Folliculo-stellate Cells in Pituitary Adenomas of Patients with Acromegaly

PATHOLOGY RESEARCH AND PRAQlCE

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Folliculo-stellate Cells in Pituitary Adenomas of Patients with Acromegaly Doris Volt', Wolfgang Saeger' and Dieter K. Ludecke/ 1 Institute of Pathology, Marienkrankenhaus, Hamburg; 2Neurosurgical Clinic, University of Hamburg, Hamburg, Germany

Summary Pituit ary adenoma tissue from patients with acrome galy (n = 286) was obtained by surgery and examined for folliculo-stellate cells by immunostaining for S100 protein. The number of immunostained cells varied from one adenoma to another. A hundred and ninety-eight pituitary adenomas (69%) contained S100 protein positive cells corresponding to folliculo-stellate cells (FSC): in 100 cases (35%), only few sparse FSC were found , in 43 cases ( 15%) FSC were scattered throughout the adenoma, and in 55 cases (19%) there was an abundance of FSC. There were no significant differences regarding sex or age of the patients. The relative amount of FSC varied among different adenoma types . Plurihormonal adenomas showed the highest FSC density , whereas the majority of monohormonal adenomas contained only few or no FSC. Patients with pituitary adenomas containing scattered FSC had a significantly higher preoperative mean GH level than patients with pituitary adenomas not having FSC. There was a negative correlation between the FSC density in adenoma tissue and the preoperative mean PRL level. There was no correlation between the tumour size and the amount of FSC . Our data indicate no effect of the duration of symptoms on the FSC density and do not suggest a correlation between FSC density and the percentage of a certain hormone-secreting cell type.

Key words: S100 protein - Immunohistochemistry Folliculo-stellate cells - Pituitary adenomas - Acromegaly

6, 10, 11, 19, 22]. In 1957, Farquhar found agranular follicle-composing cells, the so called "follicular cells ," in the adenohypophysis of the rat [5 J. Ever since, these cell s have been investigated in various species of animal s and reported in the human normal adenohypophysi s with an undefined biological role [3, 6, 8, 12, 13, 17,20,22,23, 24J. According to Bergland and Torack [3 J, who have described the electron microscopical appearance in detail, 3%--4% of the total pituitary cell population is made up of stellate cells . For a long time, FSC in pituitary adenomas were reported as an incidental finding in sporadic cases [6, 25]. The investigations of Lauriola et al. confirmed the relatively frequent existence of FSC in adenoma tissue [18 J. For the studies of FSC, not only S 100 protein was used , but also GFAP [11 , 26J. Tsuchida et al. [25J found that FSC react both to GFAP and to CK (due to specific co-expression of these intermediate filaments by FSC). The sustentacular cells found in the adrenal medulla and its tumours [21J, as well as in paragangliomas [14J and some carcinoids [2J, share many similarities with the FSC of the pituitary and may be of the same type and origin. The aim of our present study was to determine the distribution of FSC in pituitary adenomas of patients with acromegaly and to stud y their relationship with clinical parameters.

Material and Methods Pituitary adenoma tissue from 286 patients with acromegaly was obtained by surgery via transsphenoidal and in

Introduction Folliculo-stellate cells (FSC) in the normal anterior pituitary gland represent a group of agranular cells among the various endocrine parenchymal cells [I , 3, 4, Pathol. Res. Pract. 195 : 143-147 (1999 )

Address for correspondence: Professor Dr. med. Wolfgang Saeger, Institut fur Pathologie des Marienkrankenhauses, Alfredstr. 9, D - 22087 Hamburg, Germany. Tel.: +49-(0)402546-2701, Fax: +49 -(0)40-2546-2730 0344-0338/99/195/3 -143 $12.00/0

144 . D. Voit et al.

very few cases via transcranial approach. The total collection comprised 1517 pituitary operations with 1259 adenomas and 403 cases with acromegaly. A hundred and seventeen cases had to be excluded due to lack of sufficient clinical data or too small specimens for further pathological evaluations . Among the 286 patients, there were 145 males and 141 females. The diagnosis of active acromegaly was based on the classical clinical findings as well as on laboratory findings, such as preoperative hormone levels. The mean age of the patients was 43.3 years, ranging from 7 to 69 years. The mean duration of symptoms was 7.7 years, ranging from 1 to 25 years. The preoperative mean growth hormone level was 54.9 Jlg/l (range 4.5 to 440 Jlg/l). The preoperative mean prolactin level was 26.1 Jlg/l (range I to 434 ug/l), All tumours were diagnosed and classified by histology, immunohistochemistry and, in part, by electron microscopy. For immunohistochemistry the tissue was immunostained using the Avidin-Biotin-Peroxidase Complex method applying primary antibodies against all anterior pituitary hormones and the alpha-subunit. Our primary antibodies applied in the Avidin-Biotin-Peroxidase Complex method were: Anti-GH (Sigma Chemie, Deisenhofen) with dilution 1:1200, anti-PRL (Immunotech Hamburg) with dilution 1:100, anti-ACTH 2439 (Dako Hamburg) with dilution 1:300, anti-TSH (lmmunotech Hamburg) with dilution 1:100, anti-FSH (Immunotech Hamburg) with dilution 1:200, anti-LH (lmmunotech Hamburg) with dilution 1:200, and anti-alpha subunit (lmmunotech Hamburg) with dilution 1:100. Normal anterior pituitary glands from autopsies were used as a control.

