Ultrastructural diagnosis of human pituitary adenomas.

MICROSCOPY RESEARCH AND TECHNIQUE 20~107-135 (1992)

Ultrastructural Diagnosis of Human Pituitary Adenomas EVA HORVATH AND KALMAN KOVACS Department of Pathology, St. Michael's Hospital, University of Toronto, Toronto, Ontario M5B 1 W8, Canada

KEY WORDS

Electron microscopy, Endocrine neoplasms, Pituitary adenomas

ABSTRACT Electron microscopy, which has been instrumental in the characterization of normal pituitary cell types, has also played a crucial role in the morphologic classification of pituitary adenomas arising in the presently known 5 cell types, and in the recognition of 3 adenoma types with yet undisclosed cell derivation. This review deals with the application of electron microscopy for study of pituitary adenomas in order to provide specific pathological diagnosis and aid the clinician in selecting appropriate postoperative treatment. In addition to the ultrastructural appearance and diagnostic features of 15 adenoma types, the morphology of hyperplastic proliferations and that of known normal counterparts of various adenoma types are also discussed. Specific morphologic diagnosis of pituitary lesions is important not only for adequate postoperative management of patient, but is also a prerequisite for study of the natural history and biological behaviour of various adenoma types. INTRODUCTION Ultrastructural investigation played a crucial role in the morphologic classification of pituitary adenomas and resulted in the description of several distinct adenoma types (Foncin and LeBeau, 1963; Cardell and Knighton, 1966; Peake et al., 1969; Kovacs and Horvath, 1973; Landolt and Oswald, 1973; Robert, 1973; Horvath and Kovacs, 1974; Kovacs and Horvath, 1986a). Electron microscopy was also instrumental in revealing 3 well-differentiated adenoma types which did not have their derivation in any of the known adenohypophysial cell types (Kovacs et al., 1978a; Horvath et al., 1980, 1988a). Now, after having studied more than 3,000 surgically removed pituitary tumors, we do not anticipate to find more new types. We wish to stress the continuing importance of electron microscopy in this field for it can provide definitive diagnosis in many cases when immunohistochemistry gives inconclusive or, sometimes, misleading results. There is no doubt that immunohistochemistry is an invaluable asset in diagnostic pathology in general and in recognition of hormone producing tumors in particular (Kovacs et al., 1981; Heitz et al., 1987; Bishop et al., 1988). Yet immunohistochemistry of pituitary tumors should be assessed critically, keeping in mind that the immunohistochemical profile of several tumors may overlap. For instance, immunohistochemical findings may be indistinguishable in mixed GH cell-PRL cell adenoma and mammosomatotroph adenoma. The clinical course of the former tumor, however, is more aggressive. Immunoreactivities may be very similar in the null cell adenoma/oncocytoma group, in gonadotroph adenoma, and in the silent adenoma subtype-3. The silent adenomas can only be conclusively diagnosed by electron microscopy (Horvath et al., 1980,1988a).A special emphasis should be directed to the seemingly non-functioning adenomas of young individuals. In our experience patients under 30 years of age are not likely to have null cell adenoma and

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hormonally virtually inactive tumors represent in the younger age group one of the silent adenomas or gonadotroph adenoma. In such cases only ultrastructural investigation can provide definitive diagnosis and vital information concerning prognosis and postoperative treatment. The purpose of this review is to provide a compendium on the electron microscopic diagnosis of pituitary adenomas. Our classification of pituitary tumors and their frequency in surgical material is shown in Table 1. Fine structural features of non-neoplastic proliferation (hyperplasia) and of normal pituitary cell types will be also dealt with. GROWTH HORMONE PRODUCING PITUITARY LESIONS While causing the same clinical syndrome, acromegaly, or gigantism, growth hormone secreting adenomas are divided to two morphologically distinct types (Robert, 1973; Kovacs and Horvath, 1986a). The densely granulated tumor is acidophilic by histology and displays intense immunoreactivity for GH. This tumor often shows signs of plurihormonal differentiation exhibiting positive immunostaining mainly for a-subunit and TSH, less commonly FSH and LH (Scheithauer et al., 1986a). The sparsely granulated form is chromophobic by light microscopy. GH immunoreactivity is documented in varying proportions of adenoma cells and when present it is often noted only in the Golgi region. Only fine structural analysis reveals that the morphologic difference between the two forms far exceeds variations in granularity. Densely granulated GH cell adenoma consists of uniform, polyhedral, or elongate cells with predominantly

Received August 9, 1990;accepted in revised form September 7, 1990. Address reprint requests to Dr. E. Horvath, Department of Pathology, St. Michael's Hospital, 30 Bond St., Toronto, Ontario, M5B 1W8,Canada.

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TABLE I . Classification and frequency ofpituitary adenomas in unselected surgical material Adenoma type Densely granulated GH Sparsely granulated GH Densely granulated PRL Sparsely granulated PRL Mixed (GH cell-PRL cell) Mammosomatotroph Acidophil stem cell Corticotroph Thyrotroph Gonadotroph Silent “corticotroph” subtype 1 Silent “corticotroph”subtype 2 Silent subtype 3 Null cell Oncocytoma Unclassified plurihormonal

Frequency (9%) 6.85 6.44 0.48 26.85 4.04 1.44 1.98 10.14 0.96 9.04 1.50 2.26 1.37 14.11 11.44 1.10

spherical or ovoid, centrally placed nuclei (“rouillas et al., 1980; Kanie et al., 1983; Kovacs and Horvath 1986a,b; Scheithauer et al., 1986b; Saeger et al., 1987). The RER is usually well developed, forming parallel cisternae at the periphery of cells. The prominent globoid Golgi apparatus possesses moderately dilated sacculi and harbors immature secretory granules. The mature cytoplasmic storage granules, in general, are numerous (Fig. 1).They are likely to be spherical and evenly electron dense with tightly fitted limiting membrane, and their size encompass a wider range than in any other adenoma types. Most commonly they range between 150 and 600 nm, the majority being 350-500 nm. Mitochondria show regular features and extensive oncocytic change is seldom noted. Morphologicvariations are fairly common, especially in regard to granularity. In several tumors varying numbers of adenoma cells may harbor secretory granules ranging up to 1,500 nm (Kovacs et al., 1979). In these unusually large granule cells the morphology of secretory granules is also different: among spherical ones ovoid and irregular forms occur. Some of these large granule cells may also harbor secretory granules with ruffled limiting membrane and mottled low density core and may engage in granule extrusions-fine structural signs of mammosomatroph differentiation (Horvath et al., 1983b). An unusual feature in some densely granulated GH cell adenomas (and rarely found even in normal GH cells) is the crystallization of secretory material starting mostly within the Golgi sacculi or occasionally within the RER (Fig. 2) (Horvath et al., 198313). As a result, secretory granules with geometric shapes and odd proportions are seen, signifying growth in privileged directions. This anomaly can be considered a specific marker for densely granulated GH producing cells. Although it is rarely noticeable by histology, production of endocrine amyloid is not a rarity in all variants of GH secreting tumors (Fig. 3) (Mori et al., 1985; Saitoh et al., 1985; Landolt et al., 1987a). The fibrillar

