has been estimated that only 10% to 20% carry germline mutations in the proto-oncogene GCM2 (144, 559), leaving some 80% to 90% of prescreened FIHP kindreds with yet to be discovered genetic risk factors for their PHPT. Somatic mutation in PTH gene was detected in a PA; however, deeper molecular understanding of the parathyroid gland remains to be established. Lack of parathyroid cell lines severely limits our ability to understand the molecular mechanism of parathyroid in state of health and disease as parathyroid cells in primary culture are hard to maintain and the expression of CASR can be severely reduced in primary parathyroid culture after 24 hours.

Localization of Small Parathyroid Tumors

Metabolically significant parathyroid tumors can be very small, so more sensitive, and specific parathyroid tumor location methodologies remain a major future challenge. This is particularly true in reoperative cases of PHPT, since the surgical risk is much greater, and the benefit of focused, targeted surgery is even more evident. Beyond ^99mTc-sestamibi, 4-dimensional CT and ¹⁸F-fluorocholine positron emission tomography/CT have shown promise, but even deeper investment in discovering and optimizing superior parathyroid specific imaging modalities is warranted. Localization and management of patients with normocalcemic PHPT is particularly challenging as these tumors can be smaller than typical adenomas. It is unknown if the molecular spectrum of tumors causing normocalcemic PHPT or ectopic parathyroid tumors differs from sporadic adenomas.

Near-infrared autofluorescence used label-free or in combination with indocyanine green is an emerging tool for real-time intraoperative detection of parathyroid glands or to confirm parathyroid origin of tissue in combination with (or in lieu of) frozen section (560). Potential usefulness of near-infrared autofluorescence in reducing post-thyroidectomy hypoparathyroidism or in patients with unrevealing preoperative localization warrants further investigations.

Management of Primary Hyperparathyroidism in Pregnancy

Management of PHPT in pregnancy remains an area with sparse data and little evidence. Current practice is to manage pregnant women conservatively with consideration of parathyroidectomy in second trimester for severe or symptomatic hyperparathyroidism. Preoperative imaging in pregnant women should be limited to ultrasonogram. Cinacalcet, denosumab, or bisphosphonates are contraindicated in pregnancy, limiting available medical therapy primarily to intravenous fluids. Furthermore, symptoms of hypercalcemia can be difficult to distinguish from clinical features of pregnancy. Retrospective data indicates that patients treated with parathyroidectomy have lower rates of preeclampsia and preterm delivery compared to pregnant women treated medically (561). Maternal hypercalcemia can suppress fetal parathyroid glands and result in neonatal hypocalcemia.

Clinical Relevance of FGF23 in Primary Hyperparathyroidism

Studies in mouse models and humans indicate an elevated FGF23 level in PHPT (562). Elevated FGF23 in these patients is associated with greater weight of the PA and greater PTH levels as well as metabolic markers associated with long-term cardiovascular risk (563). It has been suggested that this is an adaptive response to counteract the PTH-induced increase in 1,25(OH)₂D levels (564). Whether the elevated FGF23 has a role either alone or in conjunction with PTH in some of the little-known associations of PHPT like cardiometabolic complications remains unknown.

Management of Parathyroid Cancer

Another outstanding future challenge is to develop effective therapies for inoperable PC. PC is generally considered insensitive to irradiation, although little research has explored potential radiosensitizing agents. In general, conventional cytotoxic chemotherapy has been ineffective in PC with survival benefit shown. There is a need for large scale prospective clinical trials that employ tyrosine kinase inhibitors for the treatment of PC, since therapy with multitarget tyrosine kinase inhibitors that potentially inhibit both angiogenesis and tumor cell proliferation have shown some efficacy in limited studies. The potential use of biologic drugs, such as vaccines or antibodies, for the treatment of inoperable PC is a relatively unexplored area, worthy of deeper investigation. There are case reports, for example, of prolonged clinical response in a patient with inoperable PC immunized with fragments of PTH (256, 565). Thus, future research to enhance our understanding of parathyroid gland and PTH is warranted.

Management of PHPT in heritable forms of PHPT

There is little evidence to guide the management of recurrent or index PHPT presentation in heritable forms like MEN1 or HPT-JT wherein patients may manifest with PHPT as early as age 5 (335). Our current practice is to extrapolate the target organ–based indications (kidney stones, hypercalciuria, or osteoporosis) recommended for asymptomatic patients with sporadic PHPT (566). Recent studies challenge the notion of bilateral exploration in patients with MEN1 or HPT-JT (567).

