2.3.3 Histopathology of pituitary tumours

The human pituitary gland consists of two major components: the adenohypophysis comprising the hormone producing cells of the pars anterior, pars intermedia, and pars tuberalis, and the neurohypophysis, also called pars nervosa or posterior lobe (1). In contrast to most mammalian species, the human gland has no anatomically distinct pars intermedia (2). The exclusively proopiomelanocortin (POMC)-producing cells of the pars intermedia are sandwiched between the anterior and posterior lobes in the majority of mammals, whereas in the human they are incorporated within the pars anterior, thereby constituting the pars distalis (3). The pars tuberalis is a minor upward extension of the adenohypophysis attached to the exterior of the lower pituitary stalk. In this chapter we deal only with adenohypophyseal tumours.

Histologically, the adenohypophysis consists of a central median (or mucoid) wedge flanked by the two lateral wings. The hormone-producing cell types are distributed in an uneven, but characteristic manner. The cells are arranged within evenly sized acini surrounded by a delicate but well-defined reticulin fibre network giving the pituitary its distinct architecture (4). In the center of the acini is the long-neglected pituitary follicle composed of the agranular nonendocrine folliculo-stellate cells (5).

Growth hormone cells or somatotrophs take up about 50% of the gland occupying chiefly the lateral wings. They show strong acidophilia by histology and growth hormone immunoreactivity by immunocytochemistry. Contingents of somatotrophs also express prolactin or the α-subunit of the glycoprotein hormones. Prolactin cells or lactotrophs account for 10–30% of cell population. The chromophobic or slightly acidophilic and prolactin-immunoreactive lactotrophs are evenly scattered with focal accumulation in the posterolateral rim of lateral wings. The majority of corticotrophs or adrenocorticotropic (ACTH) cells reside within the median wedge. They are basophilic, positive with the periodic acid–Schiff (PAS)-method and immunoreactive for 1-39 ACTH as well as for other POMC -peptides (β-endorphin, β-lipotrophic hormone (LPH), corticotropin-like intermediate peptide (CLIP), etc). Approximately 12% of adenohypophyseal cells are immunopositive for POMC-peptides, which include a small but undetermined percentage of pars intermedia-derived POMC cells as well. Thyroid-stimulating hormone (TSH) cells or thyrotrophs, occupying mainly the anterior one-third of the median wedge, represent only about 5% of adenohypophyseal cells. The slightly basophilic, angular thyrotrophs are strongly immunoreactive for β-TSH and α-subunit. Gonadotrophs producing follicle-stimulating hormone (FSH) and/or luteinizing hormone account for an estimated 15–20% of cells distributed quite evenly throughout the pars distalis. The basophilic, PAS-positive gonadotrophs are immunoreactive for β-FSH, β-luteinizing hormone and their α-subunit. The hormonal function of the adenohypophyseal cell types displays considerable flexibility depending on the functional demand placed on them. Even a reversible transdifferentiation is proven to occur between members of the Pit1 group: growth hormone cells to prolactin-cells (pregnancy) and growth hormone cells to TSH cells (hypothyroidism) (6, 7).

Pituitary cell types differ not only in their function, structure, and hormone content, but also in their morphological responses to functional stimulation or suppression. Thus, no extrapolation of findings from one cell type to another is appropriate (4). The folliculostellate cells have no hormonal function but both in vitro biochemical and morphological studies have documented that the small simple cells are not only unexpectedly versatile but frankly indispensable (5, 8).

Adenomas—benign neoplasms arising in all types of hormone-producing cell of the adenohypophysis—are common, accounting for approximately 15% of intracranial tumours. Adenomas occur as small incidental findings in 5–20% of pituitaries at autopsy (1). Regardless of their size, a total dissolution of the normal acinar architecture (Fig. 2.3.3.1a) is evident in every adenoma. Pituitary adenomas are not encapsulated, but, if large enough, the tumours compress the reticulin framework of the surrounding normal gland into a pseudocapsule (Fig. 2.3.3.1b). By histology, adenomas are acidophilic, basophilic, or chromophobic. The tinctorial properties of tumours are largely unrelated to their hormonal function. Demonstration of hormone content (immunohistochemistry), assessment of ultrastructure (electron microscopy), and the increasingly popular molecular/genetic techniques are utilized for the functional classification of pituitary adenomas (9–11).