amyloid is usually noted as asteroid clusters in lumina formed by adenoma cells. The substance is elaborated and discharged by the tumor cells, although the presence of amyloid fibers within the cytoplasm has not been convincingly demonstrated. It appears that the fibrillar protein exists in soluble form within the cells, whereas the extracellular milieu favors its polymerization. Sparsely granulated GH cell adenoma consists of cells which often show considerable variations in shape and size (Robert, 1973; Kovacs and Horvath, 1986a,b; Scheithauer et al., 1986b). Spherulation and thus loss of cohesion between cells is commonly seen. The nucleus is characteristically eccentric and flattened or crescent shaped. Markedly pleomorphic nuclei and multinucleated cells are frequent. The prominence of RER and Golgi membranes varies considerably from one case to another. In some tumors the RER may be abundant and well organized with Nebenkern formation but mostly it is well or moderately developed and is present in the form of randomly scattered profiles. The secretory granules may be extremely scanty and usually measure less than 250 nm. The most recognizable structure and the marker of the tumor type is the “fibrous body” (Cardell and Knighton, 19661, consistently located in the Golgi region, adjacent to the concave or flattened side of the nucleus (Fig. 4). The spherical body consists of varying proportions of type-2 filaments which appear to represent a class of cytokeratin, and of tubular SER (Horvath and Kovacs, 1978; Neumann et al., 1985). SER also is present in the immediate vicinity of the fibrous body but not in other parts of the cytoplasm. The fibrous body displaces or engulfs the Golgi sacculi and traps other cytoplasmic constituents such as secretory granules, mitochondria, and lysosomes within the filamentous mass. Centrioles are often seen within or adjacent to fibrous bodies. It is of note that centrioles are frequently supernumerary which is another marker of this tumor type. A fairly common although poorly understood alteration occurring in all morphologic types of GH producing adenomas is the presence of tubulo-reticular aggregates in the capillary endothelium (Landolt et al., 1976). Two rare variants of sparsely granulated GH cell adenoma are known. One is the clinically silent form, unassociated with acromegaly and elevated GH blood levels (Kovacs et al., 1989). In these rare cases the adenoma has typical ultrastructure. As attested by in situ hybridization, the GH gene is expressed in a varying number on cells, but tissue immunoreactivity for GH is slight or minimal if detectable at al. As possible reasons for the abnormality, lack of translation or defects in posttranslational processing of gene products are suggested. The other unusual lesion is the association of hypothalamic gangliocytoma or neuronal choristoma and sparsely granulated GH cell adenoma (Fig. 5) (Asa et al., 1984; Kame1 et al., 1989; Li et al., 1989).The histogenesis of this composite lesion and the possible role of neural component in the inductiodpromotion of the GH cell adenoma are for conjecture at present. Effect of medical treatment on the ultrastructure of

ULTRASTRUCTURAL PATHOLOGY OF THE PITUITARY

Fig. 1. Densely granulated GH cell adenoma. Among spherical secretory granules there are elongate or odd-shapedforms (arrows). x 8,850. Fig. 2. Densely granulated GH cell adenoma. Dilated RER cisternae show crystallization of their content (arrows). x 26,850.

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Fig. 3. Densely granulated adenomatous GH cell producing endocrine amyloid. The fibrillar substance of originates at the base of indentations of plasma membrane (arrow). At these sites the cell membrane appears discontinuous, but no fibrils are seen in the cytoplasm. x 17,750.

Fig. 4. Sparsely granulated GH cell adenoma. Note the flattened or crescent-shaped nuclei, fibrous bodies consisting of type-2 Blaments and tubular SER (arrowheads), and the tiny (50-150 nm) secretory granules (arrow). x 6,000.

Fig. 5. Composite lesion of sparsely granulated GH adenoma cells (asterisks),cells resembling secretory neurons (N),and of neuropil. x 3,100.


GH adenomas has been insufficiently studied. The dopaminergic agonist bromocriptine and related drugs may achieve often temporary clinical and biochemical improvement, but they do not seem to result in consistent morphologic changes in the adenoma cells. In some cases increased lysosomal activity is noted. It is unclear whether or not this is related to treatment. In our material there is only one tumor with excessive lysosomal accumulation removed from a patient treated with bromocriptine. Presently a long-acting somatostatin analogue (Sandostatin, SMS 201-995) is used in the medical treatment of acromegaly (George et al., 1987; Landolt et al., 198713;Barkan et al., 1988; Beckers et al., 1988b).Morphologic findings in GH cell adenomas exposed to somatostatin analogue range from no change to lysosoma1 accumulation with crinophagy, oncocytic change, perivascular fibrosis, and, in a minority of cases, reduction of cell size and increase in size and number of secretory granules. Marked alterations in nuclear morphology and chromatin pattern, suggestive of drug effect at the transcription level, are not reported in cases of somatostatin analogue administration. GH cell hyperplasia is a rare condition. In all known cases, the hyperplasia was associated either with McCune-Albright syndrome (Kovacs et al., 1984) or it was secondary to extrahypothalamic production of growth hormone releasing hormone (GRH) (Thorner et al., 1982,1984; Horvath, 1988). In the few examples, studied by electron microscopy, the GH cell hyperplasia is a diffuse lesion consisting of densely granulated cells with unusually prominent Golgi complex (Fig. 6). Data are limited on the fine structural morphology of the non-adenomatous adenohypophysis surrounding a GH cell adenoma, since normal tissue is rarely included in the EM specimen. In our experience the extra tumoral GH cells display essentially normal features. If there are subtle quantitative differences in the amount of RER and Golgi membranes, morphometry of several cases would be required to document it. The normal counterpart of GH cell adenomas is assumed to be the familiar ovoid cell with spherical nucleus, lamellar, most often peripherally located RER, globoid Golgi apparatus, and numerous, evenly dense, mostly spherical secretory granules in the range of 150-600 nm (Fig. 7) (Horvath and Kovacs, 1988).However, several facts suggest that the truth may not be this simple. In normal adenohypophysis many GH cells possess secretory granules measuring up to 800 nm or more. Varying proportions of these secretory granules have different morphology being elongate or pleomorphic (Fig. 8). Uncommonly, some of these cells may display granule extrusions signifying mammosomatotroph differentiation. By immunohistochemistry and immunoelectron microscopy some GH producing cells contain PRL or a-subunit as well (Horvath and Kovacs, 1988). The existence of a small granule (150 nm) GH cell is also proven (Horvath and Kovacs, 1988). In situ hybridization also detects several cells expressing both GH and PRL genes (Lloyd et al., 1989). The two principal forms of GH cell adenoma vary in biological behaviour, the sparsely granulated type being more ag-

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gressive. Their immunohistochemical profiles differ as well: densely granulated tumors are often immunoreactive for a-subunit, PRL, TSH, and sometimes FSH and LH (Scheithauer et al., 1986a), whereas sparsely granulated adenomas are more likely to be monohormonal, sometimes with light, scattered PRL or a-subunit immunoreactivity. It should also be noted, that large-granule mammosomatotrophs and crystallization of secretory material are seen only in densely granulated GH adenomas, whereas clinical silence and the gangliocytic variant is associated only with sparsely granulated tumors. All these findings support the view that the GH cell population is not homogeneous and the parent cells of two GH cell adenomas belong to different subsets.

PROLACTIN PRODUCING PITUITARY LESIONS The overwhelming majority of PRL-producing tumors belong to the sparsely granulated variety, whereas the densely granulated form is very rare (Kovacs and Horvath, 1986a). By histology, the sparsely granulated tumors are chiefly chromophobic, the densely granulated ones acidophilic. PRL cell adenomas are predominantly monohormonal containing only immunoreactive PRL. Significant immunoreactivity for other hormones (usually a-subunit) is uncommon. The uniform morphology of these tumors does not reflect the great variations in biologic behavior seen clinically. By electron microscopy, cells of sparsely granulated PRL cell adenoma have the striking appearance of hormonally active PRL cells (Fig. 9) (Horvath and Kovacs, 1974,1986; Robert and Hardy, 1975; Kovacs and Horvath, 1986a). The nucleus is often irregular but predominantly euchromatic with large, dense nucleolus. At one side of the nucleus, the cytoplasm harbors masses of RER in forms of parallel cisternae or concentric whirls (Nebenkern). The other half of the cytoplasm is dominated by the large, prominent Golgi apparatus holding several immature secretory granules. In hormonally active adenoma cells the majority of secretory granules are located within the Golgi area, whereas the mature granules are rapidly released. Among spherical or ovoid immature secretory granules there are always pleomorphic forms, a characteristic feature. The cytoplasmic storage granules are likely to be chiefly spherical measuring up to 300 nm in most cases. They consistently engage in granule extrusion, which is the specific morphologic marker of prolactin production in the human pituitary. Granule extrusions may occur at the basal portion of cell facing the perivascular space (orthotopic exocytosis),or at the lateral cell surfaces far from the basement membrane (“misplaced exocytosis”) (Horvath and Kovacs, 1974). PRL cell adenomas have no significant variations from one case to another. A few especially aggressive tumors may consist of smaller, less developed cells, but granule extrusions are invariably present. The rare densely granulated variant (Kovacs and Horvath, 1986a) consists of middle-sized, elongate, or polyhedral cells, in which the RER and Golgi membranes are less prominent than in the sparsely granu-

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Fig. 6. GH cell hyperplasia. The enlarged acini are populated chiefly by heavily granulated GH cells with extremely prominent Golgi regions (arrows). x 2,000.

