J. Endocrinol. Invest. 13: 435-454, 1990

REVIEW ARTICLE

Human pituitary adenomas. Recent advances in morphological studies G. Giannattasio and M. Bassetti *Centro CNR per 10 Studio della Farmacologia delle Infrastrutture Cellulari, Dipartimento di Farmacologia, Universita di Milano, 20100 Milano, Italy

INTRODUCTION

and cellular location of secretory granules were found to differ according to the type of hormone stored within them. These observations allowed a classification of the tumors to be made according to the hormones produced, as inferred from the ultrastructural features of the cellular organelles, mainly of the secretory granules (1, 3-5). However the electron microscopy only provided indirect and subjective criteria to recognize the different types of tumors. The turning point in the morphological studies of pituitary adenomas was the advent of the immunohistochemical techniques (immunofluorescence and immunoperoxidase methods), By using specific anti sera it was possible to identify directly the hormones stored within the cells. The application of these techniques led to a much more precise and reliable distinction of the different tumor types. Since the mid-seventies a large number of papers reporting immunohistochemical studies on pituitary adenomas have been published (for books and comprehensive articles see Refs. 6-16). The large scale immunohistochemical testing of adenomas for all known pituitary hormones as weil as the improvement in the techniques made it increasingly evident that adenomas producing more than one hormone were much more common than could be deduced from blood hormone measurements and clinical symptoms. In addition, the deeper insight into hormonal conte nt of adenomas gained by these techniques led to the finding that many "non-functioning" adenomas contained one or more pituitary hormones even though their presence did not result in an increase in their blood concentrations. All these findings were confirmed and stressed when highly sensitive immunoelectron microscopic techniques became available. Particularly the protein A-gold immunotechnique has been demonstrated over the last few years to be an extraordi-

A large number of morphological studies have been carried out on human pituitary adenomas over the last two decades primarily in order to establish structure-function correlations so as to provide the clinical endocrinologist with information of diagnostic interest on the endocrine activity of tumors. Classifications were made, based on the morphological observations, supporting and integrating those based on clinical manifestations and blood hormone concentration measurements. Great advances in morphological diagnosis and classification have been made, mainly in the eighties, due to the considerable improvement in the morphological procedures. However the first approach, made by means of the histological staining methods, was far from adequate. Pituitary adenomas were divided into acidophilic, basophilic and chromophobic tumors according to the staining affinities of the cell cytoplasm. However, the tinctorial characteristics of the cells proved to give a poor indication of the type of their endocrine activity. This classification, therefore, has no diagnostic value (1, 2).

Electron microscopic studies gave more insights into structure and activity of the tumor cells. The peculiarities of the cellular organelles were accurately considered. Thus, the development of the rough endoplasmic reticulum and the prominence of Golgi complex (the cytoplasmic organelles responsible for the synthesis and packaging of the hormones) typical of many adenoma cells were related to their high secretory activity. Number, size

Key-words: Pituitary adenomas. pituitary gland, morphology, immunohistochemistry. Correspondence: Giuliana Giannattasio, MD., CNR Center 01 Cytopharmacology, Via Vanvitelli 32, 20129 Milano, Italy.

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G. Giannattasio and M Bassetti

docrine activity) (6, 21). The improvement in morphological techniques and the systematic search for all known pituitary hormones have proved it to be otherwise and have led to the increasing recognition of adenomas with plurihormonal immunohistochemical patterns, wh ich however in most cases did not result in raised circulating levels of all hormones present in the cells (19-22). It is predictable that the frequency of plurihormonal adenomas will rise still more in the future if the protein A-gold immunoelectron microseopie technique is applied on larger scale. The most common form of plurihormonal adenoma produces GH and PRL (9, 14, 20, 23-38). As mentioned above the existence of this kind of tumor was early recognized since in many cases both hormones were hypersecreted and patients had acromegaly and hyperprolactinemia. Immunohistochemical and immunoelectron microscopic studies have demonstrated that also tumors removed from patients presenting only with acromegaly or hyperprolactinemia contain both hormones (9, 14,27,29, 31, 32, 35, 37, 38). Arecent classification indicates that the frequency of GH- and PRL- producing adenomas in unselected surgical material from more than one thousand tumors was 8-9%, which corresponded to about one-third of the total amount of tumors producing GH or GH/PRL (20). However, in recent studies using the protein A-gold technique 50% of tumors from acromegalic patients with or without hyperprolactinemia were found to contain GH and PRL immunoreactivities (35). This result suggests that the frequency of this tumor could be notably higher than that calculated using less sensitive techniques. It is reasonable to think that the association of normal hormonal levels with positive immunostaining is due to the fact that hormones are secreted in amounts insufficient to bring about detectable increases in their blood concentrations. This in turn can be due to a small number of cells engaged in the production of the hormones or to a low rate release. In keeping with the former explanation a positive correlation has been found between the basal PRL levels of acromegalic patients and the percentage of PRL positive cells in the tumors. Under 20-25% PRL positive cells the patients had no hyperprolactinemia (35). Less usual hormonal associations have also been found in pituitary adenomas by immunohistochemistry, mainly in recent years. A broad spectrum of combinations of all pituitary hormones has been

narily useful tool to detect very low amounts of hormones stored within a cell. The availability of advanced morphological procedures has also recently led to aseries of extremely interesting findings often of great interest also to biologists. By the application of the protein-A gold double labelling technique the presence in many tumors of cells containing more than one hormone has been definitely ascertained. These mixed cells, observed at the same time also in normal pituitaries, are regarded as one of the more interesting new findings concerning the pituitary gland biology of the last few years. The availability of specific antibosJies has also enabled researchers to demonstrate the presence in adenoma cells of a number of substances other than the known pituitary hormones. Some of them are at present considered of potential interest as diagnostic markers. Finally, morphological investigations have proved to be useful for shedding light on the mechanisms of action of drugs used in the management öf pituitary tumors. These interesting findings which have emerged in recent years will be dealt with in this article, which will therefore comprise the following sections: 1) New insights into the hormonal conte nt of pituitary tumors. The plurihormonal adenomas. The nonfunctioning adenomas; 2) The mixed cells; 3) Cellular products other than the known pituitary hormones. The diagnostic markers; 4) Effects of drugs on the ultrastructure of adenoma cells. The interested reader can find comprehensive and systematic depictions of all morphological features of the different types of human pituitary adenomas in a number of pertinent articles listed in the bibi iography (2, 17-20). NEW INSIGHTS INTO THE HORMONAL GONTENT OF PITUITARY TUMORS. THE PLURIHORMONAL ADENOMAS. THE NON-FUNGTIONING ADENOMAS With the exception of GH- and PRL -secreting adenomas, tumors producing two or more hormones were considered quite rare. This idea was due to the fact that hormones present in the cells without concomitant increases in their serum levels went unnoticed. Early histochemical and immunohistochemical studies also suggested that most pituitary adenomas were composed of almost homogeneous cell populations engaged in the production of a single hormone (with the obvious exception of the non-functioning tumors apparently deprived of en-

436

Morphological studies on pituitary adenomas

reported 1 : GH and/or PRL with one or more glycoprotein hormones; the full range of glycoprotein hormones; different combinations of ACTH and other pituitary hormones (15, 16, 19-22, 39-48). These plurihormonal adenomas are now recognized to be not as rare as they were once considered. According to the Kovacs' group these tumors, taken together and considered separately from G H / PRL producing adenomas, have frequencies up to 1015% of all adenomas (21 ). From results obtained by other authors it could be inferred that they are even more frequent (22). Their recognition is likely to increase in the future. A large group of these tumors is engaged in GH production associated with acromegaly (21 ). A frequently occurring variant produces GH, PRL and TSH (Fig. 1). Clinical syndromes are often related to a single hormone mainly reflecting the presence of GH and, to a lesser extent, of PRL. Clinical expression of glycoprotein hormones, when combined with GH and/or PRL is less frequent, although several cases of acromegaly and hyperthyroidism have been reported (46, 47). When only glycoprotein hormones are combined only one can be clinically expressed (43). The reasons for the lack of serum hormone increases and endocrinological symptoms in spite of the presence of specific immunostainings are still being discussed. Although several explanations have been envisaged such as hormone produced but not released, intracellular degradation of the hormone or others (21), the most obvious one remains that of

an insufficient number of cells engaged in the production of the "silent" hormones but precise correlations have not been calculated. An interesting finding of recent years is the presence of the free a-subunit of glycoprotein hormones in many pituitary tumors both functioning and nonfunctioning. Its presence in non-functioning adenomas will be dealt with below. Focusing on functioning tumors, the presence of an excess of a-subunits has been extensively documented in glycoproteinsecreting tumors (49-55), In these tumors the production of an excess of a-chains is likely to be due to an unbalanced synthesis of a- and ß-subunits (50, 52). However, free a-chains have been found also in PRL-secreting, GH-secreting and ACTHsecreting adenomas (22, 54, 56-59) and in adenomas producing hormones of both families (40-42). High percentages of these tumors are reported to contain a-subunits: 57% of tumors causing acromegaly, 35% of ACTH-secreting adenomas, 9% of PRL-secreting tumors according to some authors (58). Smaller by still noteworthy values can be inferred from other papers (54, 56). Adenomas producing a-subunits together with GH, PRL or ACTH are usually classified as plurihormonal even though a single true hormone is present, and are therefore to be included in this ever-growing group. Many tumors release a-subunit in quantities large enough to cause increases in its serum levels (42, 50, 5557, 60-65), but this does not result in any clinically recognized syndrome. A possible significance of asubunit as a tumor marker in patients with increased plasma levels of LH and/or FSH has oeen suggested. Elevated serum levels of a-subunit and its relative autonomy to stimulation with LHRH and suppression by testosterone would differentiate patients

1 Gonadotroph adenomas synthesizing FSH and LH and corticotroph adenomas producing ACTH and other derivatives 01 prooplomelanocortin are not perceived by pathologists as truly plurihormonal tumors and therelore are not included in this series (20, 21).