Pituitary tumours may be referred to as microadenomas (less than 10 mm in diameter), or macroadenomas (more than 10 mm in diameter). The growth pattern of these tumours may be expansive resulting in a slowly growing mass exerting increasing pressure on the surrounding normal gland and the bony sella. In contrast, invasive adenomas spread into the surrounding normal gland, dura or other parasellar structures (sphenoid sinus, cavernous sinus) regardless of their size. Adenomas extending into the suprasellar space may compress or infiltrate the optic chiasm causing visual disturbances, a frequent clinical manifestation of macroadenomas. Exceptionally large adenomas may grow into the anterior or posterior cranial fossa or downwards into the nasopharynx (12, 13).

The sparsely granulated form is the most common adenoma type accounting for 27–30% of pituitary tumours (1, 9–11). It is associated with hyperprolactinaemia, primary or secondary amenorrhoea, galactorrhoea, and infertility in women. The less specific symptoms in men—decreasing libido and impotence—usually mean delay in diagnosis and development of macroadenomas whereas the majority of tumours in women are small and classified as microadenoma. Prolactin cell adenoma is most frequent in the third and fourth decade of life in both sexes (14). However, the incidence of the tumour type is significantly higher in women in their main childbearing years. Prolactin cell adenomas are associated with wide-ranging biological behaviours from indolent to highly aggressive, usually not reflected in their morphology. The prolactin blood levels are roughly proportional to the tumour mass. The frequency of the tumour type in autopsy material is around 40% in both sexes.

Histologically, the large majority of prolactin cell adenomas are chromophobic with a diffuse pattern. The other two patterns (papillary and the type with abundant hyalinous connective tissue stroma) occur mostly as incidental autopsy findings suggesting slow growth rate and/or later onset. Immunostaining demonstrates strong immunopositivity tracing the sacculi of the prominent Golgi apparatus, characteristic of prolactin cells (Fig. 2.3.3.1c). Ultrastructurally the three salient features are: masses of rough surfaced endoplasmic reticulum, prominent Golgi apparatus, and extrusion of the small, sparse secretory granules. The latter is the specific marker of prolactin differentiation (Fig. 2.3.3.2a) (1, 9–11).

Although calcification is extremely rare in other adenoma types, an estimated 10–15% of prolactin-producing adenomas display varying degrees of calcification. The alteration may be extreme (‘pituitary stone‘). Deposition of endocrine amyloid occurs infrequently.

Adequate therapeutic response is associated with striking morphological changes in adenoma cells: the nucleus becomes heterochromatic, the cytoplasm shows marked shrinkage due to loss and involution of the hormone-producing apparatus (rough endoplasmic reticulum, Golgi complex) of the cells. Prolactin immunoreactivity is reduced or lost and, with the exception of granule extrusions, the cells have no ultrastructural markers of prolactin differentiation. Effects of treatment with long-acting form of bromocriptine can be exceptionally severe. Theoretically the morphological changes are reversible. However, portions of neoplasms may permanently lose their responsiveness and retain their suppressed features even when treatment is discontinued. Long-term administration of dopamine agonists may also cause varying degrees of fibrosis and calcification (15, 16) apparent as psammoma bodies.

The densely granulated form of prolactin cell adenoma is very rare and clinically behaves similarly to the sparsely granulated variant (9, 10).

Approximately 15% of surgically removed pituitary adenomas represent two forms of growth hormone cell adenoma (1, 9, 10). Most of these tumours are associated with physical stigmata and clinical signs of acromegaly, whereas tumours in children and adolescents causing gigantism are rare. Although the clinical manifestations and incidence of the two tumour types are similar, there are major differences in their morphology.