Polygenic risk score to predict risk of PHPT

Recent work investigating genetic associations with PHPT using both GWAS and candidate gene approaches in a Scottish population suggests involvement of genetic variants in the vicinity of SOX9, SLITRK5, LPAR3, and BCDIN3D-AS1(568). Findings also suggest that carriers of greater number of PHPT-risk alleles are associated with statistically significant increased risk of PHPT in men and women. While this is a novel finding in the field of population genetics for PHPT, the value of polygenic risk score in patient management remains uncertain. Furthermore, these findings have limited generalizability across non-European population as is typical with GWAS studies. Nevertheless, population genetics can enhance our understanding of parathyroid biology.

Indication(s) for genetic testing for heritable forms of PHPT

Despite a prevalence of 10% to 15% for heritable forms of PHPT, germline screening for heritable forms is infrequently performed or emphasized in guidelines (566). We suggest that the following should be an indication for genetic testing (single gene or panel-based testing):

• Positive family history of PHPT or its manifestations (occult disease) is the strongest predictor of a positive genetic test (569).

• Synchronous or asynchronous multigland disease.

• Recurrent or persistent (despite resection of histologically abnormal gland).

• Age ≤ 45 years has ∼10% yield of heritable forms (570-572). The threshold for genetic testing is controversial and varying thresholds ranging from 30 to 45 years have been used in different guidelines (573, 574).

• Parathyroid cancer: germline variants in CDC73 confer a 20% increased risk of parathyroid cancer or atypical tumors.

• Suspected FHH: patients should be counselled to avoid parathyroidectomy.

• Diagnosis of gastrinoma: American College of Medical Genetics recommends that all patients with gastrinomas or multifocal PNETs should be offered genetic testing for MEN1.

• Presence of 2 classical MEN1-associated tumors: patients should be tested for variants in MEN1.

• Presence of at least 1 classical MEN1-associated endocrine tumor in the context of positive family history of MEN1(or its manifestations in case of occult disease): presence of MEN1 variants should be assessed.

Genetic testing (germline) is useful in:

• cascade screening for family members

• patient management as in FHH (avoid parathyroidectomy), MEN1 (subtotal parathyroidectomy with bilateral transcervical thymectomy) or MEN2 (prophylactic thyroidectomy)

• instituting surveillance for associated manifestations as in multiple endocrine neoplasias

• access to preimplantation and prenatal genetic testing if desired by prospective parents at risk of transmitting the disease

• relieving family members at risk of inheriting from the burden of chronic surveillance and anxiety in case of a negative test.

Thus, while we have come a long way in management of patients with PHPT, areas of unmet needs persist. Future research to gather more evidence to optimize patient management is warranted.

Abbreviations

APT

atypical parathyroid tumor

CASR

calcium sensing receptor

CCND1

cyclin D1

CDK

cyclin-dependent kinase

CDKI

cyclin-dependent kinase inhibitor

CKD

chronic kidney disease

EZH

enhancer of zeste homolog

FHH

familial hypocalciuric hypercalcemia

GCM2

glial cell missing transcription factor 2

CT

computed tomography

FGF23

fibroblast growth factor 23

GEP-NET

gastroenteropancreatic neuroendocrine tumor

GPCR

G-protein–coupled receptor

HPT-JT

hyperparathyroidism jaw-tumor

HVDRR

hereditary vitamin D–resistant rickets

LOH

loss of heterozygosity

MEN

multiple endocrine neoplasia

MLL

mixed lineage leukemia

MTC

medullary thyroid carcinoma

NET

neuroendocrine tumor

NF-PNET

nonfunctioning pancreatic neuroendocrine tumor

OF

ossifying fibroma

PA

parathyroid adenoma

PAF1

Polymerase Associated Factor 1

PC

parathyroid carcinoma

PHPT

primary hyperparathyroidism

PNET

pancreatic neuroendocrine tumor

PTH

parathyroid hormone

PTHR

parathyroid hormone receptor

PTHrP

parathyroid hormone–related peptide

RET

REarranged during Transfection

VDDR

vitamin D–dependent rickets

VDR

vitamin D receptor

ZES

Zollinger–Ellison syndrome

Funding

The Intramural Research Program of the National Institute of Diabetes and Digestive and Kidney Diseases (ZIA DK043012-21 and ZIA DK043006-46) supported this research.

Disclosure

The authors have no conflicts of interest to disclose.