Figs. 7, 8. Morphologic variants of normal GH cells. The extremely elongate, sometimes spindle-shapedsecretory granules, seen in Fig. 8, are uncommon in the normal gland. x 6,600.

Fig. 9. Sparsely granulated PRL cell adenoma endowed with typical features: abundant RER, prominent Golgi apparatus, and misplaced exocytosis (arrow). X 9,300. Fig. 10. Densely granulated PRL cell adenoma. Extruded secretory granules are difficult to observe within tightly fit membraneous pits (arrows). x 6,800.

Fig. 11. Sparsely granulated PRL cell adenoma. Markedly distended ER profiles are filled with fibrillar-tubular endocrine amyloid within the cytoplasm of an adenoma cell. x 19,900.

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lated form (Fig. 10). The secretory granules, however, are much larger (up to 600-700 nm) and more numerous. Except for granule extrusions and sometimes uneven density of some secretory granules, this tumor has a fine structural appearance rather similar to that of densely granulated GH cell adenoma. Both types of PRL cell adenoma may exhibit varying degrees of calcifications (Landolt and Rothenbuhler, 1977; Rilliet et al., 1981) and may produce endocrine amyloid (Bilbao et al., 1975; Horvath and Kovacs, 1986; Kovacs and Horvath, 1986a; Landolt et al., 1987a). The extent of amyloid deposition ranges from occasional cells containing intracellular aggregates bound within dilated RER profiles, to formation of large extracellular globoid masses, dominating the appearance of the tumor. In most cases, the amyloid is detectable in the form of intracellular membrane bound fibrillar-tubular aggregates (Fig. 11).Cell death renders the amyloid substance extracellular, where large globoid masses are formed. The fibrillary amyloid forming extracellular asteroid masses in GH cell adenomas is uncommon in PRL-producing tumors. The introduction of bromocriptine and other dopaminergic agonists into the medical management of hyperprolactinemia opened a new chapter also in the pathology of PRL-producing adenomas (Tindall et al., 1982; Bassetti et al., 1984; Horvath and Kovacs, 1986; Kovacs and Horvath, 1986a; Horvath et al., 1988b). As opposed to the uniform morphology of untreated tumors, PRL cell adenomas exposed to dopamine agonists display a great variety of appearances well reflecting the wide-ranging effects seen clinically (Thorner et al., 1980,1981; Horvath et al., 1988b). Adenomas responding with significant size reduction and marked fall in serum PRL levels become extremely cellular owing to the sharp decrease of cell size. Some tumors display more moderate or uneven response, and a few tumors appear to be unresponsive to dopamine agonists. In responsive tumors, the PRL immunoreactivity is also reduced. In extreme cases, especially following treatment with the long-acting form of drug (Parlodel-LAR), neither PRL immunoreactivity nor PRL gene expression (by in situ hybridization) is detectable (Kovacs et al., 1991). Electron microscopy documents maximal functional suppression in highly responsive PRL cell adenomas: an irregular, markedly heterochromatic nucleus and small cytoplasm harboring only a few small RER profiles, few mitochondria, and small secretory granules which still engage in exocytosis (Fig. 12) (Tindall et al., 1982; Horvath and Kovacs, 1986; Horvath et al., 1988a). Owing to its involution, the Golgi apparatus is rarely encountered. Indeed, such cells answer the description of null cells and if no actual granule extrusion is present, they cannot be identified as PRL cells. As attested by the lack of gene expression and immunoreactivity, in such cases the dopamin agonist acts at the level of transcription (Kovacs et al., 1991). In several tumors less profound inhibition is seen with more moderate loss of cytoplasmic RER and Golgi membranes. In yet another relatively small group of tumors, the adenoma cells exhibit an appearance typical of untreated tumors, but display a marked increase in

lysosomal activity and crinophagy. In such cells the dopaminergic agonist exerts its effect through inhibition of hormone release. Finally, tumors consisting of multiple cell populations showing varying responses to dopamine agonists also occur. It is of note that prolonged treatment with a dopamine agonist may result in cellular injury and varying degrees of perivascular and interstitial fibrosis (Landolt and Osterwalder, 1984; Esiri et al., 1986; Hallenga et al., 1988). Following interruption of dopaminergic agonist medication, thus removal of inhibition, the original morphology of tumor cells should be restituted. It is of considerable practical importance that this does not always take place to a full extent (Horvath et al., 198813). Tumors removed from patients with a history of remote dopaminergic agonist treatment may contain varying numbers of cells displaying signs of functional suppression. Such cells may be scattered throughout the tumor or may form a solitary focus. The wideranging responses of PRL adenoma cells to institution or removal of dopaminergic treatment render the ultrastructure of treated tumors practically unpredictable, which always should be considered by the morphologist. PRL cell hyperplasia as a solitary pathologic lesion, causing hyperprolactinemia and sequelae, is extremely rare-we have seen only two such cases. The non-neoplastic proliferation of PRL cells may occasionally be associated with PRL cell adenoma and it is seen in some cases of corticotroph cell adenomas (Fig. 13) and thyrotroph cell hyperplasia due to hypothyroidism. By electron microscopy, hypertrophy as well as a numerical increase of the markedly stimulated PRL cells are seen. The lesion is never monomorphic; other cell types are intermingled. PRL cells in the non-tumorous gland surrounding PRL cell adenoma usually display signs of profound suppression (Horvath and Kovacs, 1986, 1988b; Jalalah et al., 1988). The small cells have a heterochromatic nucleus, poorly developed membraneous organelles, often lysosomes, and small (100-200 nm) sparse secretory granules. If granule extrusion is not present, such cells are difficult to identify as PRL cells. In some glands the ultrastructure of periadenomatous PRL cells is less uniform and cells retaining features of PRL differentiation are also encountered. The normal counterpart of the PRL cell adenoma is a cell with many appearances (Horvath and Kovacs, 1988). The normal PRL cell is a rather small, polyhedral cell with fairly well-developed membraneous organelles and sparse, spherical and pleomorphic, small (up to 300 nm) secretory granules which may be engaged in granule extrusions (Fig. 14). Under the influence of stimulation or inhibition the cells undergo profound morphologic changes (Fig. 15). Save for exocytosis, the suppressed and stimulated PRL cells have little in common. The densely granulated PRL cell is uncommon in the non-tumorous gland. It is not known whether it represents a morphologic variant reflecting differences in rates of hormone synthesis, release, and storage or, alternatively, represents a different phenotype.

Fig. 12. Effect of bromocriptine on sparsely granulated PRL cell adenoma is shown. As compared t o untreated tumors (see Fig. 9) the nuclei are heterochromatic, the small cytoplasm contains scanty organelles, and the tiny granule extrusion pits are barely visible (arrow). x 23,700.

