The Molecular Pathology of Pituitary Tumors

Transcription

1 Asa SL, Page 1 The Molecular Pathology of Pituitary Tumors Sylvia L. Asa, M.D., Ph.D. Department of Pathology, University Health Network and Toronto Medical Laboratories, Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5G 2M5 Abstract Pituitary tumors exhibit a spectrum of biology with variable growth and hormonal behaviors. They are classified based on cell differentiation that accounts for patterns of hormone synthesis and secretion. The various tumor types have unique pathogenetic mechanisms that underlie the neoplastic process. These include alterations in hormone regulation, growth factor stimulation, cell cycle control and cell-stromal interactions that are based on genetic mutations or epigenetic disruption of gene expression. Mouse models have validated the roles of these alterations. They can be targets for the development of therapies to manage these lesions that are being increasingly recognized as critical for quality of life Introduction Our understanding of pituitary development and cytogenesis, as well as tumor pathogenesis has been greatly advanced in the last 100 years. The impact of molecular technology has changed the concepts of hormone secretion and cell proliferation, the two major aspects of pituitary tumor biology. Historical Perspectives Galen ( AD) originally described the pituitary as the site of drainage of phlegm from the brain to the nose and throat. Descartes was the first to recognize that the brain integrates the functions of the mind and body in The association was reinforced by Zander who noted in 1890 the connection between the adrenals and the brain, referring to observations on the absence of the adrenal cortex in anencephaly as recorded by Morgagni in 1733, Soemmering in 1792 and Meckel in It was only in 1849 that direct evidence of a role for the hypothalamus in endocrinology was provided by Claude Bernard when he demonstrated that injury to the floor of the fourth ventricle, the piqûre diabetique, caused excessive urination; subsequently, numerous studies in the late 19th century and early 20th century confirmed that the hypothalamic-posterior pituitary system was the site of production of an important substance that regulated water retention. Soon after the description of acromegaly by Pierre Marie in 1886, the association of acromegaly with pituitary tumor was noted by Minkowski in 1887, and the recognition of endocrine functions of the pituitary followed rapidly thereafter, with major contributions by Cushing and Simmonds. The early part of the 20th century saw the identification, isolation and characterization of the hormones of the anterior pituitary. Their regulation by the hypothalamus was the subject of the landmark monograph by Harris in 1948, Neural control of the pituitary gland. Since these major discoveries, the identification of hypothalamicpituitary hormones has led to three Nobel Prizes, in 1954 for du Vigneaud and for Guillemin and Schally in Despite these tremendous advances in physiology, however, the study

2 Asa SL, Page 2 of pituitary pathology lagged well behind the volumes of information that clarified the basis of disease in many other organs. The 1960 s saw the first efforts to identify the pituitary cells involved in various pathological processes by Dr. Calvin Ezrin in Toronto. The 1970 s and 1980 s were the era of tumor classification by the renowned husband-and-wife team, Dr. Kalman Kovacs and Dr. Eva Horvath. They devised a classification scheme that remains the basis for our understanding of pituitary tumors. Today we recognize the various cell types of the pituitary and we are able to study their hormonal activity at the individual cell level with such tools as immunohistochemistry and reverse hemolytic plaque assays. We can accurately determine their structural alterations with the electron microscope. We can dissect the reasons for their ability to produce one hormone or another by analyzing the intricacies of transcription factors at the molecular level. We have been able, by careful analysis at autopsy and more recently by MRI, to determine that pituitary tumors are very common. Although classically considered to be rare, systematic assessments have shown a prevalence of 17% of the population (1). The sex incidence is equal. They are rare in childhood (2;3) and the incidence increases with age (4). Classification Hormonal activity characterizes pituitary adenomas. Corticotroph adenomas produce adrenocorticotropin (ACTH) and other pro-opiomelanocortin (POMC)-derived peptides and are associated with Cushing's or Nelson's syndromes. Somatotroph adenomas produce growth hormone (GH) and cause acromegaly and/or gigantism. Prolactin (PRL)-producing adenomas cause hyperprolactinemia, resulting in hypogonadism. Thyrotropin (TSH)-producing tumors are associated with thyroid dysfunction. Rare clinically-detectable gonadotroph adenomas cause elevations of circulating levels of follicle stimulating hormone (FSH) and/or luteinizing hormone (LH). The group of "endocrinologically-inactive adenomas are not associated with hormone excess syndromes. Anatomic/radiographic classification is based on tumor size and degree of invasion, information that is critical to guide the surgical approach when indicated. Morphologic classification is based on histology, immunohistochemistry and electron microscopy. This integrated approach allows identification of cell type and tumor subtype. Immunohistochemistry allows identification of transcription factors and hormone content that usually correlates with the clinical presentation (Table 1). Transcription factors that regulate hormone expression are used to classify cytogenesis even in hormone-negative adenomas (5-10). Ultrastructural classification by electron microscopy allowed identification of subcellular variants that now can be identified with immunohistochemical markers, such as keratins (11). It has become clear that there are three main pathways of cell differentiation in the anterior pituitary. ACTH-producing corticotrophs are determined by the expression of Tpit that binds with corticotropin upstream transcription-binding element (CUTE) proteins including neurod1/beta 2. Bihormonal gonadotrophs require expression of steroidogenic factor (SF)-1, GATA-2 and members of the Lhx gene family, particularly Lhx4. The complex family of Pit-1 expressing cells can mature into somatotrophs, mammosomatotrophs, lactotrophs or thyrotrophs with the additional expression of estrogen receptor (ER) alpha, which enhances PRL secretion, or thyrotroph embryonic factor (TEF), which stimulates TSH-beta production. The recognition of these molecular determinants of adenohypophysial cytodifferentiation has clarified the cytogenesis of a number of unusual tumors and has explained patterns of plurihormonality that