To detect FSC, the Avidin-Biotin-Comp lex method was employed using anti-S100 protein (Dako Hamburg, polyclonal) with dilution 1:800. They presented with the characteristic cell type and were found isolated or grouped, often in close relationship with adenoma cells. The sections were then reviewed by two authors (DY, WS) and FSC were identified on the basis of star-like structures

Fig. 2. Scattered FSC (Category S2) in a densely granulated GH cell adenoma: medium number of stellate cells between adenoma cells (S100-protein-ABC, magnification 440 x).

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Fig. 1. Few dispersed FSC (Category S 1) in a densely granulated GH cell adenoma: Few stellate cells within adenoma tissue (S100-protein-ABC, magnification 220x) .

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Fig. 3. Abundant FSC (Category S3) in a plurihormonal adenoma (S100-protein-ABC, magnification I lOx).

Table I. Folliculo-stellate cells in various adenoma types (n = 286)

Densely granulated GH cell adenomas Sparsely granulated GH cell adenomas Mixed GHlPRL cell adenomas Mammosoma totroph adenomas PIurihormonal adenomas type I

NoFSC

FewFSC

Scattered FSC

Abundant FSC

Total

9 (42%) 18 (29%) 16 (43%) 7 (4 1%) 38 (26%)

9 (38%) 24 (39%) 11(30%) 6 (35%) 50 (34%)

2 (8%) 13 (21%) 3 (8%) I (6%) 24 (16%)

3 (13%) 6 (10%) 7 (19%) 3 (18%) 36 (24%)

23 (100%) 61 (100%) 37 (100%) 17 (100%) 148 (100%)

Folliculo-stellate Cells in Pituitary Adenomas . 145 and S 100 protein-reactivity.The number and distribution of FSC result in the following four categories: SO =Complete absence of folliculo-stellate cells in the adenoma tissue S 1 = Few widely dispersed folliculo-stellate cells S2 = Scattered folliculo-stellate cells S3 =Abundant folliculo-stellate cells Seven per cent of adenomas were classified as densely granulated GH cell adenomas expressing only GH. Twenty-one per cent were sparsely granulated GH cell adenomas with typical fibrous bodies and sparse granulation in semithin sections and expression of GH only. Thirteen per cent were mixed GHlProlactincell adenomas showing one part of cells with GH expression and one part with Prolactin expression and different cell structures in semi-thin sections. Six per cent had to be classified as mammosomatotroph adenomas which show one cell type expressing both hormones. Most adenomas were plurihormonal adenomas (51%) expressing GH, mostly Prolactin and always one of two gonadotropic hormones.

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There was no obvious relationship between the age of patients and FSC density in their adenomas. Mean age in the four categories ranged from 41.2 to 45.1 years. There were no differences regarding the sex of the patients. In all four categories, the distribution of age and sex was almost equal. Relationship of hormone levels with FSC

Patients with pituitary adenomas containing scattered FSC had a significantly higher preoperative mean GH level than those without FSC (Tab. 2). Furthermore, there was an inverse correlation between FSC density and the preoperative mean PRL level (Tab. 2): The more

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With the use of anti-human S 100 protein antibody, a variable number of cells were immunostained. These cells showed the characteristic star-shaped appearance and were arranged not only in the peri phery of adenoma cell cords, but also between adenoma cells. In some cases, the expanding cytoplasmic processes partly or completely surrounded adenoma cells. In some specimens, the S 100 reactive cells were focally accumulated or formed network-like structures. A hundred and ninety-eight of the pituitary adenomas studied contained FSC: few widely dispersed FSC (category S1) were found in 100 cases (35%) (Fig. 1), scattered FSC (category S2) were seen in 43 cases (15%) (Fig. 2), and abundant FSC (category S3) were demonstrated in 55 cases (19%) (Fig. 3). In 88 cases, the specimens had no FSC (31%). The number of immunostained cells varied from one adenoma to another, but without any significant difference between the various adenoma types. Monohormonal adenomas generally presented a lower FSC density, whereas plurihormonal adenomas showed a higher FSC density than other adenoma types (Tab. 1). Among those seven adenomas with additional immunopositivity for ACTH, four did not contain any FSC and three were within category S3 (abundant FSC).

