Pituitary cells producing more than one hormone human pituitary adenomas

Pituitary Cells Producing More Than One Hormone Human Pituitary Adenomas K. Kovacs, E. Horvath, S.L. Asa, L. Stefaneanu,

and T. Sano

The existence of cells capable of producing more than one hormone in nontumorous human adenohypophyses and pituitary adenomas has been conclusively proved. In light of the evidence, current concepts on pituitary structure, function, and regulation as well as adenoma cytogenesis and classification have to be reconsidered.

Thirty years ago, pituitary cytology seemed to be simple and noncontroversial. For histologic studies, tissues were fixed in formalin, embedded in paraffin, and the sections were stained with hematoxylin-eosin and various trichrome technics. Based on the available information, it was claimed that six distinct cell types existed in the anterior pituitary, each of them producing one of the six known adenohypophyseal hormones. It was thought that acidophilic somatotrophs secreted GH, acidophilic lactotrophs secreted PRL, basophilic corticotrophs secreted ACTH, basophilic thyrotrophs secreted TSH, basophilic gonadotrophs secreted FSH, and basophilic gonadotrophs secreted LH. Chromophobic cells were believed to be hormonally inactive. This one cell-one hormone theory, which was accepted by every authority and dominated the field of pituitary morphologic studies for many years, emphasized the classic dogma of endocrinology: one endocrine cell can synthesize and release only a single hormone. However, the initial one cell-one hormone theory was modified in subsequent years when several studies provided evidence that the two gonadotropins, FSH and LH, were produced by the same cell. Thus, only one gonadotroph was recognized, reducing the

K. Kovacs, E. Horvath, S.L. Asa, L. Stefaneanu. and T. Sano are at the Department of Pathology, St. Michael’s Hospital, University of Toronto, Toronto, Ontario MSB lW8, Canada.

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number of adenohypophyseal cell types to five. Other advances necessitated modification of this scheme with respect to corticotrophs. It was revealed that corticotrophs synthesize a large glycoprotein molecule, a prohormone, called proopiomelanocortin (POMC), which is cleaved to several smaller fragments, including ACTH, endorphins and other peptides. Corticotrophs, therefore, came to be regarded as cells capable of secreting several peptides. As a result of these alterations, a modified one cell-one hormone theory recognized five clearly distinguishable cell types in the adenohypophysis: GH-secreting somatotrophs, PRL-secreting lactotrophs, corticotrophs that produce ACTH and other POMC-derived peptides, TSHsecreting thyrotrophs, and gonadotrophs synthesizing the two gonadotropins, FSH and LH. The morphologic classification of pituitary tumors also followed the simplistic view. Based on the staining affinities of the cell cytoplasm, pituitary adenomas were classified into acidophilic (eosinophilic), basophilic, and chromophobic types. Acidophilic adenomas were assumed to produce GH and to be associated with acromegaly or gigantism. Basophilic adenomas were linked with ACTH excess and were claimed to cause Cushing’s disease. Chromophobic adenomas were thought to represent inactive tumors unaccompanied by oversecretion of any known adenohypophyseal hormones. With increasing knowledge, it became obvious that a pituitary adenoma classification based on the tinctorial

characteristics of the cell cytoplasm was not satisfactory, because it fails to take into account structure-function correlations. It was conclusivelv demonstrated that acidophilic adenomas can secrete not only GH, but also PRL, or they can be hormonallv inactive. It was shown that basophilic adenomas are not invariably associated with ACTH excess, and that they can be silent (i.e., unaccompanied by oversecretion of any known adenohypophyseal hormones). It also became apparent that chromophobic adenomas frequently produce various hormones (such as GH, PRL, ACTH, or one or more glycoprotein hormones) and can be responsible for a variety of hypersecretory endocrine phenomena (such as acromegaly, gigantism, the amenorrhea-galactorrhea syndrome, hyperadrenocorticism or hyperthyroidism). It was not possible to correlate adenohypophyseal structure with endocrine activity and resolve the uncertainties regarding pituitary adenoma classification during the era of hematoxylin-eosin and trichrome stains. The breakthrough came with the introduction of immunocytochemistry. This technique permitted, for the first time, the specific localization of hormones in the cell cytoplasm. Electron microscopy revealed the fine structural feaof the cell, provided a deeper insight into the details of the secretory process, and proved to be reliable in cell identification as well as tumor diagnosis. Electron microscopic immunocytochemistry allowed the detection of hormones in subcellular structures. The immunogold technique with doublelabeling was especially useful, because it made possible the electron microscopic demonstration of two hormones in the same cell, even in the same secretory granule. Other methods that were valuable in assessing whether cells produce or release multiple hormones include tissue culture with measurement of hormones in the culture medium by radioimmunoassay (RIA) (Asa et al. 1986; Malarkey et al. 1989), the reverse hemolytic plaque assay (Yamada et al. 1989), and in situ hybridization, which discloses gene expression by demonstrating mRNA (Kovacs =t al. 1989; Lloyd 1987; Lloyd et al. 1989). How true is the quote: “A grand hypothesis is not the usual path for advancement of medical knowledge. As a rule, first comes a