Fig. 13. PRL cell hyperplasia in non-adenomatous portion of pituitary harboring corticotroph cell adenoma. x 6,250.

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Fig. 14. Normal PRL cell (asterisk) in the adenohypophysis of a patient with no endocrinopathy. X 7,850.

small cytoplasmic process, probably of the same PRL cell, displays sign of granule extrusion (arrow). x 7,000.

Fig. 15. Suppressed PRL cell (asterisk) in the non-tumorous portion of adenohypophysis harboring PRL producing adenoma. The

Fig. 16. Densely granulated GH cells and sparsely granulated PRL cells alternate in a mixed GH-PRL cell adenoma. Note granule extrusion in a PRL cell (arrow). X 5,150.


ADENOMAS PRODUCING GH AND PRL The mixed GH cell-PRL cell adenoma is a well-established cause of acromegaly and hyperprolactinemia (Guyda et al., 1973; Corenblum et al., 1976; Bassetti et al., 1986, 1988; Kovacs and Horvath, 1986a; Zurschmiede and Landolt, 1987). It is a bimorphous tumor type consisting chiefly of densely granulated GH cells and sparsely granulated PRL cells. Other combinations are rare. As attested by the varying patterns of GH and PRL immunoreactivities, the two cell types may be relatively evenly intermingled or they may exhibit uneven, almost clonal distribution showing predominance of either GH or PRL cells. As documented by immunoelectron microscopy, bihormonal mammosomatotrophs may also be present. Electron microscopy most often reveals small groups of densely granulated GH cells and sparsely granulated PRL cells with typical features already described (Fig. 16). There are tumors, especially in young individuals, which include also less typical cells. As demonstrated by immunoelectron microscopy, such adenomas are likely to contain bihormonal cells. Special features characterizing GH or PRL cell adenomas, such as endocrine amyloid, calcification, exocytosis and, less frequently, fibrous bodies, are documented in mixed adenomas. Mammosomatotroph adenoma is a well-differentiated, usually slowly growing tumor associated with acromegaly and mild or no overt hyperprolactinemia (Horvath et al., 1983b; Beckers et al., 1988a). By immunohistochemistry, the strong positivity for GH is coupled with scattered, less intense PRL immunoreactivity. Immunoelectron microscopy reveals bihormonal cells containing specific labeling for both hormones within the same cell and even within the same secretory granule. Ultrastructural study documents monomorphous tumors, the majority of which appear similar to densely granulated GH cell adenoma. The secretory granules, however, are usually very large, often ranging up to 1,000 nm, the majority being 600 nm or more. A substantial percentage of secretory granules is ovoid or pleomorphic. The markers of the tumor type are the large secretory granules with asymmetrical, low density mottled cores and often extrusion of secretory granules (Fig. 17). As opposed to the rapid dissolution of secretory granules in the extracellular space in PRL cell adenomas, extruded secretory material in mammosomatotroph tumors appears to be more resistant. It is of note that geometrically shaped secretory granules are fairly common in this adenoma group. Fibrous bodies may also occur. There is a small group of mammosomatotroph adenoma, occurring in children or adolescents, which has less well-defined ultrastructure. These adenomas comprise cells with less and smaller secretory granules usually in the range of 100-300 nm and numerous, but small, exocytoses. Notwithstanding their monomorphous appearance, the cells of these adenomas contain varying mixtures of GH and PRL, as documented by immunoelectron microscopy (Felix et al., 1986). The acidophilic stem cell adenoma is a rare mono-

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morphous bihormonal tumor type (Horvath et al., 1981; Kovacs and Horvath, 1986a). It produces predominantly PRL and GH is usually expressed as scanty immunoreactivity. Elevation of serum GH level and clinical acromegaly are rarely associated with this adenoma. In the majority of cases the ultrastructure of acidophilic stem cell adenoma is highly distinctive. In most cases it is dominated by the diffuse, advanced oncocytic change coupled with conspicuous mitochondria1 gigantism, which does not occur in any other pituitary tumor type (Fig. 18). The giant mitochondria, some of which may reach the size of the nucleus, are filled with fine granular substance and have no cristae. They always retain their double limiting membranes. Electron dense tubular structures of unknown origin and significance frequently occur within these altered mitochondria. The oncocytic transformation also obscures the RER and Golgi membranes and the small (up to 250 nm) secretory granules are often confined to the cell periphery. Granule extrusions, the hallmarks of PRL differentiation, andlor aggregates of SER, which are markers of sparsely granulated GH cell adenomas, also occur. In a few acidophilic stem cell adenomas the association of exocytoses and fibrous bodies is combined with relatively poorly developed RER and Golgi complex but no oncocytic change. No sufficient data are available on the morphology of various cell types in the non-adenomatous gland adjacent to the 3 bihormonal tumors discussed above. The normal counterparts of mixed adenoma, mammosomatotroph, and acidophilic stem cell adenoma have yet to be clarified. It is conceivable that plurihormonal subsets within the GH and, in case of acidophil stem cell adenoma, PRL cell populations give rise to these neoplasms. Alternatively, plurihormonal differentiation during tumor progression could also be entertained.

THE PATHOLOGY OF PITUITARY DEPENDENT CUSHING’S DISEASE The morphologic diagnosis of Cushing’s disease often poses a great challenge to the pathologist. While the overwhelming majority of other pituitary hypersecretory syndromes are caused by well-defined solitary adenoma, Cushing’s disease may be induced by either autonomous adenoma or non-neoplastic proliferation of corticotrophs-that is, hyperplasia and even, a combination of neoplastic and hyperplastic lesions (Kovacs and Horvath, 1986a; Lloyd et al., 1986; Robert and Hardy, 1986; Horvath, 1988). The most common pathological change is a basophilic adenoma. These tumors often measure only a few millimeters in diameter, not causing alteration of sellar configuration. If the adenoma is not clearly identified or is poorly demarcated or hyperplasia is suspected, the surgeon may perform resection of the median wedge, the most common site of corticotroph tumors, hemihypophysectomy, or even total hypophysectomy.Because of difficulties in diagnosis of corticotroph lesions, it is of utmost importance to preserve and process every bit of tissue removed at surgery, since serial sectioning of specimens is often necessary.

Fig. 17. Mammosomatotroph adenoma with ultrastructural appearance similar to that of densely granulated GH cell adenoma, but exocytoses are also present. Note funnelling of secretory material into the extracellular space (arrow). x 16,250.

Fig. 18. Acidophilic stem cell adenoma. The oncocytic tumor shows mitochondria1 gigantism (M), tubular structures in mitochondria (arrow), some SER (arrowhead), and granule extrusions (circles). x 8,500.


