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Immunohistochemistry remains an indispensable tool in diagnostic surgical pathology. In parathyroid tumours, it has four main applications: to detect (1) loss of parafibromin; (2) other manifestations of an aberrant immunophenotype hinting towards carcinoma; (3) histogenesis of a neck mass and (4) pathogenetic events, including features of tumour microenvironment and immune landscape. Parafibromin stain is mandatory to identify the new entity of parafibromin-deficient parathyroid neoplasm, defined in the WHO classification (2022).
- parathyroid carcinoma
- parathyroid adenoma
- multiglandular parathyroid disease
- atypical parathyroid tumour
- WHO classification
1. Introduction
Primary hyperparathyroidism [1,2,3,4], the classic manifestation of parathyroid tumours, represents the third most common endocrine pathology with an estimated prevalence of 3/1000 [5,6,7]. There is close bidirectional association between primary hyperparathyroidism and neoplasms of the parathyroid glands. In most cases, primary hyperparathyroidism is caused by parathyroid tumours. In turn, almost all parathyroid neoplasms present with primary hyperparathyroidism although the existence of non-functional parathyroid tumours, mainly carcinomas, has been suggested in few case reports [8,9,10]. Thus, the clinical and laboratory manifestations of the primary hyperparathyroidism represent the mainstay for the diagnostics of parathyroid tumours [1,2,3,4].
Significant progress has been achieved in the diagnostics and treatment of parathyroid mass lesions. First, the growing awareness of parathyroid pathology and increased availability of laboratory and radiological evaluation have shifted the diagnostic paradigm from clinically based suspicion [11] or even difficult diagnosis [19] in symptomatic patients to almost incidental findings [20,21] via routine biochemical laboratory assessment of serum calcium and parathyroid hormone (PTH) levels. Indeed, the incidence of primary hyperparathyroidism raised sharply after standard serum calcium tests were invented [22]. The next surge of incidence has been associated with screening and in-depth evaluation of osteoporosis patients via bone density measurements in combination with assessment of calcium and PTH levels to identify secondary osteoporosis [7,22].
Second, the parathyroid surgery is currently benefitting from its golden age [21]. The indications, technologies and steps of operative intervention have been well-defined, supported by intraoperative assessment of parathyroid hormone. Currently, parathyroid surgery is considered safe and curative in 97–98% of cases [21].
Wider application of surgical intervention has expanded pathologists’ experience in diagnostic evaluation of parathyroid tissues. A stable basis for parathyroid research was set as well. This led to the third major achievement in parathyroid pathology—the current (2022) WHO classification that is based on deeper understanding of the pathogenesis of parathyroid disease, bringing at least three revolutionary innovations [12] in regard to (1) multiglandular parathyroid disease in primary hyperparathyroidism; as well as (2) atypical parathyroid tumour and (3) the novel concept of parafibromin-deficient parathyroid neoplasms.
2. Morphological Diagnostic Criteria of Parathyroid Tumours by WHO Classification (2022)
To discuss the features of any clinicopathological entities, solid foundation is mandatory, namely, the definitions and diagnostic criteria, set by WHO and/or professional associations. This is particularly relevant to parathyroid tumours, as a new edition of WHO classification is released on 2022 [12], bringing some significant changes.
Historically, primary hyperparathyroidism was mostly attributed to three pathologies. The most frequent cause of primary hyperparathyroidism was parathyroid adenoma comprising 80–85% of cases; followed by primary parathyroid hyperplasia, found in 10–15% patients and the few cases of parathyroid carcinoma, responsible for less than 1% cases of primary hyperparathyroidism [32,33]. Upon typical presentation, these entities were easily recognised. Parathyroid adenoma was diagnosed if a single encapsulated or demarcated, non-invasive parathyroid neoplasm lacking intralesional adipose tissue was found in a patient experiencing surgery-related decrease of the parathyroid hormone level [34,35,36]. The diagnosis of adenoma was further supported by an adjacent peripheral rim of residual gland. Parathyroid hyperplasia presented as a multiglandular pathology showing mixture of parenchymal and fat cells with increased parenchyma-to-fat ratio [34,36,37]. Unequivocal invasive growth and/or presence of metastases justified the diagnosis of parathyroid carcinoma [38].