3 Asa SL, Page 3 have been recognized in pituitary adenomas, thereby providing a framework for classification of these tumors. Table 1: Pituitary cells, hormones, tumors and hormone excess syndromes Pituitary cell type Transcription Factor Hormone Tumor types Associated syndromes Densely granulated (basophilic) Sparsely granulated (chromophobic) Densely granulated (acidophilic) Sparsely granulated (chromophobic) Lactotroph Pit-1, ER PRL Sparsely granulated (Densely granulated) Cushing s and Nelson s syndromes Acromegaly, Gigantism Amenorrhea and galactorrhea (usually restricted to females), sexual dysfunction, infertility Acromegaly, Gigantism with hyperprolactinemia Hypo- or hyperthyroidism Corticotroph Tpit ACTH and other POMCderived peptides Somatotroph Pit-1 GH, α-subunit Mammosomatotroph Pit-1, ER GH, PRL, α-subunit Mammosomatotroph Thyrotroph Pit-1, TEF, TSH, Thyrotroph GATA-2 α-subunit Gonadotroph SF-1, ER, FSH, LH, Gonadotroph, null Lhx-4 α-subunit cell, oncocytoma GATA-2 *mass effects, hypopituitarism can occur with any tumor type when large Hypogonadism, mass effects, hypopituitarism* Hormonally inactive adenomas have been classified as null cell adenomas and oncocytomas in the past, but at the time of their description, Kovacs et al predicted that the terms would become obsolete as new markers emerged to classify them further (12). The expression of SF-1 by the majority of these adenomas has allowed them to be classified as members of the gonadotroph adenoma family (10). This is not a surprise, since it has been known for some 20 years that null cell adenomas and oncocytomas release gonadotropins in vitro (13-15), but this hormonal activity is often not detected by conventional immunohistochemistry. Thus, the application of new antisera for transcription factors allows cytogenetic classification in the absence of hormone positivity. Prognostic Factors The proliferative activity of pituitary tumors has been extensively investigated (16-18). Studies of proliferating cell nuclear antigen (PCNA), Ki-67/MIB-1, and Bcl-2 have unfortunately demonstrated no consistent correlation with tumor invasiveness or recurrence (17). Although invasive pituitary adenomas and carcinomas exhibit a high DNA topoisomerase II alpha (Topo II alpha) index, this indicator has no significant advantage over MIB-1 as a