The most common corticotroph microadenomas are usually intensely basophilic, PAS-positive tumors displaying immunoreactivity for ACTH and other proopiomelanocortin derived peptides (Saeger, 1978; McNicol, 1981; Charpin et al., 1982; Kovacs and Horvath, 1986a; Robert and Hardy, 1986). The fine structural diagnosis is easy and reliable. The well-vascularized adenomas consist of middle-sized, elongate angular cells. The ovoid or slightly irregular nuclei contain small to moderate amounts of heterochromatin and a prominent nucleolus situated close to the inner nuclear membrane. The randomly arranged RER is often slightly, unevenly dilated; the spherical Golgi apparatus harbors immature granules. The secretory granules are usually numerous. In less granulated cells they are confined to the cell periphery. The secretory granules possess characteristic morphology being spherical as well as dented, drop-shaped, and heartshaped with differing electronopacity. They measure 150-450 nm, most commonly 300-350 nm. The most important diagnostic markers are a few bundles of type-1 filaments located chiefly in the perinuclear region (Fig. 19). Since excessive accumulation of type-l filaments (Crooke’s hyalinization) signifies functional suppression, according to widely accepted views, it should not occur in adenomas. Nevertheless, corticotroph adenomas consisting mainly or exclusively of cells showing massive Crooke’s hyalinization do exist (Felix et al., 1982; Horvath et al., 1983a; Kovacs and Horvath, 1986a). These neoplasms are characterized by the ringlike accumulation of type-1 filaments representing cytokeratin (Neumann et al., 1984), which occupy large areas of the cytoplasm displacing organelles and secretory granules to the cell periphery (Fig. 20).Secretory granules are also trapped in the Golgi region. Apart from Crooke’s hyalinization, this group of tumors is not uniform, displaying considerable variations especially in their cellularity. The heterogeneity of these tumors is even more obvious clinically; they may cause florid Cushing’s disease or may show very low hormonal activity. A rare variant of corticotroph adenoma is the chromophobic form with scanty immunoreactivity for ACTH (Kovacs and Horvath, 1986a).As opposed to the basophilic microadenomas inducing florid clinical syndrome, the chromophobic tumors are usually macroadenomas when discovered and are associated with a more subtle form of hypercorticism. By electron microscopy, sparsely granulated tumors with uncharacteristic features are seen. Only a minority of cells show corticotroph differentiation, including characteristic secretory granule morphology and the presence of type-1 filaments. The non-tumorous adenohypophysis in pituitary Cushing‘s disease should be subjected to careful morphologic analysis for it may provide essential information. The optimal scenario is the presence of an autonomous adenoma and functional suppression of corticotrophs outside the tumor. The negative feedback effect of circulating glucocorticoids is expressed in a unique form in the human corticotroph in what is known as Crooke’s hyalinization (Horvath and Kovacs,

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1988; Halliday et al., 1990). Crooke’s cells are often large, spherical cells containing masses of type-1 filaments and often a large lysosomal body, but very few RER and Golgi membranes. Secretory granules are present only in the Golgi region and a t the cell periphery. In advanced stage of Crooke’s hyalinization an unusual ultrastructural feature is the virtual absence of the cell membrane. Since all the other cell constituents appear well preserved and viable, the loss of plasmalemma must be an artefact. One can speculate that the filamentous mass may take up fluid during fixation leading to expansion of cytoplasm and rupture of cell membrane. Then, during dehydration, it retracts again, leaving behind an electron lucent rim containing scattered secretory granules and broken membranes, as often observed. If the specimen consists solely of non-tumorous adenohypophysis and the corticotrophs exhibit Crooke’s hyalinization without change in frequency and distribution, it might mean that a corticotroph adenoma was removed but lost during surgery or tissue processing, that a corticotroph adenoma was left behind, or that the Crooke’s change developed due to extra-pituitary causes (ectopic ACTH-syndrome, iatrogenic origin). Corticotroph hyperplasia is uncommon, but it does occur and its diagnosis may be extremely difficult (Lamberts et al., 1980; Schnall et al., 1980; McNicol, 1981; McKeever et al., 1982; Saeger and Ludecke, 1983; Kovacs and Horvath, 1986a; Lloyd et al., 1986; Horvath, 1988; Young et al., 1988). It requires a sufficiently large, well-preserved specimen and considerable experience on the part of the morphologist. Serial sectioning is often necessary. Tissues are usually fragmented and only the immunohistochemical pattern by light microscopy and the frequency and cytology of cells by electron microscopy suggest the anatomical site of biopsy. Since normally most of the corticotrophs reside within the median wedge, their frequency has to be related to the site at which they are observed. Pituitary hyperplasias may be diffuse or nodular. In cases of Cushing’s disease, the two types usually occur together. The diffuse component is represented by an increase in number of large corticotrophs displaying Crooke’s hyalinization, without major distortion of acinar architecture. Enlarged acini, populated predominantly by uniform, relatively small corticotrophs with no advanced Crooke’s change, account for the nodular hyperplasia. Occasionally adenoma and diffuse and nodular hyperplasia are simultaneously present. In such cases removal of the adenoma will not result in the cure of Cushing’s disease, if hyperplastic foci are left behind. It is of note that corticotroph adenoma and even hyperplasia may be accompanied by hyperplasia of PRL cells (Horvath, 1988; Wowra and Peiffer, 1984). This phenomenon is not rare, but it is not a consistent concomitant of Cushing’s disease either. Since nontumorous tissue is not always available or the sample is small, neither the incidence nor the extent of PRL cell hyperplasia can be assessed. The normal counterpart of corticotroph lesions is the ovoid, deeply basophilic, and PAS-positive corticotroph. By electron microscopy the cell is character-

Fig. 19. Corticotroph cell adenoma with typical features: angular, densely granulated cells with fairly well-developed membraneous organelles and bundles of type-1 filaments (arrows). Note characteristic morphology of secretory granules. x 9,000.

Fig. 20. Corticotroph cell adenoma consisting of cells displaying extensive accumulation of type-1 filaments (Crooke’s hyalinization). The RER and Golgi membranes are obscured, and the secretory granules are displaced by the filamentous mass. x 5,900.


ized by the distinct morphology of its secretory granules, the presence of type-1 filaments (Fig. 21) and a large, heterogenous lysosomal body (“enigmatic body”) (Horvath and Kovacs, 1988). No particular appearance for the stimulated corticotroph is known. Crooke’s hyaline change of the functionally suppressed corticotroph has been described above (Fig. 22). It should be added that individual differences in the degree of hyalinization are considerable. Crooke’s hyalinization also appears to be reversible.

PITUITARY LESIONS CONSISTING OF THYROTROPH CELLS Thyrotroph tumors comprise the least frequent group among pituitary adenomas, yet they are not free of controversies. Thyrotroph adenomas may cause hyperthyroidism, may develop in patients with longstanding hypothyroidism, or may also occur in clinically euthyroid subjects unassociated with elevated serum TSH levels (Afrasiabi et al., 1979; Kovacs and Horvath, 1986a). There is also considerable morphologic variability among thyrotroph tumors, especially in terms of immunoreactivity and ultrastructure. The difference, however, does not correlate with clinical presentation and serum TSH levels, the reason for which is poorly understood. By histology, thyrotroph adenomas are chromophobic or may rarely exhibit varying degrees of basophilia. The extent of TSH immunoreactivity varies from strong generalized immunopositivity to scattered positive cells or even negativity, regardless of clinical correlates. Positivity for a-subunits is variable. It is of note that a varying number of cells usually exhibit GH immunoreactivity. Electron microscopy documents in the majority of cases well-differentiated adenomas consisting of moderately polar, often angular cells (Fig. 23) (Saeger and Ludecke, 1982; Girod et al., 1986; Kovacs and Horvath, 1986a). The nuclei may be uniform, although focal nuclear pleomorphism is not uncommon in thyrotroph tumors. The RER is abundant, displaying mild to moderate, fairly even dilatation. The Golgi apparatus is prominent with several immature granules. Occasionally SER and aggregates of intermediate filaments may also be noted. In most tumors the secretory granules are sparse and form a single row under the plasmalemma, marking the outlines of cells. They usually measure up to 250 nm. Cells containing larger (up to 400 nm) secretory granules tend to be more densely granulated. In some areas the cytoplasmic organization is much simpler, resembling that of null cell adenomas despite obvious hormonal activity (Katz et al., 1980; Saeger and Ludecke, 1982; Kovacs and Horvath, 1986a). Such apparent contradiction may be caused by the vagaries of sampling. As also shown by patterns of often patchy immunostainings, in glycoprotein hormone producing adenomas strongly positive areas may alternate with regions devoid of immunoreactivity, indicating differences in functional differentiation or activity. This finding underlines the importance of studying several tissue blocks. Cytological features of the non-tumorous gland

