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Molecular Oncology, Markers, Clinical Correlates |
1 Institute of Science and Technology in Medicine, School of Medicine, Keele University, Stoke on Trent, Staffordshire; 2 University Department of Pathology, Glasgow Royal Infirmary, Glasgow; and 3 Departments of Endocrinology and 4 Neuropathology, Radcliffe Infirmary Oxford, Oxford, United Kingdom
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design: Tissue from 31 corticotroph adenomas and 16 nonadenomatous pituitaries were subject to methylation-sensitive PCR to determine the methylation status of the p16 gene CpG island. The proportion of methylated versus unmethylated CpG island was determined using combined bisulphite restriction analysis. Methylation status was correlated with immunohistochemical detection of p16.
Results: Seventeen of 31 adenomas (54.8%), 4 of 6 cases of corticotroph hyperplasia, and 7 of 10 apparently normal pituitaries showed p16 methylation. Ten of 14 (71%; P = 0.01) adenomas and 2 of 3 cases of corticotroph hyperplasia, which were methylated, failed to express p16 protein. However, only 2 of 7 apparently normal pituitaries that were methylated failed to express p16 protein. Quantitative analysis of methylation using combined bisulphite restriction analysis showed only unmethylated CpG islands in postmortem normal pituitaries; however, in adenomas 80–90% of the cells within a specimen were methylated. The reverse was true for corticotroph hyperplasia and apparently normal pituitaries where only 10–20% of the cells were methylated. Thus, the decreased proportion of cells that were methylated, particularly in those cases of apparently normal pituitary, is the most likely explanation for the lack of association between this change and loss of cognate protein in these cases.
Conclusions: To our knowledge this is the first report that describes an intrinsic molecular change, namely methylation of the p16 gene CpG island, common to all three histological patterns associated with Cushing’s disease. Thus, the use of molecular pathology reveals abnormalities undetected by routine pathological investigation. In cases of "apparently" normal pituitaries it is not possible to determine whether the change is associated with adenoma cells "scattered" throughout the gland, albeit few in number, or with the ancestor-clonal origin of these tumor cells.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The evidence for a fundamental pituitary origin for tumors that arise within this gland, including those of the corticotroph lineage, is persuasive implying an intrinsic molecular defect within these cells and has been the subject of several recent reviews (10 , 11) . The assumption has been that pituitary tumors, in common with other tumor types, arise by de novo molecular changes that confer a selective growth advantage and result in a monoclonal expansion of a single cell to produce a discrete adenoma. However, the role of hypothalamic hormones and/or growth factors as initiators, facilitators, or promoters of tumor 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 nonadenomatous tissue associated with Cushing’s disease, the role of hypothalamic or rarely ectopic factors would seem intuitively attractive but lacking formal proof. Moreover, recent investigations support this view and have challenged the concept of invariant monoclonality (14 , 15) . These authors have proposed that a monoclonal expansion might, in some cases, arise on a background of cell subtype-specific hyperplasia consistent with involvement of extra- or intrapituitary factors. Indeed, animal models show this to be the case (16, 17, 18) . The nearest human counterpart to the animal models is Cushing’s disease where apparently normal pituitary tissue, hyperplasia, or adenoma may be associated with the disease.
Methylation-associated gene silencing is a frequent finding in numerous tumor types (reviewed in Ref. 19 ) including those of pituitary origin. We showed recently methylation of the tumor suppressor genes CDKN2A (p16) and RB1 CpG islands that was associated significantly with loss of their cognate proteins in nonfunctioning pituitary tumors and somatotrophinomas, respectively (20 , 21) . In nonfunctioning tumors p16-associated methylation occurred early in pituitary tumorigenesis (20) , and in other tumor types this epigenetic change has been described in the preceding preneoplastic tissue (22) . In addition, a mechanistic role for p16 in pituitary tumorigenesis is suggested by the findings that reintroduction of this gene into a corticotroph cell line AtT20, in which the endogenous gene is homozygous deleted, inhibits cells proliferation and is associated with a G1 arrest (23) . Taken together these findings prompted us to investigate methylation-associated p16 gene silencing in adenomas and nonadenomatous tissue from patients with Cushing’s disease, to determine whether molecular pathology would reveal pathogenetic changes, especially in nonadenomatous tissues, which cannot be revealed immunohistologically.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The patients were from two centers, Oxford and Stoke-on-Trent, United Kingdom, where surgery was performed by a single experienced pituitary surgeon in each center. The opinion of the surgeon regarding tumor visualization (though not size) was recorded in most instances. As in previous reports there was poor correlation between preoperative tumor visualization and what was observed at operation (reviewed in Ref. 25 ). Similarly, surgical opinion of abnormal pituitary tissue correlated poorly with histological reports (vis-à-vis: normal, hyperplasia, and adenoma).
Pituitary Histology.
Pituitary tissue removed at operation was examined by one experienced neuropathologist (M. M. E.) and was subject to systematic examination. Adjacent sections were stained with: (a) H&E; (b) Gordon and Sweet’s reticulin stain; and (c) polyclonal antibodies to ACTH, PRL, ß human chorionic gonadotropin, ß thyrotropin, ß follicle-stimulating hormone, and ß luteinizing hormone (Dako). 
