1780 Vol. 10, 1780–1788, March 1, 2004 Clinical Cancer Research Molecular Pathology Shows p16 Methylation in Nonadenomatous Pituitaries from Patients with Cushing’s Disease David J. Simpson,1 Anne M. McNicol,2 methylated, particularly in those cases of apparently normal David C. Murray,2 Adil Bahar,1 pituitary, is the most likely explanation for the lack of Helen E. Turner,3 John A. H. Wass,3 association between this change and loss of cognate protein in these cases. Margaret M. Esiri,4 Richard N. Clayton,1 and 1 Conclusions: To our knowledge this is the first report William E. Farrell that describes an intrinsic molecular change, namely meth- 1 Institute of Science and Technology in Medicine, School of ylation of the p16 gene CpG island, common to all three Medicine, Keele University, Stoke on Trent, Staffordshire; 2University Department of Pathology, Glasgow Royal Infirmary, histological patterns associated with Cushing’s disease. Glasgow; and 3Departments of Endocrinology and 4Neuropathology, Thus, the use of molecular pathology reveals abnormalities Radcliffe Infirmary Oxford, Oxford, United Kingdom undetected by routine pathological investigation. In cases of “apparently” normal pituitaries it is not possible to deter- mine whether the change is associated with adenoma cells ABSTRACT “scattered” throughout the gland, albeit few in number, or Purpose: The majority of cases of Cushing’s disease are with the ancestor-clonal origin of these tumor cells. due to the presence of a corticotroph microadenoma. Less frequently no adenoma is found and histology shows either corticotroph hyperplasia, or apparently normal pituitary. In INTRODUCTION this study we have used molecular pathology to determine Cushing’s disease is a rare disorder with an annual inci- whether the tissue labeled histologically as “normal” is in- dence estimated to be between 0.7 and 2.4 cases per million (1, deed abnormal. 2). Most cases are due to the presence of a corticotroph mi- Experimental Design: Tissue from 31 corticotroph ade- croadenoma, and selective removal of the adenomatous tissue nomas and 16 nonadenomatous pituitaries were subject to results in correction of the biochemical abnormalities and clin- methylation-sensitive PCR to determine the methylation sta- ical remission of the disease (3–6). However, several reports tus of the p16 gene CpG island. The proportion of methyl- have described patients with Cushing’s disease in whom remis- ated versus unmethylated CpG island was determined using sion was achieved where no adenoma could be identified in the combined bisulphite restriction analysis. Methylation status surgically resected sample (7–9). In these cases, and in the was correlated with immunohistochemical detection of p16. absence of an adenoma, pituitary histology revealed either nor- Results: Seventeen of 31 adenomas (54.8%), 4 of 6 cases mal pituitary tissue or was interpreted as corticotroph hyperpla- of corticotroph hyperplasia, and 7 of 10 apparently normal sia. ؍ pituitaries showed p16 methylation. Ten of 14 (71%; P The evidence for a fundamental pituitary origin for tumors 0.01) adenomas and 2 of 3 cases of corticotroph hyperplasia, that arise within this gland, including those of the corticotroph which were methylated, failed to express p16 protein. How- lineage, is persuasive implying an intrinsic molecular defect ever, only 2 of 7 apparently normal pituitaries that were within these cells and has been the subject of several recent methylated failed to express p16 protein. Quantitative anal- reviews (10, 11). The assumption has been that pituitary tumors, ysis of methylation using combined bisulphite restriction in common with other tumor types, arise by de novo molecular analysis showed only unmethylated CpG islands in postmor- tem normal pituitaries; however, in adenomas 80–90% of changes that confer a selective growth advantage and result in a the cells within a specimen were methylated. The reverse monoclonal expansion of a single cell to produce a discrete was true for corticotroph hyperplasia and apparently nor- adenoma. However, the role of hypothalamic hormones and/or mal pituitaries where only 10–20% of the cells were meth- growth factors as initiators, facilitators, or promoters of tumor ylated. Thus, the decreased proportion of cells that were growth is also widely accepted (for recent reviews see Refs. 10, 12, 13). Indeed, in those cases where disease is associated with abnormal trophic and/or secretory activity, for example in non- adenomatous tissue associated with Cushing’s disease, the role of hypothalamic or rarely ectopic factors would seem intuitively Received 7/31/03; revised 11/17/03; accepted 11/19/03. attractive but lacking formal proof. Moreover, recent investiga- The costs of publication of this article were defrayed in part by the tions support this view and have challenged