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around thyrotroph adenoma are not known. These tumors are usually macroadenomas a t the time of surgical intervention, diminishing the chances of receiving periadenomatous pituitary tissue with the specimen. Thyrotroph hyperplasia, developing in patients with long-standing hypothyroidism and leading to pituitary enlargement, is a well-defined clinical entity. This condition is perceived as fully reversible by appropriate replacement therapy and therefore thyrotroph hyperplasia is not likely to occur in surgical material. However, in some cases the hyperplastic lesion mimics prolactin producing tumor. In others the pituitary enlargement is not amenable to replacement therapy, creating the impression of true neoplasm. Surgical biopsy of such cases made possible the histologic and ultrastructural analysis of thyrotroph hyperplasia (Khalil et al., 1984; Pioro et al., 1988; Horvath, 1988). These lesions consist of large ovoid or rounded cells populating massively enlarged acini. The hyperplastic thyrotrophs have the description of so-called thyroid deficiency cells possessing spherical euchromatic nucleus, abundant, evenly dilated RER, and large, spherical Golgi complex (Fig. 24). The dilatation of RER is progressive and its degree is the same within individual cells. The granularity of cells shows inverse correlation with the dilatation of RER. The small (up to 250 nm) secretory granules are very sparse in the most advanced phase of RER dilatation. The overactivity of PRL cells and even PRL cell hyperplasia may also be associated with the lesion. The normal counterpart of thyrotroph lesions is a middle-sized or larger, characteristically angular cell with a spherical nucleus, moderately to well developed, slightly dilated RER, and ring-like Golgi apparatus with numerous vesicles (Fig. 25) (Horvath and Kovacs, 1988).The secretory granules measure chiefly around 200 nm; their number greatly varies. It is of note that some cells having the description of thyrotrophs possess spherical secretory granules, whereas others contain rod-shaped and drop-shaped granules sometimes with loosely fitting limiting membrane. The reason for such morphologic variations, which occur in other species as well, is not known. The normal pituitary also contains small granule cells with poorly developed membraneous organelles. Although such cells may show some resemblence to thyrotrophs, they cannot be positively identified without immunoelectron microsCOPY. The morphology of the stimulated thyrotroph is discussed above. The fine structure of the suppressed thyrotroph (in Graves’ disease) has not been investigated to date.

GONADOTROPH ADENOMAS Although these tumors were extensively studied in the 1980s, the clinical and morphological diagnostic criteria of gonadotroph adenomas are still not established (Trouillas et al., 1981, 1986; Nicolis et al., 1982, 1988; Beckers et al., 1985; Snyder, 1985). Although in men serum gonadotrophin levels are often elevated, some tumors are clinically silent. In women, most of the tumors are, being associated with FSH/LH levels normal for the patient’s age. Morphologic features of

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Fig. 21. Normal corticotroph cell. Note type-1 filaments in the perinuclear and Golgi areas (arrows). x 7,750.

Fig. 22. Suppressed corticotrophs (Crooke’s cells) in adenohypophysis harboring corticotroph adenoma. Note massive, ring-like accumulation of type-1 filaments displacing secretory granules. x 7,100.


Fig. 23. Thyrotroph cell adenoma associated with hyperthyroidism. Note preferred localization of secretory granules along the plasma lemma. x 8,900.

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Fig. 24. Thyrotroph hyperplasia. The large, sparsely granulated cells possess abundant, dilated RER with a low-density content, extensively developed Golgi apparatus, and large, heterogenous lysosomes. x 4.750.

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Fig. 25. Normal thyrotroph cells. Note conspicuous lysosomal bodies (arrows). x 10,000.

Fig. 26. Gonadotroph adenoma (male type) comprised of elongate, polar cells. Note uneven distribution of secretory granules (upper right corner). x 3,350.


gonadotroph tumors are also variable and no correlation exists between the clinical presentation and histology, immunoreactivity, and ultrastructure of gonadotroph adenomas. By histology, gonadotroph tumors are chromophobic or, in cases of oncocytic change, display varying degrees of acidophilia (Horvath and Kovacs, 1984; Kovacs and Horvath, 1986a). The histologic pattern in non-oncocytic tumors is diffuse or sinusoidal with a pseudorosette formation around vessels. Well-differentiated tumors may contain papillary areas. Oncocytic adenomas always show a diffuse pattern. Tumors of men usually exhibit considerable, although often patchy positivity for FSH and LH. Immunoreactivity for a-subunit may or may not be present. In the majority of gonadotroph tumors of women the immunopositivity for FSH, LH, and a-subunit is scanty or even missing. Intense, diffuse immunostaining is rarely seen but it is always present in the rare densely granulated variant. Electron microscopic study of gonadotroph adenomas unveiled the unique phenomenon of sex-linked ultrastructural dichotomy (Kovacs et al., 1978b, 1980a; Horvath and Kovacs, 1984; Murray et al., 1985; Kovacs and Horvath, 1986a). Tumors in males show a considerable variability regarding the degree of functional differentiation. About 50% of these adenomas consists of moderately polar cells with spherical nuclei and well-developed RER (Fig. 26, 27). The prominent Golgi complex displays regular features. The uneven distribution of the small (up to 200-250 nm) secretory granules is readily apparent; the majority of granules accumulates in the cell processes, whereas the nuclear pole of cytoplasm contains very few. A few scattered wellgranulated adenoma cells resembling normal gonadotrophs may occur in several tumors. The other half of adenomas in males consists of mixtures of well-differentiated cells and of elements resembling null cells. Probably this is the reason for the patchy FSH/LH immunoreactivity seen so often in these tumors. An important morphologic marker, present in many gonadotroph adenomas in males, is follicle formation. These structures are formed by tumor cells around tissue debris. They are never delineated by basement membrane. Varying degrees of oncocytic change is observed in about 50%of gonadotroph tumors in men. More than 70% of gonadotroph adenomas in women represent monotonous, highly differentiated adenomas comprised of strikingly polar, closely apposed cells with long, fine, intertwined processes. The spherical or slightly ovoid nuclei are chiefly euchromatic. Under the electron microscope, these adenomas are characterized by low electron density of the cytoplasm. The structural basis of this is the delicate network of welldeveloped, slightly dilated RER filled with a low density proteinaceous substance, a normal number of mitochondria with moderately electron dense matrix, and practically no or few secretory granules within the nuclear pole. The diagnostic marker of female-type tumors is the unique vacuolar transformation of the Golgi apparatus that we termed the “honeycomb Golgi complex” (Horvath and Kovacs, 1984; Murray et al.,

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1985) (Fig. 28). It appears that the original membrane system transform into (probably) 3 clusters of fairly uniform spheres filled with proteinaceous substance of very low electron opacity. As opposed to the developing granules usually seen in Golgi complexes with regular features, immature secretory granules are rarely noted in the honeycomb Golgi system. The transformation of the Golgi complex occurs with different frequency, from nearly 100% to less than 10% of the cells, in individual tumors. The secretory granules are sparse and very small (up to 150 nm), and their overwhelming majority is collected in the long, fine cytoplasmic processes. Formation of follicular structures by adenoma cells may occur, but less frequently than in tumors of males. In about 20% of gonadotroph adenomas in women, well-differentiated ultrastructure as described above alternates with less developed cytoplasmic organization. The latter areas still display polarity of cells, indicating glycoprotein hormone differentiation, but the RER is not prominent and the Golgi apparatus, showing regular features, is poorly developed. An unusual, rare varient of gonadotroph adenoma, observed only in postmenopausal women so far, is comprised of fairly well-granulated cells resembling normal gonadotrophs or even stimulated gonadotrophs (“castration cells”) (Fig. 29) (Horvath and Kovacs 1988).In this variant the polarity of cells is not present or not prominent. The RER is well developed, but its morphology exhibits considerable variations from one cell to another by displaying varying degrees of dilatations. The prominent Golgi complex shows regular features; vacuolar dilatation may occur only in occasional cells. The secretory granules are quite numerous, tend to be more randomly distributed, are spherical or slightly irregular with ruff led limiting membrane and variable electron density, and measure up to 450-500 nm. The ultrastructure of gonadotroph adenomas may include some rare features. The most common is the presence of so-called light bodies. These bodies of unknown origin and significance have been described in the rat pituitary and are assumed to indicate gonadotroph differentiation (Horvath and Kovacs, 1988). The membrane bound bodies exhibit a tubular-granular internal structure and measure from 800 nm up to 2,0003,000 nm. They appear to be more common in tumors of males. In our experience, light bodies are not strictly specific, since they may rarely occur in other adenoma types. In spite of this, we consider light bodies useful morphologic markers aiding diagnosis of gonadotroph differentiation. An intriguing phenomenon, seen rarely in gonadotroph adenomas, is the transformation of adenoma cells into cells practically indistinguishable from the socalled granular cells of the posterior pituitary. It appears this change starts with increased lysosomal activity and crinophagy leading to the disappearance of secretory granules and the striking accumulation of large, complex lysosomes. The reason for this peculiar transformation, seen in tumors of males so far, is not known. The third rare fine structural feature is a unique