Fig. 1 - P/urihormona/ adenoma associated with acromega/y. /mmunoreactivity tor GH (A), PRL (8) and TSH (G). Avidin-biotin-peroxidase comp/ex technique (X 170). Reproduced trom Scheithauer et al. (21) with permission.

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G. Giannattasio and M. Bassetti

with tumor from those with a primary gonadal disease (50) . Moreover, the excess production of free O'-chains in TSH-producing tumors has been used to distinguish adenomatous from non-adenomatous causes of inappropriate TSH secretion (66). New insights have also been gained from immunohistochemical studies into the hormonal content of the so-called non-functioning adenomas i.e. adenomas unassociated with clinical or biochemical evidence of hormone hypersecretion 2 . Clinical symptoms are only due to the compression of the tumoral mass on the surrounding tissues. Diagnosis is often difficult since there is no rise in any pituitary hormone in blood. The incidence of these tumors is high . Different authors report frequencies ranging from 20 to 40% in series of 100-300 patients with pituitary adenomas (9, 22, 69-73). Their frequency is still higher in autopsy studies of incidentally encountered adenomas (74). Immunohistochemical studies on different surgical series of non-functioning tumors showed that high proportions (trom 35 to 90% according to most authors) of adenomas contained immunoreactive pituitary hormones (9,

22 , 70, 72, 73, 75). In some ca ses the positivity was widespread, in others only scattered positive cells were observed in keeping with the lack of an increase in hormonal blood levels. Non-fun ctioning adenomas were found to be monohormonal or, in many cases, plurihormonal. All pituitary hormones have been found in non-functioning tumors but according to different authors the most frequently present molecules were glycoprotein hormones and / or their uncombined 0' - and B-subunits (Fig . 2) (70 , 72 , 73, 75). Analyses of pituitary hormone gene expression confirmed immunohistochemical findings (73). Moreover, recent in vitra studies showed that almost all tested non-functioning adenomas released in culture gonadotropins and their 0'- and Bsubunits (76) . Several functionless tumors have been found to contain exclusively O'-chains (22 , 54, 58, 70, 77, 78). It is evident that all thes e tumor variants contain defects in one or more steps of glycoprotein hormone biosynthesis (73). Moreover, in some cases, the secretion of uncombined hormonally inert subunits c.ould account for the apparent lack of endocrine activity (79, 80). The "pure alpha-producing (or alpha only) adenoma " may be considered a tumor wh ich has lost the ability to synthesize a complete hormone. Interestingly, a mouse pituitary tumor li ne secreting only O'-chains developed from a thyrotropic tumor after a 6-yr period of serial transplantation (81 ). An unc oupling of B-subunit gene ex pression and protein bi osynthesis might be the possible underlying mec hanism

2The terms "non-secreting". 'endocrinologica lly inactive" or "s ilent" are alten used as synonyms of " non-functioning" On the contrary the term "chromophobic " is not a synonym and only refers to the fact that tumors have no affinity for histological dyes. Since the affinity for dyes is directly related to the numbe r and size of sec retory gran ules present in the ce ll cytoplasm. many tumors composed of actively secreting cells with little sto rage (e.e. wi th spa rse and small secretory granules) do not stain . This is the ca se of many highly sec reting. poorly granulated prolactinomas (67 . 68).

Fig . 2 - Non-functioning adenoma. /mmunoreactivity for a-subunit. A vidin-biotin -peroxidase comp/ex technique (X 330) Courtesy of C Capel/a.

438

Morphological studies on pituitary adenomas

since ß-subunit mRNA has been detected in a tumor secreting only a-chains (77). Since a-subunit is often hypersecreted (82, 83), its role as a diagnostic marker for non-functioning adenomas has been put forward (82). Functionless tumors not containing immunocytochemically detectable hormones were termed null cell adenoma (20, 69). By means of sensitive procedures it has recently been demonstrated that some of them contain few and scattered cells positive for a- and ß-subunits (20). The release of small quantities of glycoprotein hormones and a-subunit from these tumors could also be demon.strated in in vitra studies (84). This group of tumors seems to represe nt the most undifferentiated stage of non-functioning adenomas and of all pituitary tumors. Fine structure observations showed that their cells contained few and poorly developed organelles and had sparse and very small secretory granules, frequently lining up along the cell membranes. In line with all this, some authors in an ultrastructural study on non-functioning tumors have recognized organelle-rich and organelle-poor adenomas. These latter tumors containing particularly small granules seem to correspond to null cell adenomas (85, 86). Variants of null cell adenomas are the oncocytomas composed of cells with abundant cytoplasm densely packed with innumerable mithocondria of abnormal structure. Oncocytic transformation seems to be a gradual process and many transitional forms are described (69, 87, 88). Alpha-subunit and glycoprotein hormones have been demonstrated also in these tumors (78, 88). Finally it is worth noting that other types of nonfunctioning adenomas have recently been reported as distinct entities, namely the three subtypes of "silent corticotroph adenomas" (89) and the "silent somatotroph adenoma" (90) composed of cells poorIy or not positive for GH where the presence of GH mRNA was detected by means of the very recent in situ hybridization technique (91, 92).

some early reports exist about the presence of this very peculiar cell type in pituitary adenomas. Besides cells secreting both FSH and LH present in gonadotrophic tumors (52, 93), in some adenomas from acromegalic patients, producing both GH and PRL, cells positive for both hormones (mammosomatotrophs) were found using double immunostaining and adjacent section immunostaining (2730, 94, 95). Mammosomatotrophs were initially interpreted as a dedifferentiated stage of somatotrophs, consequent on the neoplastic transformation, able to express both GH and PRL. Their frequency was largely underestimated. When the immunoelectron microscopic techniques began to be applied, the occurrence of mixed cells in pituitary adenomas became increasingly evident. Particularly the protein A-gold double labelling with gold particles of different sizes proved to be very suitable for identifying this type of cell allowing the simultaneous and clear-cut identification at ultrastructural level of two different hormones on the same section (96, 97). Moreover the technique is very sensitive and permits the detection of extreme minority hormones. Since the mid-eighties many studies have been performed mainly by means of this procedure and different types of mixed cells, largely distributed in plurihormonal adenomas, have been described (Table 1). The existence of mammosomatotrophs in tumors producing GH and PRL was confirmed by many authors (22, 36, 38, 98102). Extensive investigations from our group have demonstrated the presence of mammosomatotrophs (from 50 to 80% of the whole cell population) in all tumors removed fram acromegalic patients with hyperprolactinemia (33, 35). Variable proportions of mammosomatotrophic cells were also present in about a half af tumors removed fram acromegalic patients without hyperprolactinemia (Fig. 3) or from hyperprolactinemic patients without acromegaly. Almost all tumors producing both GH and PRL had mammosomatotrophs (35, 37). Figure 4 depicts the cellular composition of 22 tumors from acromegalic patients with and without hyperprolactinemia. It can be seen that besides mammosomatotrophs, mono hormonal cells positive for either GH or PRL were present. In another type of mixed cell GH colocalized with TSH (42,46,47) (Fig. 5). These cells were found in tumors producing GH and TSH removed from acromegalic patients most of whom had also hyperthyroidism. Only some cells in these tumors were

THE MIXED CELLS

It is now definitely ascertained that in plurihormonal adenomas the different hormones can be synthesized both by different types of cells and by mixed cells (able to elaborate more than one hormone), often simultaneously present in the same tumor. Although mixed cells were difficult to identify by means of light microscopy immunotechniques,

439

G Giannattasio and M Bassett!

Table 1 - Different types 01 mixed cells in human pituitary adenomas

Mixed cells*

Adenomas

Clinical presentation

References

FSH + LH

gonadotropinproducing adenomas

gonadotropins hypersecreted or normal

52, 93

GH + PRL

GH- and PRLproducing adenomas

acromegaly; hyperprolactinemia; acromegaly and hyperprolactinemia

GH + TSH

GH- and TSH-producing adenomas; GH-, TSH- and a-subunitproducing adenoma

acromegaly and hyperthyroldism

22,27-30, 33,35-38, 94,95,98-102 42,46.47

Gr+ + a-subunit

GH- and a-subunitproducing adenomas; GH-, TSH- and a-subunit -producing adenoma

acromegaly; acromegaly and hyperthyroidism

42,54,56, 59,103

PRL + a-subunit

PRL- and a-subunit producing adenomas; PRL-, TSH- and a-subunit-producing adenoma; PRL-, TSH-, FSH-and a-subunit-producing adenoma

hyperprolactinemia; hyperprolactinemia and hyperthyroidism

40,41,54

ACTH + a-subunit

Silent ACTH- and a-subunitproducing adenomas

no signs or symptoms of hormone hypersecretion

54

FSH + TSH

FSH- and TSHproducing adenomas

FSH hypersecreted or normal

43

GH + FSH or LH

LH-, FSH- and GHproducing adenoma

gonadotropins hypersecreted

45

PRL + FSH or LH

LH-, FSH- and PRLproducing adenoma

gonadotropins hypersecreted and hyperprolactinemia

48

GH+PRL + a-subunit

GH-, PRL- and a-subunit-producing adenoma

no signs or symptoms of hormone hypersecretion

54

*

The frequeneles of mixed eells in the tumors were vanable and largely dependent on the method of deteetlon

No direet evidenee exists of eells eontaining three hormones due to the teehnieal diffieulties of performing tripie labelling. However, so me authors, performing immunostaining on serial seetions, have found eells positive for GH, PRL and a-subunit (54). Mixed eells were found to be heterogeneous as regards the seeretory granules (Fig. 3). In some of them the two hormones were loealized in separate granules while in other eells they were stored within the same granules. Finally, some cells contained both mixed granules and granules positive for one or other of the hormones (33, 35, 37, 38, 47, 99). While these studies on adenomas were proeeecJing, normal anterior pituitary glands were analyzed in

mixed. Cells eontaining simultaneously GH and asubunit (42, 54, 56, 59, 103) (Figs. 5 and 6), PRL and a-subunit (40, 41, 54) or ACTH and a-subunit (54) were also observed. Their frequeneies were different. In one study tumors removed from aeromegalie patients with a-subunit hyperseeretion were found to be almost entirely eomposed of eells with seeretory granules positive for GH and a-ehains (56). Other authors observed in the same type of tumors mixed GH/a-subunit cells together with cells positive for one or other of the moleeules (103). Other mixed eells were reported to be immunoreaetive for FSH and TSH (43), for GH and FSH or LH (45) and for PRL and FSH or LH (48).