The densely granulated growth hormone cell adenoma occurs with the same frequency in both sexes peaking at the same time (in the sixth decade). The tumours display slow, expansive growth resulting in the typical ‘ballooning of the sella’ and may remain intrasellar for several years. Histologically, the densely granulated form is strongly acidophilic displaying diffuse or, less frequently, trabecular pattern. An extensive immunoreactivity for growth hormone (Fig. 2.3.3.1d) is usually accompanied by similarly strong positivity for α-subunit (1, 9, 10, 17). Scattered immunopositivity for prolactin and β-TSH is usually not associated with oversecretion of these hormones. The ultrastructure of adenomatous densely granulated growth hormone cells is similar to that of the normal phenotype, featuring well-developed rough endoplasmic reticulum, prominent Golgi complex and numerous large secretory granules mostly in the 350–500 nm range (see Fig. 2.3.3.2b).

The sparsely granulated growth hormone cell adenoma is more common in women, and is also diagnosed earlier peaking in the fourth decade (14). The tumours tend to be macroadenomas at the time of diagnosis and are often invasive. Histology detects chromophobic adenomas invariably displaying a diffuse pattern. Nuclear pleomorphism may be evident and adenoma cells frequently harbour a homogeneous, spherical juxtanuclear, practically unstained structure (18). This ‘fibrous body’ is strongly immunopositive for cytokeratin (Fig. 2.3.3.1e). The striking polkadot-pattern thereby generated is the best histological marker of the tumour type, since growth hormone immunoreactivity is often scanty. As opposed to the densely granulated type, multiple immunoreactivities for pituitary hormones are rarely noted. The ultrastructural phenotype of the sparsely granulated growth hormone cell adenoma—the spherical filamentous aggregate of the fibrous body nesting in the concavity of the eccentric crescent-shaped nucleus and scanty, small (less than 250 nm) secretory granules—is not seen in the normal gland (Fig. 2.3.3.2c).

Morphologic effects of treatment with a long-acting somatostatin analogue are neither severe nor consistent (16, 18). Remarkable shrinkage of cells and marked fibrosis are infrequent. Most common findings are the increase in size and number of secretory granules and/or increased lysosomal activity whereas significant fibrosis is less frequent. The close correlation observed between clinical response and tumour morphology in cases of prolactinomas treated with dopamine agonists does not exist in examples of octreotide treatment.

Both types of growth hormone cell adenoma may engage in usually focal, rarely massive production of endocrine amyloid. Approximately 2–3% of morphologically typical sparsely granulated adenomas are clinically silent, the reason for which is unknown (9, 10). Rare examples of the sparsely granulated tumours contain variable amounts of nervous tissue (neuron-like cells and neuropil) as a likely result of neuronal differentiation within the adenoma (9, 10).

The mixed (growth hormone cell/prolactin cell) adenoma, a tumour comprising two distinct cell types, is the most important in this group (1, 9, 10), accounting for an approximate 5% of surgically removed adenomas. The tumours are associated with acromegaly and varying degrees of hyperprolactinaemia. They tend to be aggressive and are difficult to treat. Mixed adenomas usually consist of densely granulated growth hormone cells and sparsely granulated prolactin cells. Other combinations may occur but they are rare. Accordingly, they are composed of acidophilic and chromophobic cells by histology displaying immunoreactivity for growth hormone and prolactin, α-subunit positivity is common, as well. By electron microscopy, the cell types constituting mixed adenomas have the features of densely granulated growth hormone cells and sparsely granulated prolactin cells as described earlier in this chapter. The infrequent (2%) mammosomatotroph cell adenoma is monomorphous, that is, it consists of one cell type displaying markers of both growth hormone and prolactin differentiation (1, 9, 10). Clinically they are associated with acromegaly and variable, usually mild hyperprolactinaemia. The acidophilic tumours are immunoreactive for growth hormone and α-subunit and, to a much lesser extent, for prolactin. At the ultrastructural level the densely granulated cells possess unusually large (up to 1000 nm and over) secretory granules and display granule extrusions, a prolactin cell marker. The slow-growing mammosomatotroph adenomas show biological behaviour similar to that of densely granulated growth hormone adenomas. They should be considered a morphological variant of densely granulated growth hormone cell adenoma with no difference in the clinical presentation.