However, the historical classification faced difficulties, which mainly focused on two areas: the clinically significant distinction between carcinoma and adenoma, and the differential diagnosis between adenoma and primary parathyroid hyperplasia.
Currently, the WHO classification of parathyroid tumours includes the entities of multiglandular parathyroid disease, adenoma, atypical parathyroid tumour, and carcinoma [12].
Atypical parathyroid tumour is defined as a parathyroid neoplasm of uncertain malignant potential. It shows some cytological or histological features that increase suspicion of carcinoma, but the diagnostic criteria of carcinoma cannot be identified although sufficient number of tissue samples has been submitted for microscopy. The worrisome features that constitute the diagnostic criteria of atypical parathyroid tumour, include the following:
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Trabecular or sheet-like architecture;
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Band-like fibrosis in the absence of history of fine needle aspiration (FNA) that might induce scarring via needle track or at the site of FNA-induced necrosis. Secondary or tertiary hyperparathyroidism are also associated with fibrotic bands and should be considered clinically;
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Cytological atypia, enlarged nucleoli;
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Mitotic activity exceeding 5 mitoses/50 high power fields;
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Atypical mitoses;
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Coagulation necrosis in the absence of history of FNA;
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Adherence to the surrounding tissues but not frank invasion into these tissues;
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Tumour cells located within the capsule, but lacking full-thickness penetration through the capsule [12].
The presence of the listed traits should induce active search for the definitive criteria of parathyroid carcinoma. However, the characteristics of atypical parathyroid tumour themselves are not sufficient to justify the diagnosis of carcinoma.
Parathyroid carcinoma is a clear-cut malignancy, manifesting with either invasion or metastasis. To classify a parathyroid tumour as carcinoma, any of the following diagnostic criteria [12] must be present:
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Angioinvasion in a blood vessel located either outside the tumour or in the capsule; the tumour growth through vascular wall and/or carcinoma cells within thrombus must be visible. Considering the fenestrated endothelium, a mere presence of neoplastic cells in an intratumoural vessel does not qualifies for true invasion. Vascular invasion must also be distinguished from artificial displacement (“seeding”) of tumour cells into blood vessel lumen, that can happen during grossing. True vascular invasion is recognised by verified tumour penetration through vessel’s wall or by presence of the tumour cells in a thrombus, showing a biological reaction to invasion;
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Invasion in lymphatics provided that retraction phenomenon is excluded. Immunohistochemistry for endothelial markers is highly recommended for this;
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Perineural or intraneural invasion;
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Invasion into surrounding soft tissues, thyroid, oesophagus, skeletal muscle. Presence of neoplastic cells within the tumour capsule does not qualify for the diagnosis of carcinoma. The mere presence of parathyroid tissues within the thyroid also is not sufficient to justify the diagnosis of parathyroid carcinoma, because ectopic location of a parathyroid gland, adenoma or carcinoma is a well-known phenomenon [44,45]. Invasion must also be distinguished from parathyromatosis—a rare condition characterised by a presence of multiple microscopic islets of benign parathyroid tissue scattered throughout the soft tissues of neck and/or superior mediastinum [46,47,48,49,50];
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Metastases in lymph nodes or distant organs [12]. However, considering the indolent course of parathyroid carcinoma, metastatic spread is not always present. In a recently published large, SEER-based study, evaluating 609 cases of parathyroid carcinoma (1975–2016), lymph node metastases were found in 25.2% of all patients and 29.2% of cases where lymph node status was reported. Distant metastases were present in 2.2% of all patients and 3.8% of cases with a known stage [17].