440

Morphological studies on pituitary adenomas

order to seek mixed cells (besides the al ready known FSH/LH cells). Immunoelectron microscopic techniques were here flanked by the reverse hemolytic plaque assay performed on cells in culture (104). The results came up to expectations. A large number of mammosomatotrophs were found in rat, cow and human pituitaries (105, 109). Ir) addition, in normal human pituitary tissues different types of mixed cells have recently been observed (FSH, LH and PRL; ACTH and PRL) by means of serial section immunoelectron microscopy (110). Finally, in cow pituitaries cells positive for a number of hormones (GH, PRL, TSH and LH) have been found (110 bis). It has therefore been concluded that the ability to expreSs more than one hormone is not a consequence of the neoplastic transformation, but exists also in normal pituitary cells. Moreover, mixed cells seem to be able to elaborate biochemically unrelated hormones contrary to the general idea of the existence of three different ceillines stemming from distinct progenitor cells, producing the three different classes of pituitary hormones. A great deal of attention is now paid to mixed cells and a number of questions have been raised ranging from the mechanisms of sorting of the two hormones (when targeted to distinct secretory granules) to their physiological role and to the mech-

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Fig. 4 - Cellu/ar eomposition ot GH-seereting adenomas from aeromega/ie patients with (eases 16, 18-22) or without hyperprolaetinemia. Dark hatehed bars = eells positive on/y tor GH; open bars = eells positive only tor PRL; light hatehed bars = eells positive tor both GH and PRL. Reprodueed trom Bassetti et a/. (35) with permission.

anisms controlling the release of the two distinct hormones from the same cell (can release be dissociated and differently regulated when the two hormones are stored in separate granules?). Most of these questions are still unanswered. As regards the role of mixed cells it has been postulated that in normal pituitary they might represent multipoten-

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"_ .. Fig. 3 - Adenomas trom aeromegalie patients " without hyperprolaetinemia. Doub/e immuno/abelling with anti-hGH and anti-hPRL sera and protein A-gold partie/es ot different sizes. Smal! gold particles = GH; /arge gold partie/es = PRL. A, portion ot a mammosomatotrophie eell with granules positive tor GH and granu/es positive for PRL (X 58,000). B, Portions ot a eell eontaining granules positive only tor PRL (*), ot a eell with granules positive only for GH (.) and of a mammosomatotrophie eell eontaining granu/es positive only tor PRL and granules positive for B both GH and PRL (*) (X 25,000) Reprodueed from Bassetti et al. (37)

441

G, Giannattasio and M Bassetti

tial stem cells imparting "plasticity" to the gland (109-110), In any event, the recognition of mixed cells in pituitaries introduces a new element of complexity into pituitary cytology and makes the one cell-one hormone theory, which dominated pituitary cytophysiology and cytopathology for several decades, untenable, 00 the adenomatous mixed and monohormonal cells of plurihormonal tumors originate from the concomitant proliferation of normal counterparts? Or might the tumors result from the aberrant multiplication of multipotential stem cells capable of divergent differentiation? At present we cannot choose between the two alternatives, From a functional point of view, the ,existence of mixed cells is to be borne in mind when interpreting the dynamics of hormonal secretion , The anomalous responses of a hormone to the releasing or inhibiting factors specific for another hormone in patients with pituitary tumors could be, at least in some ca ses, accounted for by the presence in the tumors

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of a large number of mixed cells storing the two hormones within the same secretory granules (33, 56), CELLULAR PRODUCTS OTHER THAN THE KNOWN PITUITARY HORMONES. THE DIAGNOSTIC MARKERS

Immunohistochemical and immunocytochemical techniques have permitted the analysis of pituitary adenomas in order to seek the presence of a number of molecules different from the known pituitary hormones or their subunits, A broad spectrum of peptides has been sought and some have been found, such as gastrin in some null cell adenomas (111) and vasoactive intestinal polypeptide (VIP), a putative PRL-releasing factor, in many functioning and non-functioning adenomas (112), In functioning tumors indirect evidence seemed to indicate that VIP colocalized with the classic pituitary hormones and was probably associated with secretory granules. Many peptides, including gastrin and VIP, have been found in normal pituitary glands, where they might have paracrine and autocrine effects, As regards adenomas, their significance is unknown, A possible involvement of VIP in the tumor growth has tentatively been proposed (112), Neurophysins, the carrier proteins of oxytocin and vasopressin, produced in the hypothalamic nuclei have been reported to be present in human pituitary corticotrophs, in Cushing 's ade-

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Fig, 5 - Monomorphous adenoma produeing GH, TSH and ll'subunit, RER = rough endoplasmie retieulum, Go = Golgi eomplex: arrows = seeretory granules: arrowheads = lysosomes (X 5,000). Insets, double immunoloealization of GH/ll'-subunit and GH /ßTSH with protein A-go/d partie/es of different sizes, Small gold partieles = GH, large gold partieles = ll'-subunit (A) or ßTSH (B). Some eells had granules positive for GH and ll'subunit (A X 51 ,000), Other eells had granules positive for GH and ßTSH (B, X 46,000). Reprodueed from Beek-Peeeoz et al. (42) with permission

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442

Morphological studies on pituitary adenomas

nomas and in ACTH - positive cells of GH-producing adenomas. Their role is at present not identified (113). Components of renin-angiotensin system have also recently been found in normal human PRL cells as weil as in prolactin omas (114, 115). No immunoreactivity was observed in other types of pituitary adenomas. The colocalization of renin and prolactin in the same secretory granules was demonstrated by means of the protein A - gold double immunostaining (115). Since it has been found that angiotensin II promotes PRL release both in rat and in human pituitaries, a possible autocrine action of the renin-angiotensin system on PRL release from normal and adenomatous mammotrophs has been envisaged (114, 115). A great deal of interest is at present focused on a group of proteins termed neuroendocrine markers and extensive investigations have been carried out to assess their distribution in pituitary adenomas. A variety of endocrine cells and neurons are able to synthesize and secrete substances of the same type which include amines, peptides and granuleassociated proteins. Moreover, many cellular components such as enzymes and cytoskeletal proteins are present selectively both in neurons and in endocrine cells (116). Finally, a population of small, clear synaptic-like vesicles distinct from the typical peptide- containing secretory granules has recently been described in a number of different endocrine cells (116-118). These facts support the current idea of a diffuse neuroendocrine system with common cytological and functional features (116, 119). Many of the molecules expressed by the cells of the neuroendocrine system are also expressed by the related neuroendocrine tumors. Anterior pituitary gland and pituitary adenomas are currently included in the neuroendocrine system. In line with this idea in normal pituitary cells and in functioning and nonfunctioning pituitary adenomas immunoreactivity for norepinephrine was found (120). In addition, both in normal pituitary cells (117) and in all types of pituitary adenomas (116) ultrastructural studies revealed the presence of small, clear vesicles similar to synaptic vesicles (their function is still unknown). Among the substances expressed by the neuroendocrine system cells those present in a large number of neurons and neuroendocrine cells are considered neuroendocrine markers and have the potential to be used as diagnostic markers for neuroendocrine tumors. Their possible usefulness in the diagnosis of pituitary adenomas has recently

been investigated. The most studied neuroendocrine markers are neuron-specific enolase, synaptophysin and chromogranins (Table 2). Antibodies against these molecules are currently available. Neuron-specific enolase (y-enolase), a specific form of the glycolitic enzyme enolase, is present in high concentrations in neurons and neuroendocrine cells and has therefore been considered a broad neuroendocrine marker (121) although its presence also in non-neuroendocrine cells and tumors limits its usefulness in the diagnosis of neuroendocrine tumors (122). Neuron-specific enolase immunoreactivity has been found in any type of normal pituitary cell and in almost all adenomas of large series which include functioning tumors, null cell adenomas and oncocytomas (Table 2) (111, 123, 124). The positivity was localized in the cell cytoplasm and va ried from light to intense but no correlation was found with the granularity or the differentiation of the cells (123). The enzyme appears therefore of no use in distinguishing the various types of pituitary adenomas and could only be of some help in the diagnosis of null cell adenomas which lack any immunohistochemical hormonal marker (123, 124). Synaptophysin (also referred to as protein p38) is an intrinsic membrane protein of synaptic vesicles of neurons with Mr 38,000 (117). It has also been found in the membranes of the small clear vesicles in a variety of endocrine cells (117). It has been shown to be a broad range and sensitive immunohistochemical marker for normal and neoplastic neuroendocrine cells (116, 125, 126). Synaptophysin immunoreactivity was detected in human pituitaries and in almost all pituitary adenomas tested (Table

