Arthur S. Tischler, MD Humana Press

Vol. 16, No. 3, Fall 2005 ISSN: 1046-3976

NDOCRINE E ATHOLOGY P The Official Journal of the Endocrine Pathology Society

Editor:

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Arthur S. Tischler, MD

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Humana Press EP_16_3_cvr, spine

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11/8/05, 8:01 AM

Clinical Research

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Double Pituitary Adenomas

Double Adenomas of the Pituitary: Transcription Factors Pit-1, T-pit, and SF-1 Identify Cytogenesis and Differentiation R. A. Jastania, MD,1,3 K. O. Alsaad, MD,1,3 M. Al-Shraim, MD,1,3 K. Kovacs, MD, PHD,2,3 and S. L. Asa, MD, PHD1,3 Abstract The diagnosis of double adenomas of the pituitary can be very complex and is usually suspected on histological assessment of a specimen and confirmed by immunohistochemical and ultrastructural studies. The most commonly applied technique is currently immunohistochemical staining to localize the six pituitary hormones. Application of this technique may fail to identify double adenomas when hormone immunoreactivity is weak or absent in one or both cell populations. We examined specimens from eight patients diagnosed with double adenomas over a 15-yr period. We tested the ability to detect the difference in the two adenomas in each case using three immunostains for the pituitary transcription factors Pit-1, T-pit, and SF-1. We conclude that immunohistochemical localization of the transcription factors Pit-1, T-pit, and SF-1 accurately detects and classifies the distinct cytodifferentiation of double adenomas of the pituitary. Key Words: Double pituitary adenomas; transcription factors; cytogenesis; differentiation.

Introduction

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Department of Pathology, University Health Network & Toronto Medical Laboratories, 2 Department of Laboratory Medicine and Pathology, St. Michael’s Hospital, and 3 Department of Laboratory Medicine and Pathobiology, University of Toronto. Address correspondence to Dr. Sylvia L. Asa, Department of Pathology, University Health Network, Toronto Medical Laboratories, 610 University Avenue, Suite 4-302, Toronto, Ontario, M5G 2M9 Canada. E-mail: [email protected] Endocrine Pathology, vol. 16, no. 3, 187–194, Fall 2005 © Copyright 2005 by Humana Press Inc. All rights of any nature whatsoever reserved. 1046–3976/05/16:187–194/ $30.00

Although it is easy to recognize multiple adenomas, up to hundreds, in some organs like the colon, it is often difficult to diagnose even double adenomas in the pituitary gland. The importance of making this diagnosis is to correlate the patient presentation with the type of hormone production in the tumor [1]. Part of the difficulty is due to the failure of diagnostic imaging techniques, even MRI, to demonstrate more than one lesion. CT scan and MRI can detect microadenomas of 2–3 mm with a sensitivity of only 60% and 85% respectively [2]. Furthermore, post-operative imaging is more difficult to interpret, and it is often a challenge to detect residual adenoma tissue [3]. For this reason, the task of diagnosing double adenomas of the

pituitary usually falls to the pathologist. However, the fragmented nature of surgically resected pituitary tumors makes the issue extremely complex. The classification of pituitary adenomas is currently based on the profile of hormone production. The recent WHO classification classifies pituitary adenomas into Growth Hormone (GH)–producing adenomas, Prolactin (PRL)–producing adenomas; Thyroid Stimulating Hormone (TSH)–producing adenomas, Adrenocorticotrophic (ACTH)–producing adenomas, Follicular Stimulating Hormone/Luteinizing Hormone (FSH/LH) gonadotropin– producing adenomas, and null cell adenomas [4]. These lesions can be further subclassified into subtypes of each adenoma based on granularity and other subcellular features. 187

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By and large, this classification is based on the pathological assessment of pituitary adenomas including the results of immunohistochemical localization of hormones. Although the classification is clear, the interpretation of the results of immunohistochemistry is not always definitive. In some cases, electron microscopy (EM) may play a role to accurately determine the cell type comprising a tumor. However, in practice, EM is not done in all cases, and, frequently, the specimen sent to EM is not sufficient or does not contain any tumor. The first difficulty in immunohistochemistry for hormones is the often focal positivity of tumors, especially among the sparsely granulated adenomas (PRL-producing and GH-producing) [5]. Another problem is the cross-reactivity of antisera to more than one hormone. Typical examples are cross-reactivity of GH with PRL, and contamination of antibodies to the common α-subunit in antisera to β-TSH, β-FSH, and β-LH [6]. However, the currently available monoclonal antisera [6] have minimized or completely abolished this problem.