The acidophil stem cell adenoma is a rare (2%) monomorphous type with morphological signs of prolactin and growth hormone differentiation (1, 9, 10). The tumour is associated chiefly with hyperprolactinaemia, but the serum prolactin levels may be disproportionately low for the size of the tumour. Physical stigmata of acromegaly and significant elevation of growth hormone levels are infrequent. These tumours grow aggressively in young subjects with tendency to invade infrasellar areas. Histology demonstrates chromophobic adenomas with moderate to strong immunoreactivity for prolactin. Immunopositivity for growth hormone is weak or negative, but immunostain for cytokeratin reveals the dot-like positivity of fibrous bodies. The striking ultrastructure is characterized by oncocytic change with formation of giant mitochondria, sparse, small secretory granules with extrusion (prolactin marker) and fibrous bodies (growth hormone marker).

Corticotroph or ACTH-cell adenomas responsible for pituitary dependent Cushing’s disease account for 10–12% of surgically removed adenomas. The tumours show marked (4–5:1) female preponderance. The age-related occurrence of corticotroph adenoma is similar in the two sexes peaking in the fourth decade (14). Most corticotroph lesions are small microadenomas causing florid Cushing’s disease (1, 9, 10, 19). The tumours, often measuring only a few millimetres in diameter, may be too small to conclusively detect by imaging or to clearly identify by the neurosurgeon (20). Therefore serial sectioning of the biopsied tissue fragments is often needed and it may not result in the demonstration of the tumour in every case.

Histologically, corticotroph adenomas are basophilic and PAS positive with a sinusoidal or diffuse pattern (1, 9, 10, 19). Immunoreactivity can be demonstrated not only for 1-39 ACTH, but for other POMC-derived peptides, (β-endorphin, β-LPH, CLIP, etc.) as well. Electron microscopy documents cells densely granulated with secretory granules ranging up to 450–500 nm similar to those of normal ACTH cells. The best markers are: (1) the morphology of the secretory granules being spherical as well as notched, drop-shaped, and heart-shaped often displaying variable electron density; (2) perinuclear bundles of cytokeratin filaments, characteristic for the human corticotroph (Fig. 2.3.3.2d).

In a minority of cases pituitary dependent Cushing’s disease is brought about by larger tumours. These neoplasms are often associated with a milder form of hypercorticism, but the tumours grow aggressively, and they often invade and are frequently macroadenomas at the time of diagnosis (21). Histologically they exhibit variable, often weak PAS positivity and immunoreactivity for ACTH. A few examples of aggressive macroadenomas display immunoreactivity for luteinizing hormone and/or α-subunit. Morphological features of corticotroph adenomas in cases of Nelson’s syndrome are similar to those of densely granulated corticotroph adenomas in Cushing’s disease with few or no cytokeratin filaments.

Crooke’s hyalinization, i.e. excessive accumulation of cytokeratin filaments, is the ubiquitous response of the normal human ACTH cell to longlasting elevation of circulating glucocorticoid levels (4). Accordingly, Crooke’s hyalinization is noted in (1) nontumorous corticotrophs adjacent to corticotroph adenomas, (2) in ectopic ACTH/corticotrophin releasing hormone (CRH) syndrome, (3) in patients with glucocorticoid secreting adrenocortical tumours, (4) and in subjects having been treated with pharmacological doses of glucocorticoids. Crooke’s hyalinization is not expected to develop in corticotroph tumours. Yet, a minority of such adenomas contains variable percentage of adenoma cells displaying the alteration (9, 10). Crooke’s cell adenomas do not represent an entity and have no clinical correlates. Such tumors may be associated with mild, moderate, or severe hypercorticism and with variable biological behaviour (22).

A mere 1% of surgically removed adenomas derive in TSH cells (5, 9, 23, 24). These tumours are associated either with hyperthyroidism and inappropriately elevated levels of TSH or they develop in hypothyroid subjects, probably preceded by thyrotroph hyperplasia. Inexplicably, some adenomas bearing immunohistochemical characteristics of thyrotroph adenomas occur in euthyroid subjects. At the time of diagnosis, these tumours are often macroadenomas with a tendency to invade. The morphology of the small group of thyrotroph adenomas exhibit surprising diversity. Histologically, the adenomas are chromophobic and negative or mildly positive with PAS. They may be highly differentiated comprising elongate polar cells forming pseudorosettes around vessels. Alternatively, the pattern may be diffuse in some cases with considerable nuclear pleomorphism. Yet another variant is markedly fibrotic. Minute calcifications may be evident as well. Immunoreactivity for TSH is variable; it is often patchy or scattered, rarely extensive. Scattered cells may exhibit immunoreactivity for α-subunit, growth hormone, and prolactin. No specific ultrastructural markers exist for the tumours. They are sparsely granulated (granule size: up to 250); the secretory granules are often confined to the cell periphery outlining cell contours.