4 Asa SL, Page 4 prognostic marker (19). Cyclooxygenase-2 (COX-2) expression correlates with patient age, but not with tumor size or invasiveness (20). Detection of telomerase expression may predict recurrence in pituitary adenomas (21). Galectin-3, a beta-galactoside-binding protein implicated in cellular differentiation and proliferation as well as angiogenesis, tumor progression and metastasis, may play a role in pituitary tumor progression (22). Unfortunately, none of these is a marker of biological behavior. The best predictive markers remain those that subclassify adenomas accurately based on hormone content and cell structure. For example, among acromegalics who fail surgical resection, response to long-acting somatostatin analogues is best predicted by the subtype of somatotroph adenoma as densely or sparsely granulated (23;24). This finding renders the value of a Cam 5.2 keratin stain to identify fibrous bodies as more important than any stain in this setting. A silent corticotroph adenoma will recur more often and more aggressively than a silent gonadotroph adenoma. And a silent subtype 3 adenoma will almost certainly behave invasively, infiltrating the base of the skull, while a silent adenoma of the gonadotroph lineage will usually grow by expansion upwards. It remains to be established whether growth factors and/or their receptors, oncogene products, or loss of tumor suppressor gene products will prove to be reliable predictors of invasive growth, recurrence, or metastasis. Pathogenesis Our understanding of pituitary tumorigenesis is largely derived from experimental studies in animal models and from molecular analyses of human pituitary tumors. Alterations of protooncogenes and tumor suppressor genes have been implicated in the pathogenesis of these tumors (25;26) (Table 2). Activating mutations of the Gsα gene on chromosome 20 have been demonstrated mainly in a subset of densely granulated somatotroph adenomas. Overexpression of the epidermal growth factor receptor (EGF-R) has also been implicated in the more aggressive behavior of recurrent somatotroph adenomas. Another member of the EGF family, TGF-α, is overexpressed in some pituitary adenomas and TGF-alpha overexpression in lactotrophs has been shown to result in prolactinoma formation in transgenic mice. FGF-2 (bfgf) and FGF-4 have been implicated in prolactinoma development. FGF-2 is also produced by other adenomas and elevated circulating levels are associated with aggressive tumors. A novel FGF antisense gene GFG regulates cell proliferation and hormone secretion. FGF receptor expression is altered in pituitary adenomas and one member of this family, FGFR4, undergoes alternative transcription initiation, giving rise to an oncogenic protein in almost half of pituitary adenomas of various subtypes. Expression of this pituitary tumorderived (ptd)-fgfr4 protein is more frequent in macroadenomas than in microadenomas and correlates with the Ki-67 labeling index. It appears that ptd-fgfr4 alters cell adhesion in a mechanism that explains the loss of reticulin that is the hallmark of pituitary adenomas. Other putative oncogenes include cyclin D1 overexpression and upregulation of PTTG (pituitary tumor transforming gene), a member of the securin family of proteins involved in chromosome separation during mitosis. Mutations of the ras genes have been implicated in the development of rare pituitary carcinomas. Loss of tumor suppressor gene expression is a critical event in pituitary tumorigenesis as shown by animal studies. p27 kip1 null mice develop multiorgan neoplasia, including pituitary tumors. Mice lacking both p18 ink4 and p27 kip1 succumb to lethal pituitary adenomas by three months of age. The expression of p27 is reduced in pituitary adenomas, mainly in ACTH-

5 Asa SL, Page 5 producing tumors. Hypermethylation of p16 is detected in adenomas of gonadotroph lineage. In contrast to these models, another mouse model of pituitary tumorigenesis, deficiency of the retinoblastoma (Rb) gene, does not seem to have human application. While the mice develop pituitary corticotroph adenomas arising in the intermediate lobe, human tumors have no mutation or loss of heterozygosity (LOH) at the Rb locus. This may be attributed to unique features of intermediate lobe corticotrophs that do not apply in humans. GADD45γ (growth arrest and DNA damage-inducible gene) is another tumor suppressor that is significantly reduced in clinically nonfunctioning pituitary adenomas using cdna-representational difference analysis; this too has been shown to be due to promoter methylation. Table 2: Summary of pituitary tumour dysregulated signaling and genetic events Misexpression Experimental Model Dysregulated hormone signaling GHRH Overexpressed GHRH transgenic mouse SST Underexpressed GH/IGF-1 GHR inactivating mutations Estrogen Dopamine D2R-KO Prolactin PRL-R-KO Dysregulated growth factor signaling TGF-α Overexpressed TGF-α transgenic mouse FGF/FGFRs Ligand overexpression Receptor truncation ptd-fgfr4 transgenic mouse Dysregulated signaling proteins Gsα Activating mutation gsp trangenic mouse Protein kinase A Mutation in Carney complex Cell cycle control Menin Mutation and LOH in MEN-1 Menin-KO mouse Rb Methylated Rb-KO mouse P27 Methylated p27-ko mouse P18 p18-ko mouse Other GADD45 Methylated MEG3a Since pituitary adenomas form an important part of the syndrome of multiple endocrine neoplasia type 1 (MEN-1), the menin gene was considered to be a critical tumor suppressor for pituitary. Although familial pituitary adenomas associated with MEN-1 have mutation and LOH of the menin locus at 11q13, downregulation of menin is rare or absent in the more common sporadic adenomas.

