Basic-Clinical Analysis of Parathyroid Cancer

1. Clinical Approach

1. Primary Hyperparathyroidism

Primary hyperparathyroidism (PHPT) is a common endocrine disorder characterized by excessive and autonomous secretion of parathyroid hormone (PTH) by one or more parathyroid (PT) glands, resulting in asymptomatic or symptomatic hypercalcemia [1].

Regarding the etiology of PHPT, a spectrum of proliferative disorders in the PT gland is described: solitary parathyroid adenoma (80%), multiglandular parathyroid disease (previously known as parathyroid hyperplasia, 10-15%), multiple parathyroid adenomas (5%), and parathyroid carcinoma (~1%) [2,3].

Epidemiologically speaking, PHPT is traditionally a disease diagnosed in postmenopausal women, with a predominance in African American and Caucasian populations. Its prevalence and incidence depends mainly on the availability of PTH and serum calcium measurement, associated with screening protocols, which has favored the transition to early and asymptomatic diagnosis [4]. American studies estimate an age-adjusted prevalence of 233 and 85 per 100,000 inhabitants for female and male populations, respectively; showing a 2.5:1 female predominance and a tripling of prevalence over a 15-year period (1995-2020). In addition, with advancing age (>45 years), a pronounced increase in sex-specific incidence differences is described, with a significant female predominance [5].

Clinically, most patients are asymptomatic or present with nonspecific symptoms such as fatigue, depression, or mild cognitive impairment [6]. Persistence of the disease favors symptomatology secondary to hypercalcemia, characterized by renal involvement (nephrolithiasis and renal insufficiency), gastrointestinal disease (peptic ulcer and pancreatitis), musculoskeletal disorders (fractures on pathological bone, fibrous osteitis cystica, osteoporosis, chondrocalcinosis, and arthritis), neuropsychiatric symptoms (lethargy, confusion, psychosis, and coma), among other symptoms such as polydipsia, polyuria, abdominal pain, nausea, and vomiting [6,7]. Simultaneous hypercalcemia and elevated PTH levels (or inappropriately normal levels) represents the most important factors to diagnosticate PHPT [2]. The diagnosis of PHPT requires biochemical analysis revealing.

Surgical treatment (parathyroidectomy) is the only definitive treatment for PHPT, and it is indicated in symptomatic patients or asymptomatic patient who meets the criteria determined by the Fifth International Workshop (<50 years, serum calcium >1.0 mg/dl above the upper normal level, bone mineral density showing T Score <2.5 in those over 50 years, fracture on pathological bone, nephrolithiasis, nephrocalcinosis estimated glomerular filtration rate (eGFR) <60 ml/min, or hypercalciuria) [8,9]. Classically, surgical management of PHPT involves bilateral cervical exploration; however, advances such as intraoperative PTH measurement and improved preoperative localization methods have favored the implementation of minimally invasive techniques, remote approaches (retroauricular, axillary, submammary, transoral) and robotic-assisted surgery. Thus, parathyroidectomy is an active area of research, where various modern techniques claim better aesthetic outcomes, surgical times, or postoperative recovery [7].

2. Parathyroid Cancer

As previously mentioned, parathyroid carcinoma (PC) is the least common etiology in the context of PHPT (about 1%). It is an uncommon endocrine neoplasm, currently accounting for 0.005% of malignant neoplasms in western population [10]. The estimated annual incidence of this condition is 3.5-5.7 cases per 10,000,000 individuals, with a peak incidence in the fifth decade and no gender differences. A recent retrospective study of the SEER database analyzed 609 cases of PC, determining a higher incidence in males (52.2%), predominance in Caucasian population (75.4%), average age at diagnosis of 62 years, mainly adenocarcinoma histology (99.7%), with 36.8% of cases being organ-confined at the time of diagnosis. Risk factors for mortality include older age (>40 years), tumor size >4 cm, male gender, metastasis, and poor histological differentiation [11].

The etiology of PC is unknown, but an increased incidence has been associated with cervical irradiation and stage V chronic kidney disease [12]. This cancer typically presents as spontaneous disease; however, it can also be associated with specific syndromes such as hyperparathyroidism-jaw tumor syndrome (HPT-JT) or multiple endocrine neoplasia (MEN) types 1 and 2A. Genetically, a relationship has been established between mutations in the CDC73 gene (which codes for parafibromin) and spontaneous PC (present in up to 70% of cases) and familial forms [10,12]. Parafibromin is a tumor suppressor protein that can be detected by immunohistochemistry, and its decrease is associated with increased cell proliferation, apoptosis, and chromosomal instability [13].