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Fig. 27. High-power view of gonadotroph adenoma (male type). Note accumulation of secretory granules in cell processes (arrows). One cell (asterisk) has larger secretory granules and it resembles normal gonadotroph. x 8,550.

Fig. 28. Gonadotroph adenoma (female type). The elongate cells with long slender processes harbor the multipolar “honeycomb Golgi complex” (arrowheads). The large majority of tiny secretory granules is seen within the cell processes. x 7,350.


Fig. 29. Uncommon type of gonadotroph adenoma in a postmenopausal woman. The adenoma cells are similar to normal gonadotrophs. The cells at right lower corner (F) are parts of a follicle. x 8,700.

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Fig. 30. Normal gonadotrophs in a female. Note euchromatic nuclei and the dull, low-contrast cytoplasm. In these cells the rnorphology of secretory granules is similar to that of corticotroph granules, but has much lower electron density. x 8,800.

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alteration of the Golgi apparatus which is probably the forerunner of the honeycomb Golgi complex (Kontogeorgos et al., 1990). As deduced from ultrastructural images, at first the long Golgi complex is folded up into a tripartite, clover-leaf-like structure with a pair of centrioles appearing at the base, adjacent to the nucleus. Then the 3 loops separate and with the help of microtubules radiating from the pair of centrioles, they move away from each other. In the center of the loops an interconnected tubular system, continuous with the innermost Golgi saccule, develops. This is the area where the first vacuoles of the honeycomb Golgi complex appear. There is little doubt that the unique alterations of the Golgi apparatus in adenomas of women have functional implications. However, the cause, the genesis, and the functional significance of these changes are entirely unknown. Gonadotroph hyperplasia to our knowledge has never been studied by electron microscopy in the human pituitary. The lesion appears to be exceptionally rare and may be observed incidentally a t autopsy in cases of long-standing primary hypogonadism. The non-tumorous part of adenohypophysis harboring gonadotroph adenoma is practically unexplored. Since these tumors are almost always diffuse macroadenomas at the time of surgery, periadenomatous tissue is not likely to be included in the specimen. Among the more than 150 gonadotroph tumors studied in our laboratory, we encountered only 2 which contained small areas of non-tumorous tissue-not enough to draw valid conclusions concerning the cytology of various cell types. The normal counterpart of gonadotroph tumors is an ovoid or rounded cell having a large interface with the basement membrane (Fig. 30) (Horvath and Kovacs, 1988). The spherical nucleus is euchromatic. Most of these cells have a characteristically dull appearance when compared to the high contrast of other surrounding cell types. The RER is slightly dilated, and the ring-shaped Golgi complex harbors immature granules. The secretory granules of varying electron density may be spherical or irregular, resembling the morphology of corticotroph granules. This is, however, where the similarity ends. The corticotroph with its high contrast and the specific type-1 filaments is easily distinguishable from the gonadotroph. In the case of primary gonadal failure or ablation of gonads, the stimulated gonadotrophs undergo marked changes leading to development of “castration cells.” The simultaneous proliferation and dilation of the RER result in the enlargement of the cell (Fig. 31) (Horvath and Kovacs, 1988). The Golgi apparatus is also hypertrophic, creating a well-known “negative Golgi image” seen in histology specimens. On the other hand, the granularity of cells gradually decreases with the progression of the castration change. Information is inadequate concerning the morphology of the inactive or suppressed gonadotroph. Based on a case of Kallmann’s syndrome (hypothalamic hypogonadism) (Kovacs and Sheehan, 1982) and a periadenomatous hypophysis adjacent to gonadotroph adenoma, the suppressecVinactive gonadotroph is a cell smaller than normal with a more heterochromatic nu-

cleus, poorly developed RER and Golgi membranes, few secretory granules, and an abundance of large, heterogenous lysosomes.

SILENT ADENOMAS In the course of our pituitary adenoma studies we have encountered 3 adenoma types endowed with welldifferentiated distinct ultrastructure reflecting high hormonal activity (Kovacs et al., 1978a; Horvath et al., 1980; Kovacs and Horvath, 1986a). However, none of these tumors is associated with a recognizable endocrine hypersecretory syndrome and they cannot be associated with any of the known pituitary cell types. We assume that these adenomas arise in cell types which are not yet characterized and neither their hormonal products nor their target tissues are known. At present, combination of electron microscopy and immunohistochemistry is the only way to conclusively diagnose silent adenomas. The silent “corticotroph” adenoma subtype-1 displays all histologic, immunohistochemical, and ultrastructural features of corticotroph adenomas associated with Cushing’s disease (Fig. 32) (Horvath et al., 1980; Serri et al., 1987). The 2 tumors cannot be distinguished on a morphological basis unless specific immunohistochemical markers are found for the 2 adenomas. Silent “corticotroph” adenoma subtype-1 is not associated with signs and symptoms of Cushing’s disease; the tumors are discovered at the macroadenoma stage and show unusual tendency to hemorrhage and apoplexy. When comparing the ultrastructure of corticotroph adenomas and silent “corticotroph” subtype-1 adenomas in a large material, there appears to be one difference: formation of follicles is more frequent in the silent type. However, follicular structures may occur in corticotroph tumors as well; thus this finding has no value in differential diagnosis. Little is known about the periadenomatous gland around silent “corticotroph” adenoma subtype-1. We had the opportunity of studying such tissue only in one case-not enough to draw valid conclusions. It is of note, however, that the corticotrophs displayed no signs of Crooke’s hyalinization. The normal counterpart of the tumor type is not known. However, it is suggestive that the basophils of intermediate and posterior lobes possess similar tinctorial characteristics, immunohistochemical profiles, and ultrastructures as anterior lobe corticotrophs. Yet there is no indication that they produce biologically active ACTH. This POMC-producing cell type is the best candidate for giving rise to silent subtype-1 adenomas. The silent “corticotroph” adenoma subtype-2, occurring mainly in men (male/female ratio = 4:1), also appears to be a POMC-producing neoplasm (Horvath et al., 1980). The chromophobic or slightly basophilic tumors may show varying degrees of PAS positivity and consistently exhibit immunoreactivity for ACTH, p-endorphin, P-lipotropin, and other POMC peptides. In 2 aggressively growing tumors positivity for a-subunit was documented as well. Electron microscopy detects tumors consisting of polyhedral angular cells with centrally placed nuclei


Fig. 31. Gunadectomy cell in the pituitary of an ovariectomized woman. The markedly enlarged cell possesses abundant, dilated RER and enormous Golgi appartus (arrowheads). x 6,250.