Human chorionic gonadotropin, (Biogenex) and growth hormone (kind gift from Dr. Andrew F. Parlow, National Hormone and Pituitary Program, University of California Los Angeles, Los Angeles, CA). Histological classification was as follows: (a) corticotroph adenoma, uniform ACTH-immunoreactive cells with absence of, or fragmented, reticulin staining; (b) corticotroph hyperplasia, expanded clusters comprising mainly ACTH-immunoreactive cells and that distend the reticulin baskets; and (c) apparently normal pituitary tissue, ACTH-immunoreactive cells showing a similar frequency and distribution to that seen in postmortem normal pituitary gland, occupying normal-sized reticulin baskets.
Tissue and DNA Preparation.
Ten 5 µm unstained sections were taken from the pituitary tissue described above together with 6 postmortem normal pituitaries processed in the same manner and obtained within 12 h of death. Before molecular and immunohistochemical analysis histological classification was confirmed in a preceding and subsequent section to ensure analysis corresponding to the histology described. For the molecular analysis sections were subject to microdissection and DNA extraction as described previously (20)
, and stored at 4°C.
Sodium Bisulphite Modification.
DNA (
2 µg) was denatured with NaOH (final concentration 0.2 M) in a total volume of 30 µl for 10 min at 37°C. Urea/sodium bisulphite solution (final concentration 0.5 mM hydroquinone, 5.36 M urea, and 3.44 M sodium bisulphite) at pH 5, freshly prepared, was added, mixed, and incubated at 55°C, in the absence of light for 4 h (26)
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DNA samples were purified using the GeneCleanII purification kit according to the manufacturer’s protocol (Bio 101 Vista 101, San Diego, CA) and eluted in 50 µl of water. Modification was completed by desulphonation with NaOH (final concentration 0.3 M) for 15 min at 37°C. DNA was ethanol precipitated and resuspended in 10 µl of water. Samples were stored at -20°C.
Methylation Status of CDKN2A/p16 and RB1 by Methylation-Sensitive PCR (MS-PCR).
Oligonucleotides specific for the p16 CpG island were as described previously (27)
. The specificity of the methylation target (p16) was determined by assessing the methylation status of the RB1 gene CpG island as described previously (21)
. Oligonucleotide sequences are shown in Table 2
. To confirm the PCR amplification of methylated p16/RB1 CpG islands after bisulphite modification we in vitro methylated genomic DNA with the CpG methylase enzyme SssI. This DNA was then subjected to urea/sodium bisulphite modification as described above and served as a positive control.
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PCR products were run on 8% nondenaturing polyacrylamide gels, fixed in 10% methylated spirit/0.5% acetic acid for 6 min, and then incubated in 0.1% aqueous silver nitrate for 15 min. After two brief washes in distilled water, products were visualized by development in 1.5% sodium hydroxide/0.1% formaldehyde.
Combined Bisulphite Restriction Analysis.
To quantitate methylated and unmethylated p16 CpG island sequences contained in DNA extracted from archival specimens we used combined bisulphite restriction analysis (COBRA; Ref. 28
). Because the oligonucleotides were designed to specifically amplify the p16 CpG island (see Table 2
) and contain no CpG dinucleotides, both methylated and unmethylated sequences are coamplified by a single primer pair after bisulphite modification. Amplification is achieved at an annealing temperature of 60°C using PCR parameters as described above except that PCR cycles were reduced to 24 to ensure amplification within the linear range.
The bisulphite conversion of the DNA results in the methylation-dependent retention of a BstUI (CGCG) restriction site within the p16 CpG island. Five-µl of each PCR product was digested with the BstUI restriction enzyme and the resulting fragments (147, 104, and 43 bp) run on 8% polyacrylamide gels and visualized as described above (see Fig. 3
). The percentage of fully methylated BstUI sites in the DNA sample can then be calculated from the ratio between the BstUI cleaved (methylated) and the undigested (unmethylated) PCR product relative to the product generated in the same reaction not subject to restriction digest. Quantitation was carried out using a GS-700 scanning densitometer (Bio-Rad, High Wycomb, United Kingdom).
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Tumor immunopositivity was classified using criteria identical to that described by Geradts et al. (29) . According to their criteria and our own investigations of p16 expression in pituitary tumors, only nuclear staining was defined as positive (20 , 29 , 30) . Where no staining of nuclei was observed in normal pituitary tissue surrounding/within the tumor section as an inbuilt control, the case was deemed inconclusive.
Statistical Analysis.
Statistical analysis was performed using the Stata v.5 statistical package (Stata Corp. TX). 
2 tests were used to compare variables. Significance was taken at the 5% level.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In addition as controls we included 6 postmortem normal pituitaries from patients with no history of endocrine disease or steroid therapy. As an outcome measure postsurgical remission rate was not significantly different (P = 0.5) between those patients in whom an adenoma was identified and those in whom disease was associated with nonadenomatous pituitaries (Table 1)
. The nonadenomatous specimens were subject to systematic examination of all of the available pituitary tissue to confirm initial observations. This examination confirmed the absence of adenomatous tissue and revealed in 6 of the 16 cases evidence of corticotroph hyperplasia and in the remaining 10, apparently normal pituitary tissue. The specimens of nonadenomatous pituitary associated with Cushing’s disease all showed various degrees of Crooke’s hyaline change. This change, reflecting high circulating glucocorticoid levels, was confined to the corticotroph cell population (data not shown).