Table 2 - Immunohlstochemicallocalization of neuroendocrine markers in human pituitary adenomas

Neuronspecific enolase a

Synaptophysin b

Chromogranin

GH-producing

+

+

+/ -

PRL -producing

+

+

Gonadotropinproducing

+ >-

+

+/ -

Adenomas

N

TSH-producing

+ / -

+ / -

+ / -

ACTH-producing

+>

+

>+ >-

+

Null cell

+ >-

+/ -

>-

+. All tumors tested by different authors were positive; + > -. the maJonty 01 tumors were positive, + / -. positive and negative tumors were lound: -, no tumors were positive, ' reis. 111, 123, 124; breis. 111, 116, 124, 126, c reis. 111, 116, 124, 133, 137 -139

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found in large dense core vesicles identified in peptidergic neurons (118, 127, 131). They are released together with the other granule-associated proteins. Their function is still under investigation (130). Normal anterior pituitary glands from different species have been found to contain immunoreactivity for chromogranins (127, 128, 132-135). They appear mainly stored in secretory granules of glycoprotein hormone-producing cells (128,134,135), but have also been found in mammosomatotrophic cells of cows in a distinct population of granules (134,136) storing TSH and/or LH as weil (110 bis). In human pituitary adenomas immunohistochemical studies were mainly performed using antibodies against chromogranin A (Fig. 7). Immunoreactivity was found in many glycoprotein hormone-producing tumors and in most null cell adenomas. Also many GH- and ACTH-producing adenomas were positive or focally positive, while prolactinomas were consistently negative (Table 2) (111, 116, 124, 133, 137 -139). Chromogranin B has recently been detected in gonadotrophic and null cell adenomas as weil as in prolactinomas (138). It has been proposed that the immunohistochemical detection of chromogranins may be useful in the charaeterization of some glycoprotein hormone-produeing tumors as weil as of null eell adenomas (133). Although null eell adenomas are not immunoreactive for pituitary hormones, they possess eleetron mieroseopically demonstrable secretory granules. These typieally small granules are likely to contain ehromogranins since they are argyrophilic (70) and it has been demonstrated that immunoreaetivity for ehromogranins elosely follows the argyrophilic Grimelius' reaetion (128) Chromogranins appear therefore very good markers for null cell adenomas for whieh they represent, together with neuron-specifie enolase and synaptophysin, the only available immunohistochemieal diagnostic tools (133). An interesting development could be the use of chromogranins as plasma markers. Sinee they are released in the same way as hormones their plasma levels rise in patients with neuroendoerine tumors (140). Thus their plasma assay could be useful in clinical practice. The recent finding that ehromogranin A plasma concentrations were increased in some patients with non-funetioning pituitary adenomas (140) raises the possibility of using the protein as a plasma marker for these tumors. Another protein reeently demonstrated to be associated with secretory granules is the polypeptide

2) (111 , 116, 124, 126). However its presence could not be correlated with hormone conte nt or other cytological and clinical features of adenomas (124). Therefore, the immunohistochemical detection of synaptophysin, like that of neuron-specific enolase, cannot be used to recognize specifically the various types of adenomas (124). However its demonstration may be of help in the immunocytochemical characterization of null cell adenomas (124). Chromogranins (secretogranins/ chromogranins) are a family of acidic, sulphated and phosphorilated secretory proteins occurring in a wide variety of endocrine and neuronal cells (127 -130). The family comprises at least three distinct proteins, chromogranin h, chromogranin Band secretogranin II with Mr ranging from 75, 000 to 120, 000. Antisera and specific monoclonal antibodies have been raised for each of them. They appear to be costored with other secretory peptides in a very large number (although not in all) of peptide-containing secretory granules of endocrine cells and have also been

Fig. 7 - Non-tunctioning adenoma. /mmunoreactivity tor chromogranin A. Avidin-biotin-peroxidase comp/ex technique (X 420). /munoreactivity is otten /ocalized at the periphery of the cytop/asm (arrow). Courtesy of C. Capel/a.

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EFFECTS OF DRUGS ON THE UL TRASTRUCTURE OF ADENOMA CELLS

designated 782, widely distributed in nervous and endocrine tissues and whose distribution is similar to that of chromogranins (141 ). Many patients with pituitary adenomas were found to have elevated plasma levels of 782 (142). Its usefulness as a tumor marker is under study. Immunohistochemical studies have also been carried out to examine the distribution in pituitary adenomas of cytoskeletal proteins, such as cytokeratins, constituents of the intermediate filaments of epithelial cells and markers of the epithelial differentiation (22, 143). They appear differently distributed among the different types of pituitary tumors gut their diagnostic significance is still to be defined. Finally the immunohistochemical demonstration of S-100 protein, initially considered a marker for glial cells but subsequently recognized as present in other cell types (144), has been described as a reliable means to detect follicolo-stellate cells in norrlal pituitaries and in pituitary adenomas (144, 145).

Morphological investigations have been helpful in shedding light on the mechanisms of action of drugs employed in the treatment of pituitary adenomas. The most extensive studies concern the effects of bromocriptine, the well-known dopaminergic agonist widely used in the pharmacological management of PRL-secreting tumors. The drug not only is able to lower the serum PRL levels of patients but also quickly and dramatically reduces the size of most prolactinomas (146). Electron microscopic observations and morphometric analyses have been used to investigate the changes induced by the drug in the morphology of the tumor cells. Early effects can be clearly recognized in isolated and cultured prolactinoma cells. After hours (1-24) of exposition to bromocriptine an accumulation of secretory granules within the cells often accompanied by a decrease in the number of exocytoses was observed, reflecting the inhibitory effects of the drug on PRL

Fig. 8 - Pro/actinomas removed trom un_,.........,"", treated patients (A and C) and trom patients treated with bromocriptine tor 6 weeks (8 and 0), A and 8, X 430; C and 0, X 2,800. Note the reduction ot cytop/asmic areas and the nuc/ei with c/umped chromatin in tumors trom treated patients. RER = rough endop/asmic reticu/um; Go = Go/gi comp/ex; arrows = secretory granu/es; V = autophagic vacuo/es. Reproduced trom Bassetti et a/. (146) with permission.

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release (147, 148). In tumors from patients treated with bromocriptine for short periods (3-7 days) and more pronouncedly when the treatment was longer, the effects of the drug on PRL synthesis (149) clearly appear. Electron microscopic observations showed that the ultrastructural appearance of adenomatous mammotrophs was profoundly altered (146, 150-154). The most striking aspect was the dramatic reduction in the cytoplasmic area (Fig. 8). In one study electron microscopic morphometry showed a 57% reduction in the cytoplasmic area after a 6 week treatment. The reduction of the nuclear area was of 28% and that of the whole cell area of 47% (146). The shrinkage of the cytoplasm appears to be a consequence of the striking involution of rough endoplasmic reticulum and of Golgi complex, typically prominent in adenomatous PRL cells, reflecting the decreased synthesis and packaging of the hormone (Fig. 80). In line with the idea that bromocriptine affects PRL synthesis at transcriptional level (149) the nuclei showed smaller nucleoli and coarsely clumped chromatin (146, 150) (Fig. 80), Secretory granules increased in number in some treated tumors; in other adenomas they were sparse. The drastic reduction in cell size weil explains most of the tumor shrinkage which initially had been regarded as a puzzling phenomenon. Changes induced by bromocriptine are reversible. When the treatment is discontinued the tumor reexpands and the typical ultrastructural features of PRL tumor cells are reestablished. However, in tumors from patients with remote bromocriptine treatment some cells with the typical involution brought about by the drug were observed (20), Thus, for unknown reasons in so me cells the ultrastructural alterations can be irreversible. Some authors have found an increase in the fibrous tissue content and in necrotic and hemorrhagic areas of the treated tumors (155, 156), which could contribute to the shrinkage of the tumor, although these phenomena have not been observed by others (150), No major morphological changes were observed in GH-secreting adenomas removed from responsive patients in wh ich bromocriptine had achieved a reduction in GH hypersecretion (20, 157), thus suggesting that the drug affects GH release rather than synthesis (20), Other investigations have been carried out to assess the effects of SMS 201-995, a long-acting analogue of somatostatin on the morphology of adenomatous somatotrophs, The drug exerts a prolonged inhib-

itory effect on plasma growth hormone in acromegalic patients. A slight tumor shrinkage mayaiso occur in some cases, but does not appear to be a typical and dramatic effect of SMS 201 -995 therapy. No substantial histological or ultrastructural changes were seen in GH-producing tumors from patients treated with SMS 201-995. Nuclei, rough endoplasmic reticulum, Golgi complex and secretory granules were similar to the controls. Moderate or no changes in the size of cells, nuclei or nucleoli were observed (158). The drug does not seem, therefore, to exert effects comparable to those of bromocriptine on PRL synthesis, However, so far few adenomas have been analyzed and more studies are needed to further verify these observations, ACKNOWLEDGMENTS We wish to thank Drs, C, Capella, R, Buffa and C, Rlva (Dlpartimento di Patologia Umana ed Ereditaria, Sezione Anatomia Patologica 11, Universlta di Pavia a Varese) for providing Figures 2 and 7,