the concept of payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to invariant monoclonality (14, 15). These authors have proposed indicate this fact. that a monoclonal expansion might, in some cases, arise on a Requests for reprints: William E. Farrell, Centre for Cell and Molec- background of cell subtype-specific hyperplasia consistent with ular Medicine, School of Postgraduate Medicine, Keele University, involvement of extra- or intrapituitary factors. Indeed, animal North Staffordshire Hospital, Stoke-on-Trent ST4 7QB, United King- dom. Phone: 44-1782-555225; Fax: 44-1782-747-319; E-mail: models show this to be the case (16–18). The nearest human [email protected]. counterpart to the animal models is Cushing’s disease where Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2004 American Association for Cancer Research. Clinical Cancer Research 1781 apparently normal pituitary tissue, hyperplasia, or adenoma may Table 1 Methylation and expression status of p16 in Cushing’s be associated with the disease. disease adenomatous and nonadenomatous pituitaries Methylation-associated gene silencing is a frequent finding Methylation IHCa in numerous tumor types (reviewed in Ref. 19) including those Patient Tumor grade status CDKN2A status p16 of pituitary origin. We showed recently methylation of the 11 MϪ tumor suppressor genes CDKN2A (p16) and RB1 CpG islands 2 1 U N/A that was associated significantly with loss of their cognate 3 1 U N/A 41 Mϩ proteins in nonfunctioning pituitary tumors and somatotrophi- 51 Mϩ nomas, respectively (20, 21). In nonfunctioning tumors p16- 6 1 M N/A associated methylation occurred early in pituitary tumorigenesis 71 Uϩ ϩ (20), and in other tumor types this epigenetic change has been 81 U 91 MϪ described in the preceding preneoplastic tissue (22). In addition, 10 1 U ϩ a mechanistic role for p16 in pituitary tumorigenesis is sug- 11 1 U N/A gested by the findings that reintroduction of this gene into a 12 1 U ϩ Ϫ corticotroph cell line AtT20, in which the endogenous gene is 13 1 M 14 1 M Ϫ homozygous deleted, inhibits cells proliferation and is associ- 15 1 M ϩ ated with a G1 arrest (23). Taken together these findings 16 1 M N/A prompted us to investigate methylation-associated p16 gene 17 1 U N/A Ϫ silencing in adenomas and nonadenomatous tissue from patients 18 1 M 19 1 M Ϫ with Cushing’s disease, to determine whether molecular pathol- 20 2 U N/A ogy would reveal pathogenetic changes, especially in non- 21 2 U N/A adenomatous tissues, which cannot be revealed immunohisto- 22 2 U ϩ ϩ logically. 23 2 M 24 2 M Ϫ 25 2 M Ϫ 26 2 U N/A MATERIALS AND METHODS 27 3 U ϩ Patient Characteristics. All of the patients had pituitary 28 4 M Ϫ dependent Cushing’s disease as defined by: (a) clinical features; 29 4 U N/A 30 4 M Ϫ (b) sustained increased urine free cortisol excretion; (c) loss of 31 4 M N/A plasma cortisol diurnal rhythm; (d) failure to suppress plasma 32 Hyperplasia U ϩ cortisol with low-dose dexamethasone (0.5 mg dexamethasone 33 Hyperplasia M Ϫ Ϫ every 6 h ϫ48 h); and (e) Ͼ50% suppression of plasma cortisol/ 34 Hyperplasia M 35 Hyperplasia M NI adrenocorticorticotropic hormone (ACTH) with high-dose dex- 36 Hyperplasia U ϩ amethasone (2 mg dexamethasone every 6 h ϫ48 h). Brief 37 Hyperplasia M ϩ patient details are shown in Table 1. 38 Normal M ϩ ϩ Pituitary imaging was carried out by computed tomography 39 Normal M 40 Normal M ϩ or magnetic resonance imaging scan and the designation of 41 Normal U ϩ adenoma size used a modified Hardy classification (24) where 42 Normal U ϩ grade 1 microadenomas were Ͻ10 mm diameter; grade 2 mac- 43 Normal M ϩ Ϫ roadenomas were Ͼ10 mm diameter but without extra/parasel- 44 Normal M 45 Normal M Ϫ lar extension; grade 3 macroadenomas were as grade 2 with 46 Normal M ϩ extra/parasellar extension; and grade 4 metastatic tumors were 47 Normal U N/A within the central nervous system. The majority of patients were 1–6 Postmortem normal (X6) U ϩ young women. Clinical and biochemical status was assessed a IHC, immunohistochemistry; M, methylated; U, unmethylated; within 2–3 months postoperatively and remission defined as ϩ, IHC positive; Ϫ, IHC negative; NI, noninformative/not interpretable; normalization of cortisol production (normal urine free cortisol NA, insufficient material for analysis; Grades: 1, microadenoma; 2, intrasellar macroadenoma; 3, extrasellar macroadenoma; 4, carcinoma. excretion, normal plasma cortisol diurnal rhythm, and dexa- methasone suppressibility; low dose). The patients were from two centers, Oxford and Stoke-on- Trent, United Kingdom, where surgery was performed by a Pituitary Histology. Pituitary tissue removed at opera- single experienced pituitary surgeon in each center. The opinion tion was examined by one experienced neuropathologist of the surgeon regarding tumor visualization (though not size) (M.