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new or improved method whose application to a variety of problems leads unexpectedly to greater understanding” (Paddock 1968). The application of these novel techniques proved the existence of plurihormonal ceils in nontumorous adenohypophyses and pituitary adenomas and shattered the one cell-one hormone theory, a concept that was based on insufficient information. Conan Doyle has Sherlock Holmes say: “I had come to an entirely erroneous conclusion, which shows, my dear Watson, how dangerous it is always to reason from insufficient data.” Furth and his associates were the first to suggest that adenomas in the rodent pituitary may be plurihormonal (Furth and Clifton 1966). The existence of human plurihormonal pituitary adenomas was not demonstrated until the late 1970s. We described three new types of pituitary adenoma, mixed somatotrophic-lactotrophic

termed ade-

noma, acidophil stem cell adenoma, and mammosomatotroph cell adenoma (Corenblum et al. 1976; Horvath et al. 1977, 1981, 1983a). These three tumor types produce two hormones, namely, GH and PRL. In 1982, we reported a plurihormonal pituitary adenoma removed surgically from a young man with typical clinical and biochemical manifestations of acromegaly, but no evidence of TSH excess (Kovacs et al. 1982). By electron microscopy, we found that the tumor was composed of thyrotroph-like cells that were immunoreactive for both GH and TSH. At that time, we thought the combination of GH and TSH immunoreactivity in pituitary adenomas to be rare. However, within the next few years, several similar cases were published, indicating that plurihormonal pituitary adenomas producing GH and TSH concomitantly cannot be regarded as infrequent findings (Kovacs and Horvath 1986; Scheithauer et al. 1986). lmmunocytochemical examination of human pituitary samples obtained at autopsy showed that 68% of pituitary adenomas contained two or more adenohypophyseal hormones (Heitz 1979). Subsequent reports conclusively showed that pituitary adenomas containing more than one hormone are not uncommon in surgically removed hypophyseal tumor material (Horvath et al. 1983b; McComb et al. 1984; Schei-

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thauer et al. 1986; Kovacs and Horvath, 1988), the most frequent combination being GH and PRL. Other combinations that are not infrequent include GH and TSH, GH, PRL, and TSH, or the a-subunit of the glycoprotein hormone. Other combinations such as GH and ACTH, ACTH and LH, ACTH and a-subunit, etc., can occur; these plurihormonal adenomas are, however, rare. Plurihormonal adenomas are tumors capable of producing two or more hormones that differ in chemical composition, immunoreactivity, and biologic action. Some of the hormones present in the adenoma cells are released in excess, leading to various hypersecretory syndromes. However, discrepancies may exist between hormone content and hormone secretion, and the presence of immunoreactivity may not be reflected in clinical and biochemical alterations. Space does not permit us to speculate here on the possible causes of the absence of hormone excess in patients harboring silent tumors. Detailed immunocytochemical and ultrastructural studies revealed that plurihormonal pituitary adenomas can be divided into monomorphous and plurimorphous types. Monomorphous plurihormonal adenomas consist of one cell type that produces two or more hormones. The presence of multiple hormones can be documented by immunocytochemistry in the cytoplasm of the same cell. Monomorphous adenoma cells are often unusual and do not resemble any adenohypophyseal cell known to occur in the nontumorous pituitary gland. Plurimorphous plurihormonal adenomas are composed of two or more cell types, each of which is immunoreactive for only one hormone and is similar in ultrastructural appearance to its nontumorous counterpart. The distinction between monomorphous and plurimorphous adenomas is not possible in some cases. We have investigated tumors that were diagnosed as plurimorphous adenomas and seemed to consist of two distinct cell types by histology, light microscopic immunocytochemistry, and transmission electron microscopy. To our surprise, immunoelectron microscopy revealed two hormones in the same cells, even in the same secretory granules. In other studies, in situ hybridization demonstrated PRL gene expression in