PC presents clinically as a case of hyperparathyroidism (described earlier) which precedes local compression symptoms. Additionally, PC tends to have higher hormonal activity than PT adenomas and hyperplasias, resulting in serum PTH levels up to 10 times the normal value and hypercalcemic crisis in up to 12% of patients (serum calcium >14 mg/dL, oligoanuria, and neuropsychiatric symptoms). Among the symptoms associated with PHPT, renal (nephrolithiasis and renal insufficiency) and skeletal (bone pain, fractures in pathological bone, osteoporosis, etc.) manifestations are most common. On physical examination, a palpable cervical mass can be found in 40-70% of cases, which may be associated with compressive symptoms of the upper airway and digestive tract. It is described that up to 10% of these carcinomas are non-functioning, and in consequence are diagnosed later, usually due to compressive symptoms. Cervical lymph nodes are the main site of metastasis, present in 15-20% of cases; followed by distant metastasis to lung, liver, and bone (1%) [14,15].

Clinical suspicion of PC is important due to the similarity with other proliferative pathologies within the PHPT spectrum, and the rarity of this carcinoma. In this context, it is important to remember the preoperative suspicion criteria: male gender, young age (<50 years), concomitant renal and skeletal manifestations, palpable cervical mass, paralysis of the laryngeal nerves, plasma PTH levels >10 times the normal level, serum calcium >14 mg/dL or hypercalcemic crisis, local invasion of adjacent structures and suspicion of metastasis on imaging studies [10,14].

Despite the previously described criteria, the definitive diagnosis of PC is complex as it requires unequivocal criteria of invasion in postoperative biopsy; including: angioinvasion, lymphoinvasion, perineural invasion, invasion of neighboring soft tissues and musculoskeletal structures, thyroid, esophagus, etc., and/or presence of lymph node or distant metastasis. The aforementioned are absolute criteria, and meeting just one of them is sufficient for diagnosing PC. The cytological or histological characteristics of the tumor are not sufficient to make the diagnosis. This determines the difficulty in discriminating between PT adenoma and carcinoma. Consequently, malignancy-associated criteria have been established. The presence of these criteria indicates a high risk of malignant behavior, acquiring the name of “atypical parathyroid tumor” (however, not sufficient for PC diagnosis). These criteria include trabecular or “sheet-like” architecture, wide bands of intraglandular fibrosis, diffuse cellular atypia, enlarged nucleoli, >5 mitoses per 10 mm³, atypical mitoses, coagulation necrosis, capsular involvement without extension into the periglandular fibroadipose tissue [3,16].

Given the difficulty in confirming the diagnosis of PC, attempts have been made to address this problem through the search for biomarkers using immunohistochemistry (IHC); however, none have proven to be pathognomonic of this disease (see state of the art analysis) [16].

Within this theme, the phenomenon of epithelial-mesenchymal transition (EMT) and cancer stem cells (CSC) markers have been scarcely studied; and could eventually help discriminate between a diagnosis of parathyroid adenoma or carcinoma.

Regarding treatment, surgical management is the only curative option, prior medical management of metabolic abnormalities. This consists of an en bloc resection of the primary tumor, along with the ipsilateral thyroid lobe and isthmus, as well as involved pre-thyroid muscles and central neck lymph node dissection [10,14].

On the other hand, PC is considered radioresistant; however, some series report lower disease progression and possibly lower recurrence when used as postoperative adjuvant treatment [12]. Despite this, the use of adjuvant radiotherapy has not shown benefits in overall survival. Additionally, a better 5-year survival is described in patients treated surgically compared to isolated radiotherapy [11]. Currently, its adjuvant use is reserved for patients at high risk of locoregional recurrence [17,18].

Similarly, the use of chemotherapy in PC has historically shown low to no efficacy. A recent report describes a favorable response with the use of the multikinase inhibitor Sorafenib in a case of PC with lung metastases, although further evaluation is needed [10,19].

Reports by Christakis et al. highlight that there have been no significant changes in the management and prognosis in overall survival in the past 35 years at the University of Texas MD Anderson Cancer Center, underscoring the importance of promoting research and progress in treatments for this cancer [17].

Long-term survival is favorable, with 5- and 10-year survival rates ranging from 85-91% and 49-87.6%, respectively. In general, mortality is secondary to complications associated with hypercalcemia, rather than tumor burden [12].

3. Parathyroid Carcinoma Genomics

Advances in genomics are emerging and have enormous potential as they could be used relatively simply and reliably as diagnostic tools or decision aids.