3. Immunohistochemical Profile of Parathyroid Tumours
3.1. Parafibromin
Parafibromin, the tumour suppressor protein coded by Cell Division Cycle 73 (CDC73) gene, represents the most extensively studied immunohistochemical target in parathyroid pathology. It is the driver of parathyroid carcinogenesis and thus the only protein that is advised to be detected immunohistochemically in parathyroid tumours (at least in all carcinomas and atypical parathyroid tumours) in accordance with the current (2022) WHO recommendations [12].
Parafibromin is a tumour suppressor protein that induces cell cycle arrest by repressing cyclin D1 [56]. It is involved in the regulation of p53 pathway [51]. CDC73 mutations lead to loss of both function and immunohistochemical expression of parafibromin. Since the first discoveries, absence of parafibromin has been associated with diagnostic evidence [57,58,59] and worse prognosis of parathyroid carcinomas [59,60,61] and malignant behaviour of tumours histologically diagnosed as atypical adenomas [40]. However, controversies exist that can be attributed to technological differences and challenges [51], nuclear, nucleolar or cytoplasmic location of reactivity [62,63,64] or cases showing partial or weak expression [51,62,65,66].
3.2. Proliferation Activity by Ki-67
Ki-67 is a nuclear protein that is expressed during the active phase of cell cycle while strongly down-regulated during the G0 phase. As the presence of immunohistochemically detectable Ki-67 identifies proliferating cells, Ki-67 is widely used in morphological protocols for tumour diagnostics, including grading, molecular classification, prognostic evaluation and prediction of treatment efficacy. The biological functions of Ki-67 include mitotic, interphase and regulatory processes. During mitosis, Ki-67 participates in the build-up of perichromosomal layer: a ribonucleoprotein sheath that coats the condensed chromosomes and prevents them from aggregation. In interphase, Ki-67 protein maintains the structure of heterochromatin. Ki-67 also regulates the cell cycle via p21 protein-related pathways [38,76,77].
3.3. Cell Cycle Regulation
3.3.1. p27 Protein
The p27 protein is best-known as a cyclin dependent kinase inhibitor and tumour suppressor that slows cell cycle progression, mediating G1 arrest. It also regulates G2/M progression. Other functions of p27 include control of cellular differentiation, motility and migration, as well as the activation of apoptosis. Malignant cells can lose p27 expression due to impaired synthesis or accelerated degradation, or inappropriate intracellular localisation of the relevant protein [88,89,90].
3.3.2. p21 Protein
The p21 protein controls cell cycle progression, apoptosis and transcription. It is the key mediator of cell cycle arrest in response to DNA damage [94] and a component of p53 pathway [92]. The expression of p21 has dual effects, including suppression or enhancement of apoptosis [94,95].
In early studies, setting the cut-off threshold at the level of 10%, nuclear expression of p21 was found in 58% of adenoma and 55% of carcinoma cases [92]. Tissue microarrays were used in the given study [92]. Later, significant heterogeneity of p21 expression was observed manifesting as the hotspot pattern [38]. The remarkable heterogeneity hinted on cautious interpretation of microarray-based results although the differences and trends in p21 expression were preserved independently of the counting mode: mean vs. hotspot [38].
3.3.3. Cyclin D1
The cyclin D1 regulates transcription and acts as an important molecular switch in the proliferation control. As an allosteric activator, it forms a complex with cyclin dependent kinases 4 and 6 (CDK4 and CDK6) that phosphorylate and thus inactivate the tumour suppressor protein Rb, resulting in the cell cycle progress from the G1 to S phase [96,97]. The overexpression of cyclin D1 in parathyroid neoplasms can be caused by pericentric inversion of chromosome 11p that results in CCND1 gene control by parathyroid hormone gene promoter. However, this inversion is seen in lower frequency than the overexpression of the relevant cyclin D1 protein, e.g., 5–8% vs. 40% in adenomas [98]. Consequently, other mechanisms are involved, such as gene amplification, transcriptional activation, e.g., via Wnt or MAPK pathways [96] or deranged degradation [99,100].