6 Asa SL, Page 6 The p53 gene does not seem to be of pathogenetic significance in pituitary tumors. Although p53 expression has been detected in adenomas of all types, and may be more common in recurrent neoplasms, there is no evidence of mutation or LOH and the significance of the immunoreactivity remains to be established. The vast majority of the genetic alterations in pituitary adenomas appear to be due to promoter methylation or acetylation with silencing of tumor suppressors. A role for the chromatin-remodelling protein Ikaros has been identified in the genesis of the oncogenic ptd- FGFR4. This takes the analysis of the molecular basis of pituitary tumorigenesis to the level of epigenetic regulation. Conclusions The history of pituitary pathology is short, but there has been exponential growth in our understanding of the cytogenesis and pathogenesis of these common lesions. The advent of molecular technology has allowed careful dissection of cell differentiation and the genetic and epigenetic changes that underlie tumor formation. It is anticipated that specific interruption of several signaling cascades will afford new approaches to the therapeutic armamentarium, permitting more effective strategies in the management of pituitary tumors. References 1. Ezzat S, Asa SL, Couldwell WT et al. The prevalence of pituitary adenomas: a systematic review. Cancer 2004; 101(3): Kane LA, Leinung MC, Scheithauer BW et al. Pituitary adenomas in childhood and adolescence. J Clin Endocrinol Metab 1994; 79: Mukai K, Seljeskog EL, Dehner LP. Pituitary adenomas in patients under 20 years old. A clinicopathological study of 12 cases. J Neurooncol 1986; 4: Kovacs K, Ryan N, Horvath E, Singer W, Ezrin C. Pituitary adenomas in old age. J Gerontol 1980; 35: Asa SL, Puy LA, Lew AM, Sundmark VC, Elsholtz HP. Cell type-specific expression of the pituitary transcription activator Pit-1 in the human pituitary and pituitary adenomas. J Clin Endocrinol Metab 1993; 77: Friend KE, Chiou Y-K, Laws ER, Jr., Lopes MBS, Shupnik MA. Pit-1 messenger ribonucleic acid is differentially expressed in human pituitary adenomas. J Clin Endocrinol Metab 1993; 77: Zafar M, Ezzat S, Ramyar L, Pan N, Smyth HS, Asa SL. Cell-specific expression of estrogen receptor in the human pituitary and its adenomas. J Clin Endocrinol Metab 1995; 80: Friend KE, Chiou YK, Lopes MBS, Laws ER, Jr., Hughes KM, Shupnik MA. Estrogen receptor expression in human pituitary: Correlation with immunohistochemistry in normal tissue, and immunohistochemistry and morphology in macroadenomas. J Clin Endocrinol Metab 1994; 78: Lamolet B, Pulichino AM, Lamonerie T et al. A pituitary cell-restricted T box factor, Tpit, activates POMC transcription in cooperation with Pitx homeoproteins. Cell 2001; 104(6): Asa SL, Bamberger A-M, Cao B, Wong M, Parker KL, Ezzat S. The transcription activator steroidogenic factor-1 is preferentially expressed in the human pituitary gonadotroph. J Clin Endocrinol Metab 1996; 81: Asa SL. Tumors of the Pituitary Gland. Atlas of Tumor Pathology, Third Series, Fascicle 22, 1998 Washington, D.C., Armed Forces Institute of Pathology. 12. Kovacs K, Horvath E, Ryan N, Ezrin C. Null cell adenoma of the human pituitary. Virchows Arch [Pathol Anat ] 1980; 387:

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