REFERENCES 1. Solcia E., Capella C, Buffa R., Frigerio B" Fontana P., Usellini L. Tumori dell'adenoipofisi: diagnosi morfologica e classificazione. Pathologica 69: 333, 1977. 2, Kovacs K., Horvath E, Pathology of pituitary tumors. Endocrinol. Metab, Clin. North Am, 16 529, 1987. 3. Landolt A.M, Ultrastructure of human sella tumors. Correlatlons of clinical findings and morphology. Acta Neurochirurgica (Suppl) 22 1, 1975, 4, Racadot J" Vila-Porcile E., Olivier L" Peillon F, Electron microscopy of pituitary tumours. Progr. Neurol Surg, 6 95, 1975. 5, Horvath E" Kovacs K. Ultrastructural classification of pituitary adenomas. Can. J. Neurol Sei. 3: 9, 1976. 6, Kovacs K., Horvath E., Ezrin C Pituitary adenomas, Pathol. Annu. 12: 341, 1977, 7. Girod C., Mazzuca M., Trouillas J" Tramu G., Lhentler M., Beauvillain J.-C, Claustrat B" Dubois M,·P Light microscopy, fine structure and immunohistochemistry studies of 278 pituitary adenomas, In Derome P,J" Jedynak CP., Peilion F. (Eds), Pituitary adenomas (11 Eur. Workshop, Pans 1979) Asclepios, Paris, 1980, p, 3,

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Morphological studies on pituitary adenomas

19. McNicol A.M. Pituitary adenomas. Histopathology 11: 995, 1987.

8. Martinez A.J., Lee A., Moossy J, Maroon J.C. Pituitary adenomas: clinicopathological and immunohistochemical study. Ann. Neurol. 7: 24, 1980.

20. Horvath E., Kovacs K. Pituitary gland. Path. Res. Pract. 183: 129, 1988.

9. Esiri M.M., Adams C.B.T., Burke C., Underdown R. Pituitary adenomas: immunohistology and ultrastructural analysis of 118 tumors. Acta Neuropathol. (Berl.) 62: 1, 1983.

21. Scheithauer BW., Horvath E., Kovacs K., Laws E.R. Jr., Randall RV, Ryan N. Plurihormonal pituitary adenomas. Semin. Diagn. Pathol. 3: 69, 1986.

10. Girod C. Immunocytochemistry of the vertebrate adenohypophysis. Handbuch der Histochemie, ed. 5. Gustav Fischer Verlag, Stuttgart-New York, 1983, vol. 8, p. 291.

22. Heitz P.U., Landolt A.M., Zen klusen H.-R., Kasper M., Reubi J-C., Oberholzer M., Roth J. Immunocytochemistry of pituitary tumors. J. Histochem. Cytochem. 35: 1005, 1987.

11. Lloyd R.V., Gikas PW, Chandler W.F. Prolactin and growth hormone-producing pituitary adenomas. An immunohistochemical and ultrastructural study. Am. J Surg. Pathol. 7: 251, 1983.

23. Guyda H., Robert F., Colle E., Hardy J. Histologic, ultrastructural, and hormonal characterization of a pituitary tumor secreting both hGH and prolactin. J. Clin. Endocrinol. Metab. 36: 531, 1973.

12. Mukai K. Pituitary adenomas. Immunocytochemical study of 150 tumors with clinicopathologic correlation. Cancer 52: 648, 1983.

24. Zimmerman EA, Defendini R., Frantz G. Prolactin and growth hormone in patients with pituitary adenomas: a correlative study of hormone in tumor and plasma by immunoperoxidase technique and radioimmunoassay. J Clin. Endocrinol. Metab. 38: 577, 1974.

13. Challa V.R., Marshall R.B., Hopkins M.B., Kelly D.L. Jr., Civantos F. Pathobiologic study of pituitary twmors: Report of 62 cases with a review of the re cent literature. Hum. Pathol. 16: 873, 1985.

25. Corenblum B., Sirek A.M.T., Horvath E., Kovacs K., Ezrin C. Human mixed somatotrophic and lactotrophic pituitary adenomas. J. Clin. Endocrinol. Metab. 42: 857, 1976.

14. Randall R.V., Scheithauer BW, Laws E.R., Abboud C.F., Ebersold M.J, Kao P.C. Pituitary adenomas associated with hyperprolacttinemia: a clinical and immunohistochemical study of 97 patients operated on transsphenoidally. Mayo Clin. ProC. 60: 753, 1985.

26. Mashiter K., Van Noorden S., De Marco L., Adams E., Joplin G.F. Hormone secretion by human somatotrophic, lactotrophic, and mixed pituitary adenomas in culture. J. Clin. Endocrinol. Metab. 48: 108, 1979.

15. Irsy G., G6th M., Balint K., Siowik F., Szabolcs 1., Pasztor E., Szilagyi G. Comparison of the serum hormone levels and histological findings in pituitary adenomas. Exp. Clin. Endocrinol. 88: 224, 1986.

27. Fukaya T., Kageyama N., Kuwayama A., Takanohashi M., Okada C., Yoshida J., Osamura Y. Morphofunctional study of pituitary adenomas with acromegaly by immunoperoxidase technique and electron microscopy. Cancer 45: 1598, 1980.

16. Saeger w., Schulze C., Lüdecke D.K. Immunhistologie der hypophysenadenome - Bedeutung für klassifikation und klinik. Verh. Dtsch. Ges. Path. 70: 347, 1986.

28. Trouillas J, Girod C., Lheritier M., Claustrat B., Dubois M.P. Morphological and biochemical relationships in 31 human pituitary adenomas with acromegaly. Virchows Arch. [Al 389: 127, 1980.

17. Asa S.L., Kovacs K. Histological classification of pituitary disease. Clin. Endocrinol. Metab. 12: 567, 1983.

29. Halmi N.S. Occurrence of both growth hormone- and prolactinimmunoreactive material in the cells of human somatotropic pituitary adenomas containing mammotropic elements. Virchows Arch. [A] 398: 19, 1982.

18. Kovacs K., Horvath E. Tumors of the pituitary gland. In: Atlas of tumor pathology, ed. 2. Armed Forces Inst. of Pathology, Washington DC, 1986, vol. 21, p. 1.

447

G. Giannattasio and M. Bassetti

30. Kanie N., Kageyama N., Kuwayarna A, Nakane T., Watanabe M., Kawaoi A Pituitary adenomas in acromegalic patients: an immunohistochemical and endocrinological study with special reference to prolactin-secreting adenoma. J Clin. Endocrinol. Metab. 57: 1093, 1983.

39. Horvath E., Kovacs K, Scheithauer BW, Randall R.V., Laws EH Jr., Thorner M.O., Tindall G.T., Barrow D.L. Pituitary adenomas producing growth hormone, prolactin, and one or more glycoprotein hormones a histologic, immunohistochemical, and ultrastructural study of four surgically removed tumors. Ultrastruct. Pathol. 5: 171, 1983.

31. Nieuwenhuijzen Kruseman AC., Bots GTh.AM., Roelfsema F., Frölich M., Van Dulken H. Immunocytochemical growth hormone and prolactin in pituitary adenomas causing acromegaly and their relationship to basal serum hormone levels and the growth response to thyrotrophin releasing hormone. Clin. Endocrinol. 19: 1, 1983.

40. Jaquet P., Hassoun J, Delori P., Gunz G., Grisoli F., Weintraub B.o. A human pitUitary adenoma secreting thyrotropin and prolactin: immunohistochemical, biochemical, and cell culture studies. J. Clin. Endocrinol. Metab. 59: 817, 1984.

32. Lamberts SWJ, Klijn JG.M., van Vroonhoven C.C.J, Stefanko S.Z. Different responses of growth hormone secretion to guanfacine, bromocriptine, and thyrotropin-releasing hormone in acromegalic patients with pure growth hormone (GH) - containing and mixed GH/prolactincontaining pituitary adenomas. J. Clin. Endocrinol. Metab. 60: 1148, 1985.

41. McComb D.J., Bayley TA, Horvath E, Kovacs K., Kourides IA Monomorphous plurihormonal adenoma of the human pituitary. A histological, immunocytological and ultrastructural study. Cancer 53: 1538, 1984. 42. Beck-Peccoz P, Piscitelli G., Amr S., Ballabio M., Bassetti M., Giannattasio G., Spada A, Nisslm M, Weintraub B.o., Faglia G. Endocrine, biochemical and morphological studies of a pituitary adenoma secreting growth hormone, thyrotropin (TSH), and a-subunit: evidence for secretion of TSH with increased bioactivity. J. Clin. Endocrinol. Metab. 62: 704, 1986.

33. Bassetti M., Spada A, Arosio M., Vallar L., Brina M., Gianf')attasio G. Morphological studies on mixed growth hormone (GH) - and prolactin (PRL) - secreting human pituitary adenomas. Coexistence of GH and PRL in the same secretory granule. J. Clin. Endocrinol. Metab. 62: 1093, 1986. 34. Kovacs K., Horvath E. Pathology of growth hormone-producing tumors of the human pituitary. Semin. Diagn. Pathol. 3: 18, 1986.

43. Osamura R.Y., Watanabe K. Immunohistochemical studies of human FSH producing pituitary adenomas. Virchows Arch. [Al 413 61 , 1988.

35. Bassetti M., Arosio M., Spada A, Brina M, Bazzoni N, Faglia G., Giannattasio G. Growth hormone and prolactin secretion in acromegaly: correlation between hormonal dynamics and immunocytochemical findings. J. Clin. Endocrinol. Metab. 67: 1195, 1988.