Firstly, the CDC73 gene (also known as HRPT2) is a tumor suppressor gene that encodes a protein called parafibromin (whose function has been previously mentioned). Multiple somatic and germline mutations associated with partial or total inactivation of this gene have been identified. Somatic mutations in this gene are estimated to be the main genetic alteration in sporadic PC, present in two-thirds of cases (reports vary between 9-70%). Germline mutations are associated with HPT-JT syndrome, a population with a 20% probability of developing PC [54,55].

Secondly, the PRUNE2 gene encodes the homonymous protein, which has tumor suppressor activity by suppressing the activity of RhoA, inhibiting oncogenic transformation. Recently, Yu et al. detected several mutations via whole-exome sequencing in 18% of patients with PC, but did not describe this mutation in PT adenomas. This could represent PRUNE2 as a marker that differentiates between both conditions [55,56].

Furthermore, the CCND1 gene acts as an oncogene in PT adenomas through the PTH-CCND1 rearrangement. This gene encodes cyclin D1, whose amplification has been observed in up to 71-90% of PC and is associated with cell proliferation. Recent studies have shown that amplification of CCND1 can be mutually exclusive with somatic mutations in CDC73, suggesting alternative mechanisms of malignancy [52,55].

On the other hand, multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant endocrine disorder caused by mutations in the MEN1 gene. While this syndrome is commonly associated with PT adenomas or hyperplasia, around 1% of cases may present with PC. Recent studies indicate that mutations in this gene may be present in approximately 13 to 31% of cases of PC, so additional studies could position it as a marker of the disease [55].

Genes associated with the PI3K/AKT/mTOR and Wnt signaling pathways have also been implicated in PC. The first signaling pathway plays a physiological role in cell growth and survival, and its overactivation has been associated with various types of cancers, particularly through increased cell growth, angiogenesis, suppression of senescence and autophagy, as well as increased metastatic potential. Mutations in the PI3K/AKT/mTOR pathway have been described in 13-20% of PC cases, making it a potential target for inhibitors of this pathway [55,57].

Similarly, the Wnt signaling pathway is physiologically associated with cell proliferation, survival, and apoptosis. Aberrant activation of this pathway has also been implicated in multiple neoplasms. It has been described that CDC73 regulates this pathway through stabilization of beta-catenin. In addition, recent studies have identified a regulatory role of this pathway through genetic and epigenetic changes in APC and RNF43, observed in rare cases of PC as well as colorectal and endometrial carcinomas [55].

Finally, somatic mutations have been described in other genes such as TERT, which is associated with telomerase activity through the coding of its catalytic subunit. Additionally, mutations have been identified in genes AKAP9 (signal transduction pathway), FAT3 (cellular adhesion), and ZEB1 (EMT), the latter of which was mentioned previously [55].

4. Epigenetics: miRNAs, lncRNAs, circRNAs

Epigenetics plays a crucial role in tumorigenesis. It encompasses all intracellular mechanisms involved in the modulation of gene expression without altering the DNA sequence. Within this field, the action of non-coding RNAs (ncRNAs) at the post-transcriptional level is noteworthy. These ncRNAs can be classified based on length into small RNAs (sRNAs) or long non-coding RNAs (lncRNAs), with lengths less than or greater than 200 nucleotides, respectively. Specifically, the former group includes microRNAs (miRNAs), while the latter encompasses a circular group of lncRNAs called circRNAs. Similar to tissue biomarkers, ncRNAs have been used to differentiate adenomas from carcinomas, with variable results, and so far, no pathognomonic marker for the disease has been identified [58].

MiRNAs (or miRs) regulate gene expression at the post-transcriptional level, usually through negative regulation of mRNA, specifically by binding to complementary sites (typically in the UTR region). These miRNAs have different expression patterns in tissues and serum, with the latter location providing the theoretical basis for liquid biopsies, with potential applications in non-invasive diagnosis and monitoring. Specifically, in the context of the PT gland, miRNAs participate in the regulation of hormonal synthesis and secretion, as well as tumorigenesis [59].

At the serum level, Wang et al. described a significant up-regulation of MiR-27a-5p in serum exosomes of patients with PC; revealing an area under the curve (AUC) of 0.859. It is relevant that this specific miRNA participates in the activation of the Wnt/β-catenin signaling pathway, playing a key role in the process of EMT [60].

On the other hand, Krupinova et al. reported that miR-342-3p is significantly decreased in PC, with an AUC of 0.89 when used as a biomarker [59,61].