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somatotroph clinically, logically

adenomas

biochemically, characteristic

that

were

and morphoGH-producing

tumors. Thus, these findings suggest that the distinction between monomorphous and plurimorphous adenomas is impossible in some cases, and some apparently plurimorphous the of adenomas in fact belong to the monomorphous tumor group. Immunocytochemical and electron microscopic studies of endocrine tumors arising in other organs have revealed that plurihormonality may be a widespread phenomenon. With this information, one of the most fundamental “laws” of endocrinology has been proved invalid. The concept that only one hormone can be produced by a specific endocrine cell type can no longer be accepted. Several questions remain unanswered: What is the real incidence of adenomas? What is plurihormonal their biologic significance? Are plurihormonal adenomas more aggressive, do they have a more rapid pace of growth, do they recur more frequently than the monohormonal variants? Do plurihormonal cells occur more frequently in tumors than in normal nontumorous adenohypophyses? What is the cytogenesis of plurihormonality? It is difficult to assess conclusively the incidence of plurihormonal pituitary adenomas. In tumors, even in large size adenomas, a few nontumorous adenohypophyseal cells are often noticeable among the adenoma cells and, in some cases, it cannot be decided with certainty whether the cells containing variable immunoreactivities are intermingled normal or adenomatous cells. If interspersed nontumorous cells are regarded erroneously as adenoma cells, the diagnosis of plurihormonal adenoma is not valid, and the estimated incidence of plurihormonality will be higher than in reality. On the other hand, adenomas are tested by immunocytochemistry only for the known adenohypophyseal hormones, and it is possible that they are immunoreactive for several other peptides, some perhaps possessing biologic activity. It may well be that every pituitary adenoma is plurihormonal and capable of producing several peptides. The incidence of plurihormonality depends to a great extent on terminology. At present, there is no general

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consensus as to which tumors should be classified as plurihormonal. One possibility is to label as plurihormonal all tumors that are positive for more than one hormone by immunocytochemistry. In this case, many densely granulated somatotroph adenomas, null cell adenomas, and oncocytomas could be classified as plurihormonal tumors, since they are often immunoreactive for more than one hormone. Alternatively, only those tumors that consist of cells lacking normal counterparts and containing unusual combinations of hormones would be considered plurihormonal. In this case, plurihormonal pituitary adenomas would be regarded as uncommon. Another solution would be not to use the term plurihormonal adenoma, and classify tumors on the basis of clinical and biochemical presentations, predominant cell type, hormone content, or cell of origin. Is plurihormonality important from the practical viewpoint? Are plurihormonal pituitary adenomas more aggressive, do they grow faster, do they recur more frequently than the monohormonal variants? It is known that pancreatic endocrine tumors and medullary carcinomas of the thyroid, which are plurihormonal, may have a more malignant course than their monohormonal counterparts. Although the biologic behavior of plurihormonal pituitary adenomas is not clearly delined, recent findings seem to suggest that in contrast to monohormonal adenomas, plurihormonal tumors are more often aggressive, possess a more rapid pace of growth, and have a higher recurrence rate. These are only preliminary results, and more cases with long-range followup are needed to provide conclusive evidence regarding the biologic behavior of pituitary tumors. The question of whether plurihormonal cells are less common in nontumorous adenohypophyses than in pituitary adenomas is not fully resolved. It has been documented that GH- and PRL- as well as GH- and TSH-containing cells occur in nontumorous human anterior lobes, but less commonly than in some adenomas. These plurihormonal cells could be called mammosomatotrophs and somatothyrotrophs. Our studies, still in progress, have not yet demonstrated other combinations in nontumorous human adenohypophyses, and more work is