44. Saeger W., Geisler F, Lüdecke D.K. Pituitary pathology in Cushing's disease. Path. Res. Pract. 183 592, 1988. 45. Travaglini P, Bassetti M., Spada A, Togni M.E., Faglia A Mixed LH, FSH, GH, pituitary microadenoma: clinical and morphological studies. In: Landolt AM. (Ed.), Advances in the biosciences, Pergamon Press, Oxford, 1988, vol. 69, p. 435.

·36. Robert F., Pelletier G., Serri 0., Hardy J. Mixed growth hormone and prolactin-secreting human pituitary adenomas. A pathological, immunocytochemical, ultrastructural, and immunoelectron microscopic study. Hum. Pathol. 19: 1327, 1988. 37. Bassetti M., Brina M., Spada A, Giannattasio G. Somatomammotrophic cells in GH-secreting and PRL-secreting human pituitary adenomas. J. Endocrinol. Invest. 12: 705, 1989.

46. Trouillas J, Girod C., Loras B, Claustrat B., Sassolas G., Perrin G., Buonaguidi R. The TSH secretion in the human pituitary adenomas. Path. Res. Pract. 183 596, 1988.

38. Holm R., NesJand J.M., Attramadal A, Reinli S., Johannessen J.v. Mixed growth hormone- and prolactin cell adenomas of the pituitary gland. An immunoelectron microscopic study. J. Submicrosc. Cytol. Pathol. 21: 339, 1989.

47. Malarkey W.B., Kovacs K, O'Dorisio T.M. Response of a GH- and TSH-secreting pituitary adenoma to a somatostatin analogue (SMS 201-995) : evidence that GH and TSH coexist in the same cell and secretory granules. Neuroendocrinology 49 267, 1989.

448

Morphological studies on pituitary adenomas

58. Landolt AM., Heitz P.U., Zen klusen H.-R. Production of the a-subunit of glycoprotein hormones by pituitary adenomas. Path. Res. Pract. 183: 610, 1988.

48. Ambrosi B., Bassetti M., Ferrario R., Medri G., Giannattasio G., Faglia G. Precocious puberty in a boy with PRL-, LH- and FSH- secreting pituitary tumour: hormonal and immunocytochemical studies. Acta Endocrinol. 1990, (in press).

59. Osamura R.Y. Immunoelectron microscopic studies of GH and asubunit in GH secreting pituitary adenomas. Path. Res. Pract. 183: 569, 1988.

49. Horvath E., Kovacs K. Gonadotroph adenomas of the human pituitary: sexrelated fine-structural dichotomy. A histological, immunocytochemical and electron-microscopic study of 30 tumors. Am. J. Pathol. 117: 429, 1984.

60. Kourides IA, Ridgway E.C., Weintraub B.o., Bigos S.T., Gershengorn M.C., Maloof F. Thyrotropin-induced hyperthyroidism: use of alpha and beta subunit levels to identify patients with pituitary tumors. J. Clin. Endocrinol. Metab. 45: 534, 1977.

50. Demura R., Jibiki K., Kubo 0., Odagiri E., Demura H., Kitamura K., Shizume K. The significance of a-subunit as a tumor marker for gonadotropin-producing pituitary adenomas. J. Clin. Endocrinol. Metab. 63: 564, 1986.

61. MacFarlane IA, Beardwell C.G., Shalet S.M., Darbyshire P.J., Hayward E., Sutton M.L. Glycoprotein hormone a-subunit secretion by pituitary adenomas: influence of external irradiation. Clin. Endocrinol. (Oxf.) 13: 215, 1980.

51. Girod D., Trouillas J., Claustrat B. The human thyrotropic adenoma: pathologic diagnosis in five cases and critical review of the literature. Sem in. Diagn. Pathol. 3: 58, 1986. 52. Trouillas J., Girod C., Sassolas G., Claustrat B. The human gonadotropic adenoma: pathologic diagnosis and hormonal correlations in 26 tumors. Semin: Diagn. Pathol. 3: 42, 1986.

62. MacFarlane IA, Beardwell C.G., Shalet S.M., Ainslie G., Rankin E. Glycoprotein hormone a-subunit secretion in patients with pituitary adenomas: influence of TRH, LHRH and bromocriptine. Acta Endocrinol. (Copenh.) 99: 487, 1982.

53. Asa S.L., Gerrie B.M., Kovacs K., Horvath E., Singer w., Killinger DW, Smyth H.S. Structure-function correlations of human pituitary gonadotroph adenomas in vitro. Lab. Invest. 58: 403, 1988.

63. Peterson R.E., Kourides IA, Horwith M., Duracott E., Vaughan E.o., Saxena B.B., Fraser RAR. Luteinizing hormone and a-subunit-secreting pituitary tumor: positive feedback of estrogen. J. Clin. Endocrinol. Metab. 52: 692, 1982.

54. Jautzke G. Simultaneous production of the alpha-subunit of glycoprotein hormones and other hormones in pituitary adenomas. Path. Res. Pract. 183: 601, 1988.

64. Chapman AJ., MacFarlane IA, Shalet S.M., Beardweil C.G., Dutton J., Sulton M.L. Discordant serum a-subunit and FSH concentrations in a woman with a pituitary tumor. Clin. Endocrinol. (Oxf.) 21: 123, 1984 ..

55. Samuels M.H., Wood W.M., Gordon D.F., Kleinschmidt-DeMasters BK, Lillehei K., Ridgway E.C. Clinical and molecular studies of a thyrotropin-secreting pituitary adenoma. J. Clin. Endocrinol. Metab. 68: 1211, 1989.

65. Borges J.L.C., Ridgway E.C., Kovacs K., Rogol A.o., Thorner M.O. Follicle-stimulating hormone-secreting pituitary tumor with concomitant elevation of serum a-subunit levels. J. Clin. Endocrinol. Metab. 58: 937, 1984.

56. Beck-Peccoz P., Bassetti M., Spada A., Medri G., Arosio M., Giannattasio G., Faglia G. Glycoprotein hormone a-subunit response to growth hormone (GH) -releasing hormone in patients with active acromegaly. Evidence for a-subunit and GH coexistence in the same tumoral Gell. J. Clin. Endocrinol. Metab. 61: 541, 1985.

66. Weintraub B.o., Gershengorn M.C., Kourides IA, Fein H. Inappropriate secretion of thyroid-stimulating hormone. Ann. Intern. Med. 95: 339, 1981. 67. Kovacs K., Horvath E., Corenblum B., Sirek A.M.T., Penz G., Ezrin C. Pituitary chromophobe adenomas consisting of prolactin cells. A histological, immunocytological and electron microscopic study. Virchows Arch. [Al 366: 113, 1975.

57.lshibashi M., Yamaji T., Takaku F., Teramoto A, Fukushima T. Secretion of glycoprotein hormone a-subunit by pituitary tumors. J. Clin. Endocrinol. Metab. 64: 1187, 1987.

449

G. Giannattasio and M. Bassetti

78. Landolt A.M., Heitz PU Alpha-subunit-producing pituitary adenomas. Virchows Arch. A [Pathol. Anat.] 409: 417, 1986.

68. Kovacs K., Corenblum B., Sirek A.MT, Penz G, Ezrin C. Localization of prolactin in chromophobe pituitary adenomas: study of human necropsy material by immunoperoxidase technique. J. Clin. Pathol. 29: 250, 1976.

79. Snyder P.J., Baskey J.M., Kim S.U, Chappel S.G. Secretion of uncombined subunits of LH by gonadotroph cell adenomas. J. Clin. Endocrinol. Metab. 59: 1169, 1984.

69. Kovacs K, Horvath E., Ryan N, Ezrin G. Null cell adenoma of the human pituitary. Virchows Arch. [A] Path. Anat. Histol. 387: 165, 1980.

80. Snyder P.J. Gonadotroph adenomas of the pituitary. Endocrinol. Rev. 6 552, 1985.

70. Capella C., Buffa R., Usellini L., Frigerio B., Jehenson P., Sessa F., Solcia E. Alpha and beta subunits of glycoprotein hormones in argyrophil pituitary tumors with small granule cells. Ultrastruct. Pathol. 4: 35, 1983.

81. Ridgway E.C., Kieffer J.D., Ross OS, Oowning M., Mover H., Chin WW Mouse pituitary tumor li ne secreting only the alphasubunit of the glycoprotein hormones: development from a thyrotropic tumor. Endocrinology 113: 1587, 1983.

71. Martinez A.J. The pathology of nonfunctional pituitary adenomas. Semin. Oiagn. Pathol. 3: 83, 1986.

82. Ridgway E.C., Klibanski A., Ladenson PW., Clemmons 0., Beitins 11, McArthur JW., Martorana M.A, Zervas NT Pure alpha-secreting pituitary adenomas. N. Engl. J. Med. 304: 1254, 1981.

72. Black P. McL., Hsu OW., Klibanski A., Kliman B., Jameson L.J., Ridgway E.C., Hedley-Whyte ET, Zervas NT Hormone production in clinically nonfunctioning pituitary adenomas. J. Neurosurg. 66: 244, 1987.

83. Klibanski A., Ridgway E.C., Zervas NT Pure alpha subunit-secreting pituitary tumors. J. Neurosurg. 59: 585, 1983. 84. Asa S.L., Gerrie B.M, Singer W, Horvath E., Kovacs K., Smyth H.S. Gonadotropin secretion in vitro by human pituitary null cell adenomas and oncocytomas. J. Clin. Endocrinol. Metab. 62: 1011, 1986.

73. Jameson J.L., Klibanski A., Black P. McL., Zervas NT, LindeIl G.M., Hsu OW, Ridgway E.C., Habener J.F. Glycoprotein hormone genes are expressed in clinically nonfunctioning pituitary adenomas. J. Clin. Invest. 80: 1472, 1987.