3.3.4. p53 Protein
Regarding p53 in parathyroid tumours, facilitated degradation of the relevant mRNA can be implicated. Parafibromin can bind to mRNA of p53 and destabilise it [64]. Enhanced association with mutant parafibromin [64] might result in faster degradation of p53 mRNA. The final outcome would be absence of immunohistochemically detectable p53 expression and enhanced cellular proliferation in parathyroid carcinoma while benign lesions retained wild-type protein. The general landscape of p53 expression in parathyroid diseases thus would lack diagnostic differences, remaining invariably negative. Indeed, constant negative p53 expression in normal parathyroid as well as benign and malignant tumours has been reported previously [38,92].
3.4. APC Protein
Adenomatous polyposis coli (APC) gene is a tumour suppressor that inhibits the Wnt molecular pathway. It is known for its role in colorectal carcinogenesis and association with familial adenomatous polyposis (FAP) [107,108]. Its protein product can be detected by immunohistochemistry and has been recommended by WHO (2022) as an adjunct in the diagnostics of parathyroid carcinoma [12]. Parathyroid adenomas usually retain APC while carcinomas tend to become negative, therefore loss of APC has been listed among the biomarkers that indicate an increased risk of malignant behaviour of a parathyroid tumour [12]. Hosny Mohammed et al. observed loss of APC in 20/21 (95.2%) parathyroid carcinomas, contrasting with 38/73 (52.1%) adenomas [69]. However, Kumari et al. reported on loss of APC (<10% of cytoplasmic staining) in 9% of carcinomas, 23.5% of atypical adenomas and 22% of adenomas [72]. Loss of APC acts as a screening marker for malignant potential, but the diagnosis of carcinoma still must be proved by WHO criteria, that are based on manifestations of invasive growth and metastatic spread.
3.5. Intermediary Filaments
3.5.1. Cytokeratin 19
Cytokeratin 19 is a widely expressed intermediary filament. It is invariably present in parathyroid adenomas, carcinomas [109] and normal parathyroid glands [110]. Recently, a statistically significant up-regulation of cytokeratin 19 was found in proliferating parathyroid lesions encompassing adenoma, multiglandular parathyroid disease and carcinoma. The expression was markedly heterogeneous [38].
From the point of view of surgical pathologist, it is important to remember that thyroid tumours and cancer metastases in cervical lymph nodes also are likely to express cytokeratin 19 [111,112,113,114,115]. Hence, the diagnostic value of cytokeratin 19 in parathyroid pathology is low but this antigen could rather evoke scientific interest because of its up-regulation in carcinoma. As the diagnostic criteria of parathyroid carcinoma reflect capacity for invasion and metastatic spread, the altered expression level of intermediate filaments might have pathogenetic importance.
3.5.2. Vimentin
Vimentin is a major mesenchymal intermediate filament, controlling cellular motility, signalling and directional migration [116].
The glandular histology mostly precluded the researchers from in-depth assessment of vimentin in parathyroid tissues, except stroma [110]. In addition, the rarity of parathyroid carcinoma hampered the studies of epithelial-mesenchymal transition in parathyroid malignancies.
3.6. CD44
CD44 represents a family of integral cell surface glycoproteins. It is a single-span transmembrane adhesion molecule lacking kinase activity [118,119]. The main ligand of CD44 is hyaluronic acid that is abundantly present in extracellular matrix. The interaction between ligand-binding domain of CD44 and hyaluronic acid changes the conformation of the molecule resulting in the recruitment of adaptor proteins (ERM, Src, and others) to its intracellular domain. This, in turn, triggers downstream biological effects as proliferation, motility and migration, adhesion and invasion. CD44 is expressed during embryonic development, on mesenchymal cells and in carcinogenesis. In tumours, it frequently indicates poor prognosis and is recognised as one of the cancer stem cell markers [29,118,119,120,121,122].