Fig. 1. Transcription factors at the stages of cell differentiation in pituitary tissue.

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Our understanding of cytodifferentiation in the pituitary gland has expanded with the identification of transcription factors that regulate cell differentiation and hormone production. There are many transcription factors and co-factors that have been shown to play a role in the development and cytodifferentiation of the pituitary gland (Fig. 1). These factors have elucidated distinct lineages of cell differentiation [7,8]. The transcription factor Pit-1 was the first pituitary-specific transcription factor identified; it is known to direct differentiation along a somatotroph lineage that gives rise to differentiated somatotrophs, mammosomatotrophs, lactotrophs, and thyrotrophs. The transcription factors T-pit and SF-1 control the cell lineages of corticotrophs and gonadotrophs, respectively [7,8]. The transcription factor Pit-1, also known as GHF-1, is a 33-kDa 291-aminoacid protein. Its role in the cytodifferentiation and hormone production is well described in rodent and human pituitaries [9,10]. In rodents, Pit-1 is associated with the onset of GH and PRL production and correlates temporally and spatially with β-TSH [11–13]. In humans, Pit-1 expression is restricted to somatotrophs, mammosomatotrophs, lactotrophs, and thyrotrophs [14,15], and there is a temporal association between Pit-1 and GH expression that is followed at later stages of development by the cytodifferentiation of lactotrophs and thyrotrophs [16–19]. The transcription factor T-pit is a T box factor that was found selectively in proopiomelanocortin (POMC)–expressing corticotroph cells of the pituitary as well as in hypothalamic POMC-producing neurons. T-pit is a transcriptional activator of POMC expression that interacts with other proteins, known as corticotroph upstream transcription element-binding (CUTE) proteins, such as neuroD1/beta2.

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T-pit is considered the corticotroph-specific factor, since the other proteins are not cell-type specific [20]. Inactivating mutations of T-pit have been found in patients with isolated deficiency of pituitary POMC-derived ACTH [21,22]. These data indicate an essential role of T-pit for differentiation of the pituitary corticotroph lineage. Owing to limited antibody availability, the experience in T-pit diagnostic application has not been widely replicated. Steroidogenic factor-1 (SF-1) is a member of the steroid receptor superfamily. It regulates the expression of the steroidogenic enzymes, and is expressed in adrenal cortex, granulosa and theca cell of the ovary, and Leydig cells of the testis [23,24]. SF-1 also regulates the mullerian inhibiting substance (MIS) gene to determine mullerian duct regression [25]. Studies of the role of SF-1 in the pituitary demonstrated its restricted expression in gonadotroph lineage and mice lacking a functional SF-1 gene fail to develop gonadotrophs [26,27]. In humans, SF-1 expression correlates with gonadotropin β-subunit rather than α-subunit. SF-1 expression is characteristic of gonadotroph adenomas and null cell adenomas that are known to produce gonadotropin subunits [28,29]. With the general concept of monoclonality in adenomas, it is expected to find one lineage of cell differentiation in an adenoma [5,30]. The demonstration of more than one hormone in a tumor usually is reflective of a plurihormonal adenoma; the majority of these represent tumors of the Pit-1 lineage that allows transdifferentiation and production of any of the hormones produced by somatotrophs, lactotrophs, and thyrotrophs [5,30]. However, occasional plurihormonal adenomas have been reported to contain hormones that reflect true lineage infidelity [31–33].

In some of these, however, it is possible that the lesion would represent a mixed or double adenoma—two distinct clones of tumor cells admixed with each showing lineage fidelity. To determine if the localization of pituitary lineage-specific transcription factors can be used to perform such studies, we examined a group of patients with documented double adenomas that had been examined using immunohistochemistry for hormones as well as electron microscopy to verify the distinct hormonal and structural profiles of asynchronous or topographically separate and distinct lesions.