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required to answer the question 01 whether other plurihormonal clones exist in the nontumorous pituitary gland. The cytogenesis of plurihormonal adenomas is obscure. It may be that these tumors originate in plurihormonal clones, which are present under normal conditions and as a result of neoplastic transformation begin to proliferate and continue to produce more than one hormone. Alternatively, it is possible that plurihormonal adenomas arise from endocrinologically inactive pluripotent precursor cells, which are dormant in the normal gland but in the course of tumor genesis or subsequent tumor progression undergo multidirectional differentiation and acquire the ability to synthesize two or more hormones. The latter hypothesis is especially attractive regarding null cell adenomas and oncocytomas that focally synthesize various adenohypophyseal hormones (mainly FSH, LH, and cY-subuni t). As more facts accumulate, it is becoming increasingly evident that tumor composition may undergo substantial alterations in the course of neoplastic progression. Mutation may occur; new clones exhibiting different phenotypes may arise. It may well be that some tumors originate as monohormonal adenomas and subsequently transform to the plurihormonal variant because of emergence of new clones. Tumor cell variability (i.e., morphologic difference within one clone) and tumor cell heterogeneity (i.e., formation of a new clone by mutation) are recognized facts. Plurimorphous plurihormonal adenomas may be examples of polyclonality. The time has arrived to apply molecular techniques to the study of pituitary adenomas. Analysis of gene expression may yield a deeper insight into the development of plurihormonal pituitary adenomas. Growth factors and oncogenes may alter tumor proliferation and lead to plurihormonality. More immunocytochemical markers are needed to investigate tumors that are capable of producing more than one peptide. Further use of the reverse hemolytic plaque assay revealing hormone secretion by single cells and in vitro studies of hormone release by RIA may shed light on the nature of plurihormonality in pituitary adenomas. The demonstration of surface, cytoplasmic, and nuclear receptors may provide new infor-

mation on the regulation of endocrine activity. Human pituitary adenoma ccl1 lines and their examination combined with morphology and biochemistr!, may contribute to a better understanding of the pathogenesis of these intriguing tumors. At present, there are more questions than answers, and more work is required to unravel the mysteries of hormone secretion by adenohypophyseal cells. We agree with George Bernard Shaw: “Science is always wrong. It never solves a problem without creating ten more.” This review lists references primarily from our own laboratory. Many other relevant papers were omitted owing to lack of space. The interested reader can find additional information from the publications quoted in the References.

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Acknowledgments

This work was supported in part by Grant MT-6349 awarded by the Medical Research Council of Canada. The authors are grateful to Mrs. G. Ilse, Mrs. J. Karpenko, Mrs. D. Lietz, Mrs. N. Losinski, and Mrs. N. Ryan, whose contribution made this review possible. We also wish to thank Mrs. M. Pasnik for typing the manuscript. References Asa SL, Gerrie BM, Singer W, Horvath E, Kovacs K, Smyth HS: Gonadotropin secretion in vitro by human pituitary null cell adenomas and oncocytomas. J Clin Endocrinol Metab 1986; 62:lOll. Corenblum B, Sirek AMT, Horvath E, Kovats K, Ezrin C: Human mixed somatotrophic and lactotrophic pituitary adenomas. J Clin Endocrinol Metab 1976; 42~857. Furth J, Clifton KH: Experimental pituitary tumors. In Harris GW, Donovan BT, eds. The Pituitary Gland, Volume 2. London, Butterworth, 1966, p 460. Heitz PU: Multihormonal pituitary mas. Hormone Res 1979; 10: 1.

adeno-

Horvath E, Kovacs K, Killinger DW, Smyth HS, Weiss MH, Ezrin C: Mammosomatotroph cell adenoma of the human pituitary: a morphologic entity. Virchows Arch A Path01 Anat 1983a; 398:277. Horvath E, Kovacs K, Scheithauer BW et al.: Pituitary adenomas producing growth hormone, prolactin, and one or more glycoprotein hormones: a histologic, immunohistochemical, and ultrastructural study of four surgically removed tumors. Ultrastruct Path01 1983b; 5:171.

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Horvath E, Kovacs K, Singer W, Ezrin C, Kerenyi NA: Acidophil stem cell adenoma of the human pituitary. Med 1977; 101:594.

Arch Pathol

1987;

18:1199.

Lloyd RV, Cano M, Chandler WF, Barkan AL, Horvath E, Kovacs K: Human growth hormone and prolactin secreting pituitary adenomas analyzed by in situ hybridization. Am J Path01 1989; 134:605.

Lab

Horvath E, Kovacs K, Singer W et al.: Acidophil stem cell adenoma of the human pituitary; clinicopathologic analysis of 15 cases. Cancer 1981; 47:761.

Malarkey WB, Kovacs K, D’Orisio TM: 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 1989; granules. Neuroendocrinology

Kovacs K, Horvath E: Tumors of the pituitary gland. In Atlas of Tumor Pathology, Second Series, Fascicle 21. Washington, DC, Armed Forces Institute of Pathology, 1986.

491267.

Kovacs K, Horvath E: Pathology of pituitary adenomas. In Collu R, Brown GM, VanLoon GR, eds. Clinical Neuroendocrinology. Boston, Blackwell, 1988, p 333.

McComb DJ, Bailey TA, Horvath E, Kovacs K, Kourides IA: Monomorphous phurihormonal adenoma of the human pituitary. A immunocytologic and ultrahistologic, structural study. Cancer 1984; 53: 1538.