Thyrotroph adenomas possess somatostatin receptors and may show clinical improvement to octreotide therapy (25).

The incidence of gonadotroph adenomas in surgical material is about 10%; they occur with similar frequency in the two sexes (5, 9). The majority of FSH/luteinizing hormone tumours appear as slow-growing, expansive macroadenomas causing local symptoms (12). Discrepancy between clinical parameters and morphological signs of gonadotroph differentiation is common; tumours displaying FSH/luteinizing hormone immunoreactivity and signs of high degree of functional differentiation by electron microscopy, may be unassociated with elevated serum FSH/luteinizing hormone levels.

The morphology of gonadotroph adenomas is variable (1, 5, 9). Histology may reveal polar cells forming pseudorosettes around vessels (Fig. 2.3.3.1f) or a diffuse pattern. Oncocytic change, i.e. undue increase of number and volume density of mitochondria is frequent. Immunoreactivity for FSH and/or luteinizing hormone is variable, often patchy; α-subunit, which is a useful clinical indicator, is not a reliable morphological marker. Electron microscopy documents unique sex-linked dimorphism. Many tumours in both sexes consists of polar cells having small (100–200 nm) secretory granules accumulating in cell processes. The Golgi complex has regular features in tumours of males, whereas it shows vacuolar transformation (honeycomb Golgi) in females. Adenomas comprising nonpolar cells have regular Golgi complex in both sexes.

These two adenoma types are the morphological variants of the same tumour (5, 9). The hormonally inactive adenomas account for approximately 25% of surgically removed tumours. They are twice as common in males peaking in the sixth decade in both sexes (14). Most of the tumours are slowly growing expansive macroadenomas causing local symptoms and varying degrees of hypopituitarism (12). Low-grade hyperprolactinaemia may occur (‘stalk section effect‘).

Null cell adenoma is the nononcocytic form. Histologically it is chromophobic with predominantly diffuse pattern. Pseudorosette formation, characteristic of glycoprotein hormone producing tumours, may also occur. Immunostainings may detect scattered positivity for various pituitary hormones, particulary β-FSH, β-luteinizing hormone and α-subunit, or they may be immunonegative. Electron microscopy documents small cells having poorly developed cytoplasmic organelles and small (100–200 nm) scanty randomly distributed secretory granules, but no markers of cellular derivation (26).

Oncocytomas always show diffuse pattern by histology. The adenoma cells are larger than null cells and may display acidophilia due to non-specific binding of acidic stains by mitochondria. The pattern of immunoreactivities is the same as seen in null cells. By electron microscopy the sole ultrastructural marker is the extensive accumulation of mitochondria, whereas the other organelles are poorly developed. The secretory granules are sparse, small (100–200 nm) and are often displaced to the cell periphery by the crowding mitochondria.

The term silent adenoma refers to three types of well-differentiated, morphologically well-characterized adenomas which are unassociated with any known hormonal hypersecretory syndromes and are not derived from any of the known anterior pituitary cell types (1, 5, 9). Silent adenomas and null cell adenomas are not synonymous, although clinically they cannot be distinguished.

Silent ‘corticotroph’ adenoma subtype 1 (3) (frequency less than 2%) is unassociated with clinical signs and symptoms of Cushing’s disease. It shows a lesser degree of female preponderance and different age-related occurrence than corticotroph adenomas associated with Cushing’s disease. The tumours display high propensity for haemorrhage and may present with pituitary apoplexy. Morphologically the adenomas have the same basophilia, PAS positivity, ACTH and β-endorphin immunoreactivity, and ultrastructural features as corticotroph tumours associated with Cushing’s disease.