85. Holck S., Laursen H. Silent pituitary adenomas. An ultrastructural study. Acta Path. Microbiol. Immunol. Scand. Sect. A, 94: 13, 1986.

74. Heitz PU Multihormonal pituitary adenomas. Horm. Res. 10: 1, 1979.

86. Holck S., Wewer U.M., Albrechtsen R. Heterogeneity of secretory granules of silent pituitary adenomas. Mod. Pathol. 1: 212, 1988.

75. Heshmati H.M., Turpin G., Kujas M, Lam X., Van Efferlterre R., Racadot J., de Gennes J.L. The immunocytochemical heterogeneity of silent pituitary adenomas. Acta Endocrinol. (Copenh.) 118: 533, 1988.

87. Yamada S., Asa S.L., Kovacs K. Oncocytomas and null cell adenomas of the human pituitary: morphometric and in vitro functional comparison. Virchows Arch. [A] 413: 333, 1988.

76. Kwekkeboom O.J, Oe Jong F.H, Lamberts S.WJ. Gonadotropin release by clinically nonfunctioning and gonadotroph pituitary adenomas in vivo and in vitro: relation to sex and effects of thyrotropin-releasing hormone, gonadotropin-releasing hormone, and bromocriptine. J. Clin. Endocrinol. Metab. 68: 1128, 1989.

88. Günzl H.J, Saeger W, Oiehl S, Lüdecke OK Immunohistochemical analyses of oncocytlc and chromophobe pituitary adenomas. Exp. Clin. Endocrinol. 92 51, 1988. 89. Horvath E, Kovacs K., Smyth HS, Killinger OW, Scheithauer BW., Randall R., Laws E.R. Jr., Singer W. A novel type of pituitary adenoma: morphological features and clinical correlations. J. Clin. Endocrinol. Metab. 66: 1111, 1988.

77. Jameson J.L., LindeIl C.M., Hsu OW., Habener J.F., Ridgway E.C. Expression of chorionic gonadotropin-ß-like messenger ribonucleic acid in an a-subunit-secreting pituitary adenoma. J. Clin. Endocrinol. Metab. 62: 1271, 1986.

450

Morphological studies on pituitary adenomas

100. Zurschmiede C., Landolt AM. Distribution of growth hormone and prolactin in secretory granules of the normal and neoplastic human adenohypophysis. Virchows Arch. [B] 53: 308, 1987.

90. Kovacs K., L10yd R., Horvath E., Asa S.L., Stefaneanu L., Killinger DW, Smyth H.S. Silent somatotroph adenomas of the human pituitary. A morphological study of three cases including immunocytochemistry, electron microscopy, in vitro examination, and in situ hybridization. Am. J. Pathol. 134: 345, 1989.

101. Beckers A, Courtoy R., Stevenaert A, Boniver J., Closset J., Frankenne F., Reznik M., Hennen G. Mammosomatotropes in human pituitary adenomas as revealed by electron· microscopic double gold immunostaining method. Acta Endocrinol. (Copenh.) 118: 503,1988.

91. L10yd R.v. Analysis of human pituitary tumors by in situ hybridization. Path. Res. Pract. 183: 558, 1988. 92. Tong Y., Zhao H.F., Simard J., Labrie F., Pelletier G. Electron microscopic autoradiographic localization of prolactin mRNA in rat pituitary. J. Histochem. Cytochem. 37: 567, 1989.

102. Heshmati H.M., Kujas M., Turpin G., Van Effenterre R., Racadot J., Oe Gennes J.L. Imunocytochimie des adenomas hypophysaires mixtes (hormone de croissance et prolactine) en double marquage. Presse Med. 18: 1437, 1989.

93. Trouillas J., Girod C., Sassolas G., Claustrat B., Lheritier M., Dubois M.P., Goutelle A Human pituitary gonadotropic adenoma: histological, immunocytochemical, and ultrastructural and hormonal studies in eight cases. J. Pathol. 135: 315, 1981.

103. Osamura R.Y., Watanabe K. Immunohistochemical colocalization of growth hormone (GH) and a-subunit in human GH secreting pituitary adenomas. Virchows Arch. [Al 411: 323, 1987.

94. Kameya T., Tsumuraya M., Adachi 1., Abe K., Ichikizaki K., Toya S., Demura R. Ultrastructure, immunohistochemistry and hormone release of pituitary adenomas in relation to prolactin production. Virchows Arch. [Al 387: 31, 1980.

104. Neill J.D., Frawley L.S. Detection of hormone release from individual cells in mixed populations using areverse hemolytic plaque assay. Endocrinology 112: 1135, 1983.

95. Ishikawa H., Nogami H., Kamio M., Suzuki T. Single secretory granules contain both GH and prolactin in pituitary mixed type of adenoma. Virchows Arch. [A] 399: 221,1983.

105. Frawley L.S., Boockfor F.R., Hoeffler J.P. Identification by plaque assay of a pituitary cell type that secretes both growth hormone and prolactin. Endocrinology 116: 734, 1985.

96. Geuze H.J., Siot JW, Van Der Ley PA, Scheffer R.C.T., Griffith J.M. Use of colloidal gold particles in double-Iabeling immunoelectron microscopy of ultrathin frozen tissue sections. J. Cell Biol. 89: 653, 1981.

106. Fumagalli G., Zanini A In cow anterior pituitary growth hormone and prolactin can be packaged in separate granule of the same Gell. J. Cell Biol. 100: 2019, 1985.

97. Bendayan M. Double immunocytochemical labeling applying the protein A-gold technique. J. Histochem. Cytochem. 30: 81, 1982.

107. Nikitovich-Winer M.B., Atkin J., Maley B.E. Colocalization of prolactin and growth hormone within specific adenohypophyseal cells in male, female and lactating female rats. Endocrinology 121: 625, 1987.

98. Horvath E., Kovacs K., Killinger DW., Smyth H.S., Weiss M.H., Ezrin C. Mammosomatotroph cell adenoma of the human pituitary: a morphologic entity. Virchows Arch. [A] 398: 277, 1983.

108. L10yd R.v., Anagnostou 0., Cano M., Barkan L., Chandler W.F. Analysis of mammosomatotropic cells in normal and neoplastic human pituitarytissues by the reverse hemolytic plaque assay and immunocytochemistry. J. Clin. Endocrinol. Metab. 66: 1103, 1988.

99. Felix IA, Horvath E., Kovacs K., Smyth H.S., Killinger DW, Vale J. Mammosomatotroph adenoma of the pituitary associated with gigantism and hyperprolactinemia. A morphological study including imunoelectron microscopy Acta Neuropathol. (Berlin) 71: 76, 1986.

109. Frawley L.S. Mammosomatotropes current status and possible functions. Trends Endocrinol. Metab. 1: 31, 1989.

451

G. Giannattasio and M. Bassetti

110. Newmann G.R., Jasani B., Williams E.D. Multiple hormone storage by cells of the human pituitary. J. Histochem. Cytochem. 37: 1183, 1989.

119. Pearse A.G.E. Peptides in brain and intestine. Nature 262: 92, 1976.

110. Bassetti M., Huttner WB., Zanini A.; Rosa P. bis Colocalization of secretogranins/ chromogranins with thyrotropin and luteinizing hormone in secretory granules of cow anterior pituitary. J. Histochem. Cytochem. (in press).

120. Lloyd R.V., Sisson J.C., Shapiro B., Verhofstad AAJ. Immunohistochemical localization of epinephrine, norepinephrine, catecholamine-synthesizing enzymes, and chromogranin in neuroendocrine cells and tumors. Am. J. Pathol. 125: 45, 1986.

111. Holm R., Nesland J.M., Attramadal A., Halse J., Johannessen J.v. Null cell adenomas of the pituitary gland, an immunohistochemical study. J. Pathol. 158: 213, 1989.

121. Schmechel D., Marangos P.J., Brightman M. Neurone-specific enolase is a molecular marker for peripheral and central neuroendocrine cells. Nature 276: 834, 1978.

112. Hsu DW, Riskind P.N., Hedley-Whyte E.T. Vasoactive intestinal peptide in the human pituitary gland and adenomas. An immunocytochemical study. Am. J. Pathol. 135: 329, 1989.

122. Pählman S., Esscher T., Nilsson K. Expression of y-subunit of enolase, neuron-specific enolase, in human non-neuroendocrine tumors and derived cell lines. Lab. Invest. 54: 554, 1986.

113. Kimura N., Andoh N., Sasano N., Sasaki A., Mouri T. Presence of neurophysins in the human pituitary corticotrophs, Cushing's adenomas, and growth horinone-producing adenomas detected by immunohistochemical study. Am. J. Pathol. 125: 269, 1986.

123. Asa S.L., Ryan N., Kovacs K., Singer W, Marangos P.J. Immunohistochemicallocalization of neuron-specific enolase in the human hypophysis and pituitary adenomas. Arch. Pathol. Lab. Med. 108: 40, 1984.

114. Saint-Andre J.-P., Rohmer V., Alhenc-Gelas F., Menard J., Bigorgne J.-C., Corvol P. Presence of renin, angiotensinogen, and converting enzyme in human pituitary lactotroph cells and prolactin adenomas. J. Clin. Endocrinol. Metab. 63: 231, 1986.

124. Stefaneanu L., Ryan N., Kovacs K. Immunocytochemical localization of synaptophysin in human hypophyses and pituitary adenomas. Arch. Pathol. Lab. Med. 112: 801, 1988.