Materials and Methods Identification of Double Adenomas

We identified retrospectively from our files eight patients who had a diagnosis of double adenomas of the pituitary following surgical resection of tumors over a 15-yr period (1989–2004). The diagnostic material was identified in 14 paraffinembedded tissue blocks. The double adenomas were characterized by histology, hormonal profile using immunohistochemistry, and ultrastructural studies. Immunohistochemical Technique

To test the ability to detect the difference in the two adenomas in each case, we examined the 14 blocks for the presence of three pituitary transcription factors: Pit-1, T-pit and SF-1. Paraffin sections were dewaxed in five changes of xylene and brought down water through graded alcohols. Sections were then microwaveheated in 10 mM citrate buffer at pH 6.0 inside a pressure cooker as previously optimized. Endogenous peroxidase and biotin activities were blocked, respectively, using 3% hydrogen peroxide and Vector’s avi-

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din/biotin blocking kit (cat. no. SP-2001, Vector Laboratories Inc., Burlingame, CA). Sections were treated for 10 min with 10% normal goat serum and then incubated for 1 h or overnight with the appropriate antibody in a moist chamber. The polyclonal antiserum to Pit-1 was obtained from Berkeley Antibody Co. (Richmond, CA) and was used at a dilution of 1/700. The polyclonal antiserum to T-pit was generously provided by Dr. J. Drouin (Montreal, Quebec, Canada) and was applied at a dilution of 1/300. The polyclonal antiserum to SF-1 was purchased from Upstate Biotechnology Inc. (Lake Placid, NY) and diluted 1/3000. Incubation with primary antiserum was followed by 30 min each with a biotinylated goat anti-rabbit IgG (cat. no. BA-1000, Vector Laboratories Inc.) and HRP-conjugated Ultra Streptavidin labeling reagent (cat. no. BP2378, ID Labs. Inc, London, ON, Canada). Color development was done with freshly prepared NovaRed solution (cat. no. SK-4800, Vector Laboratories Inc.) and counterstained with Mayer’s hematoxylin. For negative controls, primary antisera were replaced with normal serum of the same species.

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Results Interpretation

In each case, the two adenomas were identified morphologically, and nontumorous pituitary tissue was noted if found, as well. The results of the three transcription factors Pit-1, T-pit, and SF-1 on immunohistochemical slides were reported as positive or negative. Positive results were reported when at least 75% of an adenoma cells showed strong nuclear staining, otherwise the case was reported as negative. Non-tumorous pituitary tissue was used as an internal control for positive staining for all the three transcription factors. Results Eight patients with a diagnosis of double adenomas were identified—five females and three male patients ranged in age from 23 to 80 (average 55 yr). In all patients, we identified double adenomas. Table 1 summarizes the clinical and pathological characteristics of each case, with the results of the transcription factor expression in each adenoma. All three transcription factors were identified in non-tumorous adenohypophysis (Fig. 2). One patient had discrete

Table 1. List of Patients, Tumor Diagnoses, and Transcription Factor Expression First adenoma

Transcription factor expression

Two separate tumors

Prolactin cell adenoma

Pit-1

F M

Composite tumor Two sharply demarcated

42

F

5

23

M

Collision, some areas the two tumors intermingled Two separate tumors

6

75

F

Composite tumor

7

62

F

Two separate tumors

8

80

M

Two sharply demarcated

Prolactin cell adenoma Prolactin cell adenoma, sparsely granulated Prolactin cell adenoma, sparsely granulated Plurihormonal adenoma (GH, PRL, TSH) Plurihormonal adenoma (GH, TSH) Silent ACTH adenoma, subtype 1 Silent ACTH adenoma, subtype 1

N

Age

Sex

1

37

F

2 3

49 62

4

Morphology

Second adenoma

Transcription factor expression

Pit-1 Pit-1

GH cell adenoma, sparsely granulated with fibrous bodies ACTH cell adenoma ACTH cell adenoma

T-pit T-pit

Pit-1

ACTH cell adenoma

T-pit

Pit-1

Silent ACTH adenoma (POMC Cells) Silent ACTH adenoma, subtype 1 Gonadotroph cell adenoma (FSH, LH) Gonadotroph cell adenoma (FSH, LH)