Silent ‘corticotroph’ adenoma subtype 2 (3) has a frequency of 1.5–2.0% and shows marked male preponderance. The tumours appear as nonfunctioning masses and are usually diagnosed at the macroadenoma stage. Histology reveals chromophobic tumours comprising small cells, which exhibit only modest PAS positivity and scattered immunoreactivity for ACTH and β-endorphins. Ultrastructurally the small cells possess small to midsize secretory granules (200–400 nm) showing similarity to POMC granules. However, no cytokeratin filaments are present.

The two adenoma types described above probably derive in cells of the pars intermedia, which in the human pituitary are incorporated within the pars distalis (2, 3). The physiological function of those cells is unknown.

The silent adenoma subtype 3 has a frequency of approximately 2% and is clinically more important owing to its fairly aggressive behaviour occurring mainly in young women (27). The tumour is equally frequent in the two sexes but it has strikingly different age-related distribution. In men, the tumour may occur at any age from the second to the seventh decade. The overwhelming majority of adenomas in women present between 20 and 40 years, peaking in the late twenties, but they rarely occur after 40 years of age. Silent adenoma subtype 3 consistently mimics prolactin cell adenoma in women, being associated with low-grade hyperprolactinaemia (usually less than 100 ng/ml) at the microadenoma stage. The serum prolactin levels do not increase proportionally with tumour size. Dopamine agonist treatment is not indicated; it returns to normal levels of prolactin (probably released from the non-neoplastic pituitary), but it does not cause tumour shrinkage and does not inhibit tumour progression.

Histologically, subtype 3 silent tumours are often acidophilic and may show mild PAS positivity. The large adenoma cells form diffuse, or lobular pattern. Immunocytochemistry may demonstrate scattered, minor positivity for various adenohypophyseal hormones owing to plurihormonal differentiation, but the majority of tumour cells are immunonegative for all known pituitary hormones. The ultrastructure of adenomas displays features of glycoprotein hormone differentiation and often marked accumulation of smooth endoplasmic reticulum. Owing to unspecific and variable immunoreactivities, electron microscopy is indispensable for diagnosis. The cell derivation of this adenoma type is unknown.

Unclassified plurihormonal adenomas are rare tumours, often with unique ultrastructure (5, 9). They may consist of one morphological cell type (monomorphous), or more than one phenotype (plurimorphous). The most common combinations are: growth hormone-TSH-prolactin or prolactin-TSH.

In 2002, Roncaroli et al. (28) described a previously unknown type of primary oncocytic adenohypophyseal tumour in five elderly patients. The neoplastic cells were immunoreactive for vimentin, S100 protein, epithelial membrane antigen, and galectin-3, immunohistochemical markers of the folliculostellate cells of the pituitary. Immunostains for endocrine markers and pituitary hormones were negative. At the ultrastructural level, the tumour cells were markedly oncocytic but having no membrane specializations (junctional complexes) consistent with follicle formation. Roncaroli et al. (28) suggested derivation of the tumour from folliculostellate cells.

We have also observed a primary pituitary tumour having histological and immunohistochemical characteristics of spindle cell oncocytoma (unpublished observation). However, at the ultrastructural level widespread follicle formation and multifocal endocrine differentiation was evident as well. These findings further support the neoplastic potential of folliculostellate cells.

Pituitary carcinoma can be diagnosed only when a pituitary neoplasm gives rise to distant, craniospinal, or, less frequently, extracranial metastasis (13, 29). Such tumours are extremely rare associated with dire prognosis. The majority of pituitary carcinomas produce either prolactin or ACTH. Other types, including those unassociated with signs of hormonal overproduction, are exceptionally rare. Pituitary carcinomas are not accompanied by specific histological features: enhanced mitotic activity, nuclear and cellular pleomorphism do not necessarily herald malignancy and vice versa, neoplasms with bland features might give rise to metastasis. Application of the proliferation marker, Ki-67 using the MIB-1 antibody is more useful; carcinomas display nuclear labelling consistently higher than adenomas (30). Immunoreactivities of pituitary carcinomas follow the pattern of the nonmalignant phenotype, although the degree of immunopositivity may be variably reduced. Relatively few cases have been investigated by electron microscopy revealing marked variability. In some carcinomas enough ultrastructural characteristics are retained to recognize the cell type, whereas other tumours have appearance of endocrine carcinoma of undetermined origin.