3.7. Neuroendocrine and Hormone Markers: Chromogranin A, Synaptophysin, CD56, PTH and TTF-1
Neuroendocrine and hormone markers are helpful to detect the histogenesis of a tumour or mass lesion. Parathyroid tumours occasionally have to be distinguished from thyroid neoplasms because of close anatomic relation [137] between both glands, including occasional intrathyroidal location of normal parathyroid gland or parathyroid carcinoma. The histogenetic diagnosis is difficult also in fine needle aspiration cytology [138].
The neuroendocrine differentiation in parathyroid tissues is limited, generally manifesting as isolated positive reaction for chromogranin A, that is observed in most (98%) cases [12,139,140]. The expression of synaptophysin is less frequent albeit variable: 11–100%, according to Li et al., 2014 and Yu et al., 2019 [109,140]. Insulinoma-associated protein 1 (INSM1) is absent [140] both from normal and pathological parathyroid tissues including multiglandular parathyroid disease in primary hyperparathyroidism, secondary hyperplasia, tertiary hyperparathyroidism, adenomas, atypical adenomas and carcinomas [140]. Neural cell adhesion molecule CD56 is another negative marker despite frequent expression in neuroendocrine tumours in other locations [38,141,142,143,144].
3.8. Immunohistochemical Profile of Parathyroid Disease in MEN Syndromes: Menin
Multiglandular parathyroid disease is a typical component of certain multiple endocrine neoplasia (MEN) syndromes, namely, MEN 1, MEN 2A and MEN 4. Loss of menin is characteristic for MEN I, and decreased expression of p27—for MEN 4. However, menin is a technologically “difficult” antigen similarly to parafibromin, and loss of p27 protein is also seen in sporadic carcinomas [93].
3.9. Calcium-Sensing Receptor (CaSR) and the Associated Molecular Pathways
Most of parathyroid tumours present with hypercalcemia that is higher and therefore more frequently symptomatic in patients affected by parathyroid carcinoma, compared to benign disease. Non-functioning parathyroid carcinoma hypothetically exists but is exceptionally rare [9,10]. Although tumour weight is strongly associated with calcium and PTH concentration in blood [164], abnormal feedback and/or disturbed sensitivity to blood calcium levels could be expected in the neoplastic cells, and the dysfunction might be more marked in carcinoma. Indeed, diminished calcium-sensing receptor expression has been reported in parathyroid carcinoma but is rare in benign tumours [165]. Thus, 31% of carcinomas showed downregulation of CaSR, contrasting with adenomas and hyperplasia. In this study [166], only a single adenoma featured a “carcinoma-like” irregular or absent CaSR staining pattern (1/104 in a mixed group of adenomas, primary multiglandular disease, secondary hyperplasia and tertiary hyperparathyroidism) [166]. More recently, global loss of CaSR has been reported in 5/10 carcinomas while all adenomas (21) showed retained expression (p = 0.001), and only a single atypical adenoma (1/14) yielded global loss of expression [66]. In contrast, Storvall et al. observed retained immunohistochemical CaSR expression in all the evaluated parathyroid tumours, including 32 carcinomas, 44 atypical adenomas and 77 adenomas; just a single carcinoma and one atypical adenoma presented weaker expression [165]. CaSR shows negative correlation with Ki-67 both in secondary hyperparathyroidism and adenoma [166,167,168].
3.10. Intratumoural Heterogeneity
Parathyroid tumours are characterised by remarkable biological heterogeneity, involving proliferative activity (Ki-67) and cell cycle regulation (p21, cyclin D1), expression of intermediary filaments (cytokeratin 19, vimentin) and different receptors, e.g., calcium sensing receptor or vitamin D receptor [175]. In addition, technological variations lead to significant intertumoural heterogeneity and differences among data obtained in various studies. The detection of parafibromin is the classic example.
This entry is adapted from the peer-reviewed paper 10.3390/ijms23136981
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