Studies evaluating miRNA expression at the tissue level have shown variable results. Corbetta et al. found that miR-296 (downregulated in PC), miR-222, and miR-503 (upregulated in PC) varied significantly between adenomas and PC. On the other hand, higher levels of HGS mRNA (hepatocyte growth factor receptor-regulated tyrosine kinase substrate) were observed in PC, which has been associated with EMT-related phenomena such as downregulation of E-Cadherin and subsequent increase in invasion and metastasis. Interestingly, HGS mRNA is a direct target of miR-296, and miR-296 undergoes significant downregulation during tumor progression. Consequently, an indirect role of miR-296 in the carcinogenesis of PT is suggested [62].

In parallel, Rahbari et al. reported significant variation of three miRNAs in PC: miR-26b, miR-30b, and miR-126. Notably, miR-126 showed better characteristics as a biomarker, with an AUC of 0.776 [63].

On the other hand, Vaira et al. described that aberrations in the expression of miRNAs from the C19MC cluster are a characteristic of PC compared to PT adenomas. This cluster of miRNAs is usually silenced by hypermethylation in adults, and its association with various human tumors has been described, promoting tumor invasion and metastasis. Specifically within this cluster, miR-517c showed the most notable difference, correlating with tumor weight, serum calcium, and PTH levels [64].

Additionally, increased expression of miRNAs from the C19MC cluster and miR-372 has been identified in PC distant metastases [59]. Finally, Hu et al. report that in a Chinese population (which has a higher incidence of PC, accounting for 5-7% of HPTP cases), the following miRNAs showed differential expression between PC and PT adenomas: miR-139, miR-222, miR-30b, miR-517c, and miR-126. Specifically, the combination of miR-139 and miR-30b was found to be the best biomarker with an AUC of 0.89 [59,65]. It is important to highlight that most of these studies are limited by small sample sizes due to the low frequency of this cancer.

On the other hand, lncRNAs and circRNAs primarily act as endogenous competitive RNAs (binding to miRNAs and inhibiting their function), while circRNAs can also be translated into functional micropeptides. There are limited studies on these ncRNAs in PC. In brief, Jiang et al. described that a profile composed of the lncRNAs: LINC00959, lnc-FLT3-2:2, lnc-FEZF2-9:2, and lnc-RP11-1035H13.3.1-2:1 had an AUC of 0.88, with Sen: 81.8% and Spe: 83.9% for differentiating adenomas from PC [66].

Furthermore, Zhang et al. recently described that alterations in the expression of lncRNAs PVT1 and GLIS2-AS1 had AUC values of 0.871 and 0.860, respectively [67]. On the other hand, Hu et al. determined that circRNA_0075005 had an AUC of 0.77 for the same function [58,65].

5. Proteomics

There are limited studies on proteomic-based biomarkers in PC. Ciregia et al. documented 33 differentially expressed proteins between PC and PT adenomas. Among these, UCH-L1 (or PGP9.5) is notably overexpressed. It is an enzyme that belongs to the deubiquitinase family and is associated with carcinogenesis pathways, cell growth, DNA repair, and apoptosis. Additionally, a significant increase in ANXA2 protein has been described; a marker of aggressiveness in multiple gastrointestinal cancers, which acts by calcium-mediated binding to membrane phospholipids. Other potential markers could be MDH1, CLIC1, and SOD2 [55,68].

6. Inflammatory Markers

In addition to the previously described markers, the use of preoperative inflammatory markers to distinguish PC from PT adenomas is noteworthy. For example, Ohkuwa et al. evaluated the neutrophil-lymphocyte ratio, platelet-lymphocyte ratio, and lymphocyte-monocyte ratio (LMR), and found that LMR is significantly decreased in PC and is an independent predictor of this cancer when <4.85 [69].

7. Use of Panels

Given that none of the mentioned markers are pathognomonic of parathyroid cancer, the creation of panels, nomograms or other systems that allow for the combination of different markers has become common. These instruments offer an increased sensitivity, specificity, positive predictive value, and negative predictive value therefore maximizing the diagnostic performance of these markers.

Briefly, we present some examples: Kumari et al. describe that a panel combination of parafibromin, galectin-3, and PGP9.5 presents a Sen: 50%, Spe: 97.9%, and a predictive accuracy of 95.4% [37] On the other hand, Truran et al. determined that the use of an immunohistochemical panel using the same markers described previously plus Ki67 is better than the isolated use of any single marker (Sen: 79% and Spe: 100%). These authors point out that a positivity greater than 5% of the cells to Ki57 is considered suspicious of being PC [70]. Finally, Silva-Figueroa et al. developed a nomogram using IHC biomarkers: parafibromin, Rb, Ki67, E-cadherin, and galectin-3, which showed an AUC of 84.9% (adjusted AUC for optimism: 80.5%) [71].