Kovacs K, Horvath E, Ezrin C, Weiss MH: Adenoma of the human pituitary producing growth hormone and thyrotropin. Virchows Arch A Pathol Anat 1982; 395:59.

Paddock FK: Familiar Medical Quotations. Boston, Little, Brown, 1968, p 463. Scheithauer BW, Horvath E, Kovacs K, Laws ER Jr, Randall RV, Ryan N: Plurihormonal pituitary adenomas. Semin Diagn Pathol 1986; 3:69.

Kovacs K, Lloyd R, Horvath E et al.: Silent somatotroph adenomas of the human pituitary. A morphologic study of three cases including immunocytochemistry, electron in vitro examination, and in microscopy, situ hybridization. Am J Pathol 1989; 134:345.

Yamada S, Asa SL, Kovacs K, Muller P, Smyth HS: Analysis of hormone secretion by clinically nonfunctioning human pituitary adenomas using the reverse hemolytic plaque assay. J Clin Endocrinol Metab 1989; 68:73. TEM

Lloyd RV: Use of molecular probes in the study of endocrine diseases. Hum Pathol

4412C Cells A Neuroendocrine Model to Study Neuropeptide, Oncogene, and Growth Factor Gene Regulation FiisQn N. Zeytin

The neuroendocrine

44-2C cells synthesize,

secrete, and manifest

differential regulation of calcitonin (CT), CT gene-related peptide (CGRP), neurotensin (NT), and somatostatin (SS). These cells maintain differentiated function when grown in serum-free, growth factor and hormone-deprived milieu. The cells continue to secrete CT, CGRP, NT, and SS and differentially respond to cellular secretagogues. In serum-free cultures, the cells produce biologically active acidic fibroblast growth factor (aFGF) and differentially regulate the expression of the protooncogene fos (&OS).

One of the fundamental questions in to the neuroendocrinology pertains identity of the mechanism(s) whereby Fusun N. Zeytin is at the UCSD Medical Laboratory, School, Neuroendocrinology Basic Science Building M-003, La Jolla, CA 92093, USA.

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cells integrate the signals that reach the cell surface. There are currently several elegant models for studying in vitro neuroendocrine cell function. Among these are the pituitary GH, cell line established by Drs. Armen Tashjian and Gordon Sato, and the PC12 pheochromocytoma cell line established by Drs.

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Lloyd Greene and Art Tischler. These cell lines have been and are of great value to investigators. For example, the demonstration of the stimulation of prolactin (PRL) secretion by thyrotropin-releasing hormone (TRH) was first shown in GH3 cells (Tashjian and Hoyt 1972). The PC12 cells have most recently been used to show that fibroblast growth factor (FGF) mimics the effects of nerve growth factor (NGF) (Leonard et al. 1987). In the presence of NGF, PC12 cells differentiate from replicating chromaffin-like cells to nonreplicating synaptic neuron-like cells (Anderson et al. 1988). In this report, a third neuroendocrine cell model is described (44-2C cells). In addition to briefly reviewing the differential regulation of peptide secretion and gene regulation, the most recent findings of our group are described. These include the effects of acidic and basic FGFs on the expression of mRNAcr8 ccRP,andC-/O;the regulation by calcitonin (CT) of the expression of its own gene product; and the positive and negative modulation of mRNA’-‘““. To study the regulation of peptide synthesis and secretion, we established in culture several rat cell lines (6-23, 44-2, 44-3C) derived from the intrathyroidal C-cells shown to be of neural crest origin by LeDouarin et al. (1972; 1988). The initial focus of these experiments was to have a cell model secreting only one peptide, in this case, CT. Soon after the establishment of the 44-2 cell line, we discovered that these cells and the clones derived from them (i.e., 44-2C) also synthesized and secreted calcitonin gene-related peptide (CGRP), neurotensin (NT), and somatostatin (SS). Most recently, we have shown that these cells express mRNA coding for the nuclear proto-oncogene fos (c-fos) and acidic FGF (aFGF) (Zeytin et al. 1988a). To date, the 44-2C cells are the only reported neuroendocrine cell line that expresses only the acidic form (i.e., the brain form) of FGF and has receptors for both acidic and basic FGFs (see reviews by Gospodarowicz et al. 1986; Thomas 1987). The ability of the 44-2C cells to grow and maintain differentiated function in culture medium devoid of serum and all hormonal supplements (Zeytin et al. 1988a) has enabled studies on the induction of endogenous growth factors and oncogenes. The 44-2C cells provide

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