115. Saint-Andre J.-P., Rohmer V., Pinet F., Rousselet M.C., Bigorgne J.C., Corvol P Renin and cathepsin B in human pituitary lactotroph cells. An ultrastructural study. Histochemistry 91: 291, 1989.

125. Wiedenmann B., Franke WW, Kuhn C., Moll R., Gould V.E. Synaptophysin: a marker protein for neuroendocrine cells and neoplasms. Proc. Natl. Acad. Sci. USA 83: 3500, 1986.

116. Buffa R., Rindi G., Sessa F., Gini A., Capella C., Jahn R., Navone F., De Camilli P., Solcia E. Synaptophysin immunoreactivity and small clear vesicles in neuroendocrine cells and related tumours. Mol. Cell. Probes 2: 367, 1988.

126. Gould V.E., Wiedenmann B., Lee 1., Schwechheimer K., Dockhorn-Dworniczak B., Radosevich JA, Moll R., Franke WW. Synaptophysin expression in neuroendocrine neoplasms as determined by immunocytochemistry. Am. J. Pathol. 126: 243, 1987.

117. Navone F., Jahn R., Di Gioia G., Stukenbrok H., Greengard P., De Camilli P. Protein p38: an integral membrane protein specific for small vesicles of neurons and neuroendocrine cells. J. Cell Biol. 103: 2511, 1986. 118. Navone F., Di Gioia G., Matteoli M., De Camilli P. Small synaptic vesicles and large dense-core vesicles of neurons are related to two distinct types of vesicles of endocrine cells. In: Thorn NA, Treiman M., Petersen O.H. (Eds.), Molecular mechanisms in secretion. Alfred Benzon Symposium 25, Munksgaard, Copenhagen, 1988, p.433.

127. Rosa P., Hille A., Lee RWH., Zanini A., De Camilli P., Huttner WB. Secretogranins land 11: two tyrosine-sulfated secretory proteins common to a variety of cells secreting peptides by the regulated pathway. J. Cell Biol. 101: 1999, 1985. 128. Rindi G., Buffa R., Sessa F., Tortora 0., Solcia E. Chromogranin A, Band C immunoreactivities of mammalian endocrine cells. Distribution, distinction from costored hormones/prohormones and relationship with the argyrophil component of secretory granules. Histochemistry 85: 19, 1986.

452

Morphological studies on pituitary adenomas

129. Winkler H., Apps D.K., Fischer-Colbrie R. The molecular function of adrenal chromaffin granules: established facts and unresolved topics. Neuroscience 18: 261, 1986.

(chromogranin B) in neuroendocrine cells and tumors. Am. J. Pathol. 130: 296,1988. 139. Sobol R.E., Memoli V., Deftos L.J. Hormone-negative, chromogranin A-positive endocrine tumors. . N. Engl. J Med. 320: 444, 1989.

130. Huttner W.B., Benedum U.M., Rosa P. Biosynthesis, structure and function of the secretogran ins/ chromogranins. In: Thorn NA, Treiman M., Petersen O.H. (Eds.), Molecular mechanisms in secretion. Alfred Benzon Symposium 25, Munksgaard, Copenhagen, 1988, p.380.

140. Deftos L.J, O'Connor D.T., Wilson C.B., Fitzgerald PA Human pituitary tumors secrete chromogranin-A. J. Clin. Endocrinol. Metab. 68: 869, 1989. 141. Benjannet S., Marcinkiewicz M., Falgueyret J.-P., Johnson D.E., Seidah N.G., Chretien M. Secretory protein 7B2 is associated with pancreatic hormones within normal islets and some experimentally induced tumors. Endocrinology 123: 874, 1988.

131. Somogyi P., Hodgson AJ., DePotter R.w., FischerColbrie R., Schober M, Winkler H., Chubb I.W. Chromogranin Immunoreactivity in the central nervous system. Immunochemical characterization, distribution and relationship to catecholamine and enkephalin pathways. Brain Res. Rev. 8: 193, 1984.

142. Natori S., Iguchi H., Nawata H., Kato K.-I., Ibayashi H., Nakagaki H., Chretien M. Evidence for the release of a novel pituitary polypeptide (7B2) from the growth hormone-producing pituitary adenoma of patients with acromegaly. J. Clin. Endocrinol. Metab. 66: 430, 1988.

132. 0' Connor D.T., Burton 0., Deftos L.J. Chromogranin A: immunohistology reveals its universal occurrence in normal polypeptide hormone pmducing endocrine glands. Life Sei. 33: 1657, 1983.

143. Ironside J.w., Royds JA, Jefferson AA, Timperley WR. Immunolocalization of cytokeratins in the normal and neoplastic human pituitary gland. J. Neurol. Neurosurg. Psychiatry 50: 57, 1987.

133. Lloyd R.V., Wilson B.S., Kovacs K., Ryan N. Immunohistochemical localization of chromogranin in human hypophyses and pituitary adenomas. Arch. Pathol. Lab. Med. 109: 515, 1985.

144. Girod C., Trouillas J., Dubois M.P. Immunocytochemical localization of S-100 protein in stellate cells (folliculo-stellate cells) of the anterior lobe of the normal human pituitary. Cell Tissue Res. 241: 505, 1985.

134. Rosa P., Fumagalli G., Zanini A, Huttner WB. The major tyrosine-sulfated protein of the bovine anterior pituitary is a secretory protein present in gonadotrophs, thyrotrophs, mammotrophs and corticotrophs. J. Cell Biol. 100: 928, 1985.

145. Tachibana 0., Yamashima T. Immunohistochemical study of folliculo-stellate cells in human pituitary adenomas. Acta Neuropathol. 76: 458, 1988.

135. Rundie S., Somogyi P., Fischer-Colbrie R., Hagn C., Winkler H., Chubb I.W. Chromogranin A Band C: immunohistochemicallocalization in ovine pituitary and the relationship with hormone-containing cells. Regul. Pept. 16: 217, 1986.

146. Bassetti M., Spada A, Pezzo G., Giannattasio G. Bromocriptine treatment reduces the cell size in human macroprolactinomas: a morphometric study. J. Clin. Endocrinol. Metab. 58: 268, 1984.

136. Hashimoto S., Fumagalli G., Zanini A, Meldolesi J. Sorting of three secretory proteins to distinct secretory granules in acidophilic cells of cow anterior pituitary. J. Cell Biol. 105: 1579, 1987.

147. Hassoun J, Jaquet P., Devictor B., Andonian C., Grisoli F, Gunz G., Toga M. Bromocriptine effects on cultured human prolactinproducing pituitary adenomas: in vitro ultrastructural, morphometric, and immunoelectron microscopic studies. J Clin. Endocrinol. Metab. 61: 686, 1985.

137. Oe Stephano D.B., Lloyd RV, Pike AM., Wilson B.S. Pituitary adenomas. An immunohistochemical study of hormone production and chromogranin localization. Am. J. Pathol. 116: 464, 1984.

148. Saeger W., Thiel M., Caselitz J., Lüdecke D.K. In vitro effects of bromocriptine on isolated pituitary adenomas cells. Ultrastructural and morphometrical studies. Path. Res. Pract. 180: 697, 1985.

138. Lloyd R.V., Cano M., Rosa P., Hille A, Huttner WB. Distribution of chromogranin A and secretogranin I

453

G. Giannattasio and M. Bassetti

154. Niwa J, Minase T., Mori M., Hashi K. Immunohistochemical, electron microscopic, and morphometric studies 01 human prolactinomas after short-term bromocriptine treatment. Surg. Neurol. 28: 339, 1987.

149. Mauer RA Transcriptional regulation 01 the prolactin gene by ergocryptine and cyclic AMP. Nature 294: 94, 1981. 150. Tindall G.T., Kovacs K., Horvath E., Thorner M.O. Human prolactin-producing adenomas and bromocriptine: a histological, immunocytochemical, ultrastructural, and morphometric study. J. Clin. Endocrinol. Metab. 55: 1178, 1982.

155. Gen M., Uozumi T., Ohta M., Ito A., Kajiwara H, Mori

S. Necrotic changes in proractinomas after long term administration 01 bromocriptine. J. Clin. Endocrinol. Metab. 59 463, 1984.

151. Landolt A.M., Osterwalder V., Landolt TA Bromocriptine-induced removal 01 endoplasmic membranes Irom prolactinoma cells. Experientia 41: 640, 1985.

156. Esiri M.M., Bevan J.S., Burke CW., Adams C.B.T. Effect 01 bromocriptine treatment on the fibrous tissue content 01 prolactin-secreting and nonfunctioning macroadenomas 01 the pituitary gland. J. Clin. Endocrinol. Metab. 63: 383, 1986.

152. Saitoh Y., Mori S., Arita N., Hayakawa T., Mogami H., Matsumoto K., Mori H. Cytosuppressive effect 01 bromocriptine on human prolactinomas: stereological analysis 01 ultrastructural alterations with special relerence to secretory granules. Cancer Res. 46: 1507, 1986.

157. Mori H., Maeda T., Saitoh Y., Onishi T. Changes in prolactinomas and somatotropinomas in humans treated with bromocriptine. Path. Res. Pract. 183: 580, 1988. 158. George S.R., Kovacs K., Asa SL, Horvath E, Cross E.G., Burrow G.N. Effect 01 SMS 201-995, a long - acting somatostatin analogue, on the secretion and morphology of a pituitary growth hormone cell adenoma. Clin. Endocrinol. 26: 395, 1987.

153. Schottke H., Saeger W., Lüdecke D.K., Caselitz J. Ultrastructural morphometry 01 prolactin secreting adenomas treated with dopamine agonists. Path. Res. Pract. 181: 280, 1986.

454