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folliculo-stellate cells were present in the adenomas, the lower was the preoperative PRL level. Relationship of tumour size with FSC

There was no correlation between the tumour size of the various adenoma types and FSC density. The adenomas without FSC (category SO) had a mean tumour size of 15.2 mm, the adenomas with few FSC (category Sl) measured 17.7 mm, the adenomas with scattered FSC (category S2) had a mean tumour size of 13.8 mm and those with many FSC (category S3) had a mean tumour size of 15.2 mm (Table 2). Relationship duration of symptoms with FSC

Our data suggest no correlation between duration of symptoms and FSC density in the various adenoma types. In all four categories, the patients had a mean duration of symptoms between 7.1 and 8.0 years (Table 2).

Discussion In the normal pituitary gland, FSC were found to have a supporting function similar to glial cells. In addition, they may playa role in the transport and exchange of pituitary cell products. Some authors suggested a possible paracrine regulatory function on secretory cells [10, 23]. Their metabolic role was already previously postulated due to the presence of cytoplasmic organelles, lysosomes and phagosomes [6]. Still, in 1984 Hofler et al. [l1J - unsuccessful in detecting S100-reactive FSC in pituitary adenomas - postulated that the immunohistochemical demonstration of S100 protein in pituitary tissue was a "reliable aid in the discrimination between adenomas and normal pituitary tissue." In our opinion, the lack of FSC is of no practical diagnostic significance in routine work. Recent studies focused on the role of epidermal growth factors (EGF) and epidermal growth factor receptors (EGFr) within the pituitary gland [15, 16]. The effects of growth factors, a group of polypeptides with growth-promoting properties, have been studied primarily in rat pituitaries and tissue cultures. Localization studies of EGF and EGFr in human non-tumorous pituitaries have been performed only recently [16J, and immunohistochemical staining revealed that EGFr were localized in stellate cells of non-tumorous adenohypophysis. While the first studies reported EGFr in human non-functioning adenomas, Kontogeorgos et al. detected EGFr in 50% of functioning tumours [16]. Others [4 J found an increased number of FSC in the normal gland adjacent to the adenoma. It was postulated that their presence may suggest an in-

volvement in tumour progression and cell proliferation [4,16]. Ishikawa et al. [12J were able to demonstrate in-vitro cultured FSC secreting S100 protein into the medium. This protein stimulated the release of PRL from a clone ofPRL secreting cells. Giometto et al. [7 J, too, supported the theory that FSC are able to influence the hormone production and to modulate the immunoendocrine connections. Studying the senescent procine pituitary, Ogawa et al. [20 J found PRL cells and, occasionally, LH cells in close association with FSC. In our study, there was an inverse correlation between FSC density in the adenoma tissue and the preoperative PRL level of the patients. On the other hand, there was a positive correlation between increasing FSC density and the mean preoperative GH level, which could indicate a relationship between GH secretion and metabolic function of FSC. The relationship between FSC and growth hormone secretion [15J confirms the findings of Shirasawa et al. [23J, who postulated that SIOO protein was a source for a variety of follicular activites requiring a carrier protein function. The occurrence of S100 reactive cells in ACTH-producing pituitary adenomas seems to be extremely rare, as only a few cases have been reported so far [6, 11]. At present, we cannot say whether the combination of FSC and ACTH-positive adenoma cells, as observed in some of our cases, is only incidental. However, the findings by Kontogeorgos et al. [16J emphasize a possible interaction of growth factors, growth factor receptors and tumour progression in corticotroph adenomas. Further studies need to be done. Is there any relationship between FSC density and the proliferative activity of the adenomas? Hayashi et al. [9 J found a positive correlation between the S 100 protein level of glial tumours and its degree of malignancy, but these findings in tumour cells cannot be compared with FSC within pituitary adenomas, as these are apparently not tumour cells. In our series, there was no correlation between tumour size and FSC density. Sparsely granulated GH cell adenomas are known to have the most unfavourable biological behaviour among the various adenoma types in acromegalic patients [27 J, but they did not show any difference in FSC density compared to the other adenoma types. To clarify the various interactions between folliculostellate cells, growth factors, pituitary hormones and tumour progression, further studies are certainly required not only in acromegaly, but also in other adenoma types.

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Received: October 28, 1998 Accepted in revised version: January 5, 1999