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Fig. 32. Silent “corticotroph” adenoma subtype-I with features similar to those of corticotroph cell adenoma. Note folicular cell (F)in the center and bundles of type-1 filaments (arrows). x 6,200.

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which often display mild pleomorphism. The fairly well-developed, randomly distributed RER and the Golgi complex show no distinguishing characteristics. The cells are usually moderately granulated. The randomly arranged secretory granules are spherical, irregular, or drop-shaped and may vary in electron density (Fig. 33). They measure up to 400 nm in their largest diameter, the majority being around 250-300 nm. The morphology of secretory granules is similar to that of corticotroph granules. It is of note that type-1 filaments are not present in this tumor type. Neither the normal gland around silent “corticotroph” adenoma subtype-2 nor the normal counterpart of the neoplasm is known at the present time. The silent adenoma subtype-3 often appears to be functioning, sending misleading signals to the clinicians (Horvath et al., 1988a). Mild to moderate hyperprolactinemia, frequently associated with amenorrhea/ oligomenorrhea-glactorrhea, is rather a rule than an exception in women having this adenoma type. In some men the tumor seems to be non-functioning; in others it causes hyperprolactinemia or even acromegaly. The excess PRL is mostly produced by the non-tumorous pituitary for unknown reasons, whereas the excess GH is probably secreted by the adenoma, which has a high propensity for plurihormonal differentiation. By histology silent adenoma subtype-3 is chromophobic or somewhat acidophilic, showing immunoreactivity for either no known pituitary hormone or displaying scattered positivity in a minority of cells for any pituitary hormone, chiefly GH, a-subunit, TSH, and POMC peptides. Electron microscopy documents adenomas comprised of usually large, polar cells. The large nuclei contain little heterochromatin, prominent nucleoli, and, in the majority of cases, conspicuous, often multiple spheridia (Fig. 34). Mild, focal nuclear pleomorphism is common. The randomly distributed RER is extensively developed and varying amounts of SER may also be seen. The Golgi complex is characteristically multifocal, represented by several cross sections within a large cytoplasmic area indicating a long, tortuous membrane system. The secretory granules are sparse, measure around 200 nm, and tend to accumulate in cell processes. The mitochondria often occur in pockets, displaced by the proliferating RER or SER. In several tumors, multiple plexiform interdigitations of the neighboring plasmamembranes are noted. The silent subtype-3 adenoma is endowed with characteristics of well-differentiated glycoprotein hormone producing tumors, such as a polar cell body and small, unevenly distributed secretory granules. The normal counterpart of the tumor, however, has yet to be found. CLINICALLY NON-FUNCTIONING ADENOMAS: NULL CELL ADENOMA AND ONCOCYTOMA Unless discovered incidentally, these are large macroadenomas occurring predominantly in older age groups. The two morphologic variants are the non-oncocytic null cell adenoma (Kovacs et al., 1980b) and the pituitary oncocytoma (Kovacs and Horvath, 1973). By definition null cell adenomas and oncocytomas are neo-

plasms unassociated with the overproduction of any known pituitary hormone and devoid of morphologic markers sufficient to indicate their derivation (Kovacs et al., 1980b; Kovacs and Horvath, 1986a; Kovacs et al., 1990a). By histology null cell adenomas are chromophobic, whereas oncocytomas may be partly or entirely acidophilic due to non-specific uptake of acid dyes by mitochondria. Immunohistochemistry either detects no immunoreactivity for any hormone or it reveals scattered positivity for various hormones, especially a-subunit, FSH, and LH, and less commonly other hormones. The ultrastructure of null cell adenoma reflects a hormonally inactive tumor consisting of small polyhedral cells with irregular, often deeply cleaved nuclei (Fig. 35). The small cytoplasm harbors poorly developed RER and Golgi membranes and scanty, small (less than 250 nm) secretory granules. The mitochondria1 content and morphology are within normal limits in most cells, but a variable number of cells may show oncocytic change in many null cell adenomas. The sole morphologic marker of the pituitary oncocytoma is the increased number and volume density of mitochondria involving the large majority of adenoma cells (Yamada et al., 1988) (Fig. 36). All the other morphologic features of this tumor type are similar to those of null cell adenomas. The most controversial issue, concerning the null cell adenoma/oncocytoma group, is the obvious overlap between these tumors and gonadotroph adenomas (Asa et al., 1986; Yamada et al., 1989; Kovacs et al., 1990a). A considerable proportion of null cell adenomas/oncocytomas display minor immunoreactivities for glycoprotein hormones and, by electron microscopy, many of these adenomas may contain cells which show general characteristics of glycoprotein hormone differentiation (polarity of cell body and uneven distribution of small secretory granules). Indeed, in some cases it is difficult or impossible to draw the line between the two entities. Demonstration of genes for a- and P-subunits of glycoprotein hormones also indicates glycoprotein hormone differentiation of many but not all tumors of the null cell adenoma/oncocytoma group (Jameson et al., 1987). Detection of gene expression for other hormones, such as ACTH or PRL, also points to the heterogeneity of null cell adenomas and oncocytomas (Sakurai et al., 1988). The presence or absence of cytologic alterations in the non-tumorous pituitary around null cell adenomas is not known. Since these tumors are invariable large at the time of surgery, no surrounding tissue is likely to be received.

PLURIHORMONALITY IN PITUITARY ADENOMAS This recently explored aspect of pituitary adenomas (Duello and Halmi, 1977; Kovacs et al., 1982; Carlson et al., 1983; Horvath et al., 1983c; Scheithauer et al., 1986a; Heitz et al., 1987; Kovacs et al., 1990b) will be dealt with briefly, since plurihormonality has no fine structural criteria. The term plurihormonal refers to tumors which are capable of producing more than one hormone. Plurihormonality is observed in several ade-


Fig. 33. Silent "corticotroph" adenoma subtype-2 consisting of polyhedral cells with centrally placed nuclei. The randomly arranged secretory granules are spherical as well as irregular or drop-shaped. x 8,950.

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Fig. 34. Silent adenoma subtype-3. The polar cells have large nuclei harboring spheridia (arrowheads) and abundant cytoplasm with well-developed RER and Golgi complex and unevenly distributed, small secretory granules. x 9,750.

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Fig. 35. Null cell adenoma having poorly developed cytoplasmic organelles and small, scanty secretory granules. x 7,800.

Fig. 36. Pituitary oncocytoma displaying marked increase in mitochondrial content. x 5,200.

ULTRASTRUCTURAL PATH(3LOGY OF THE PITUITARY

noma types. Some of them are specific entities such as the bihormonal bimorphous mixed GH cell-PRL cell adenoma, or the bihormonal monomorphous mammosomatotroph and acidophilic stem cell adenoma. In other cases plurihormonality is only one facet of the tumor morphology, such as the common a-subunit, TSH, and PRL immunoreactivity in densely granulated GH cell adenomas (Beck-Peccozet al., 1985,1986; Scheithauer et al., 1986a; Osamura and Watanabe, 1987) or the scattered, minor, but often multiple immunoreactivities in the null cell adenoma/oncocytoma group (Kovacs et al., 1980b, 1990a; Heitz et al., 1987; Saeger et al., 1990). Apart from the 3 bihormonal GHPRL producing tumor types, we diagnose plurihormonal adenoma only if the multiple immunoreactivities are associated with multiple endocrine functions and/or the adenomas consist of more than one morphologically distinct cell type. Presently it is unclear whether plurihormonality represents abnormal gene expression or abnormal amplification of genes which are already expressed at low level in the parent cell of the tumor type. It is of note that plurihormonal cells (GH-PRL, GH-a-subunit, GHTSH, FSH-ACTH) are known to exist in non-neoplastic human or animal pituitary (Horvath and Kovacs, 1988).

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Multihormonal pituitary adenomas.

Ectopic pituitary adenomas.

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