T-pit

Pit-1 T-pit T-pit

Pit-1

T-pit SF-1 SF-1


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Fig. 2. (top—left and right) Immunostaining for transcription factors in non-tumorous adenohypophysis. (A) The transcription factor Pit-1 is identified in scattered cells of a fragment of non-tumorous pituitary tissue trapped within a gonadotroph adenoma that is negative for this factor. (B) The transcription factor T-pit has typical nuclear reactivity in the same area of non-tumorous pituitary tissue. Again the gonadotroph adenoma is negative. Fig. 3. (middle—center) Pit-1 expression in an adenoma. The transcription factor Pit-1 is localized to the nuclei of a pituitary adenoma. There is minor variation in the intensity of staining but almost all adenoma cells are positive. Fig. 4. (bottom—left and right) Transcription factor localization in a double (collision) adenoma. This collision tumor has two components. One is positive for Pit-1 in an area that stained for GH and TSH (A) and the other expresses T-pit in an ACTH-producing adenoma (B).

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lactotroph and somatotroph adenomas that were both positive for Pit-1 (Fig. 3). Three patients had adenomas containing prolactin and ACTH. There was discrete expression of Pit-1 and T-pit in the individual adenomas (Fig. 4). Two patients had a silent corticotroph adenoma associated with a separate adenoma containing growth hormone/prolactin/TSH; the former expressed T-pit and the latter contained Pit-1. Two patients had gonadotroph and silent ACTH adenomas, and again each tumor contained the appropriate transcription factor, either SF-1 or T-pit (Table 1). There was no crossover staining; no transcription factors were identified as positively staining in inappropriate cell types. Discussion Pathologists consider light microscopy the gold standard for diagnosis and classification of tumors. However, routine morphologic assessment has many limitations and pathologists continue to introduce new techniques that aid in the confirmation of diagnosis and classification of tumors. Traditionally, pituitary adenomas were classified grossly as microadenomas and macroadenomas based on the size of the lesion, and they were identified histologically as acidophilic, basophilic or amphophilic, and chromophobic adenomas. Clinically, they are divided into functional and non-functional adenomas. With the introduction of immunohistochemistry and the possibility to accurately determine hormone production by adenoma cells, the classification is now based on the type of hormone that the adenoma cells produce. The addition of electron microscopy allowed subclassification of tumors producing specific hormones into subgroups, based on the degree of granularity and other subcellular features, such as the pres-

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ence or absence of fibrous bodies, and altered Golgi features [5]. This has led to a new and fairly complex classification of pituitary adenomas that has therapeutic and prognostic as well as diagnostic importance. Although this approach is still valid, sometimes there is difficulty in the interpretation of immunohistochemical stains with some overlap in hormonal profile in the same cells. Some tumors are immunonegative for hormones, but they express other factors, such as transcription factors, that can allow clarification of cell lineage commitment. In our study, we introduce the use of the cytodifferentiation lineage concept in the diagnosis of double pituitary adenomas and compare it with terminal cytodifferentiation and hormone production. Immunohistochemical stains for the transcription factors Pit-1, T-pit, and SF-1 are useful, easy to interpret, and accurately determine the cell lineage of a given tumor. As described in Table 1, the transcription factor Pit-1 marks adenoma cells showing differentiation toward somatotrophs, mammosomatotrophs, lactotrophs, and thyrotrophs. The transcription factor T-pit indicates corticotroph differentiation, and the transcription factor SF-1 shows gonadotroph differentiation. Double adenomas of the pituitary are rare, found in approx 1% of autopsy pituitaries [34]. This number is much lower in surgical specimens but the incidence is not accurately known. In the literature there are several small series of reported cases of double adenomas [35–39]. It has been said that the incidence of double adenomas is 1.6–3.3% in patients with Cushing’s syndrome [38]. Most pituitary adenomas, including double adenomas, are sporadic neoplasms [5,30]; occasionally they occur in the setting of multiple endocrine neoplasia (MEN) type 1. Tumor formation

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theories include an underlying pituitary molecular defect (intrinsic) and an overstimulation sequence by hormones and other factors (extrinsic) initiating neoplasms [5,30]. The occurrence of double adenomas always triggers a search for predisposing factors. However, there are no well-known factors described. There is a peculiar case report of a double (composite) pituitary adenoma arising in a background of hyperplasia [40]. Before making a diagnosis of double adenoma, one must verify that there are truly two distinct neoplasms. In most cases, immunohistochemistry is the tool pathologists rely on to make such an interpretation. The use of transcription factors to determine the lineage of cytodifferentiation will certainly help to clarify such situations.

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