Representative examples of the histological findings in these three categories associated with Cushing’s disease together with postmortem normal pituitary are shown in Fig. 1
. There were no significant differences in the H&E and reticulin staining patterns between postmortem normal pituitary (Fig. 1, A–C)
and apparently normal pituitary associated with Cushing’s disease (Fig. 1, J–L)
. In both cases the H&E and reticulin staining shows normal pituitary morphology with intact reticulin baskets surrounding well-defined cell nests. All of the postmortem normal pituitaries were immunopositive for p16 protein showing clear nuclear positivity in 50–60% of cells with low level cytoplasmic staining, which was considered irrelevant. Of 8 evaluable cases of apparently normal pituitary 6 stained positively for p16 protein (Table 1)
. In the representative example of corticotroph hyperplasia (Fig. 1, G–I)
the H&E staining shows enlarged cell nests with increased numbers of hypertrophic corticotrophs interspersed with smaller numbers of noncorticotroph cells. The reticulin baskets were expanded. Of 5 evaluable cases of corticotroph hyperplasia 3 stained positively for p16 protein and 2 were negative (Table 1)
. As with normals, only a subpopulation of cells were positive. In the adenomatous tissue (Fig. 1, D–F)
, H&E staining shows a complete lack of organized structure, with loss of the normal reticulin and only small irregular fragments between tumor cells. In the example shown p16 staining was negative. Overall, of 20 evaluable adenomas 10 (50%) did not express p16 protein (summarized in Table 1
). In some cases insufficient pituitary tissue from patients with Cushing’s disease was available for immunohistochemistry (IHC) analysis or was not interpretable (see Table 1
)
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; summarized in Table 1
). In 3 cases of adenoma, where the p16 gene CpG was found to be methylated by MS-PCR, sufficient juxtaposed normal pituitary was available for analysis after microdissection. In none of these cases did we detect methylation of this CpG island. MS-PCR analysis of Cushing’s associated corticotroph hyperplasia showed 4 of 6 to be methylated (66%) and in apparently normal pituitary associated with this disease 7 of 10 (70%) were methylated (Fig. 2
; summarized in Table 1
). Of the total cohort investigated for methylation of CDKN2A 20 of 47 were analyzed for methylation of the RB1 gene CpG island by MS-PCR as described previously (21)
. None of the specimens analyzed harbored methylation of RB1 irrespective of p16 methylation status (data available on request).
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. Thus, in these cases, these results suggest that not all of the cells in the specimens harbored methylation of the p16 gene. In the experiments described thus far, we were unable to exclude the possibility that methylation might represent a secondary change due to the cortisol excess that characterizes this disease. To additionally explore this possibility we used MS-PCR for the p16 gene CpG island to determine methylation of pituitary glands obtained at autopsy from 2 patients on long-term glucocorticoid therapy and from 1 patient with ectopic ACTH syndrome. All 3 showed Crooke’s hyaline change. None of the 3 cases showed methylation of this gene CpG island (data not shown).
Association between Methylation and Absence of p16 Protein.
We and others have shown previously an association between methylation of the p16 gene and absence of cognate protein in pituitary tumors (20
, 30, 31, 32, 33)
. Therefore, we looked for such associations in the pituitary tissue associated with Cushing’s disease. In the corticotroph adenomas sufficient tumor was available from 20 of 31 adenomas for p16 IHC analysis (Table 2
and summarized in Table 1
). Ten of 14 (71%) adenomas that were methylated did not express p16, and the remaining 4 (29%) showed nuclear positivity for this protein. The 6 adenomas that were unmethylated all expressed p16 protein. Methylation was significantly associated with gene silencing in adenomas (P = 0.01).
Sufficient material from 5 of 6 cases of corticotroph hyperplasia was available for p16 IHC analysis. Of 3 samples that were methylated 2 did not express p16 protein and 1 did (Table 2
and summarized in Table 1
). The 2 specimens that were unmethylated at the sites examined within this CpG island expressed p16 protein. The few specimens available for analysis precluded statistical analysis.
In those cases of apparently normal pituitary associated with Cushing’s disease 8 of 10 were available for IHC analysis. Of 7 specimens that were methylated 2 failed to express p16 protein; however, the remaining 5 stained positively for p16 in a proportion of cells (Table 2
and summarized in Table 1
). Two specimens unmethylated by MS-PCR expressed p16 protein. The number of specimens available again precluded statistical analysis; however, the data would suggest that methylation is not associated with gene silencing in this Cushing’s associated histology.
COBRA.
We reasoned that our failure to detect a clear association between methylation and loss of p16 protein expression in apparently normal pituitary associated with Cushing’s disease might reflect the proportion of cells harboring this epigenetic aberration versus those that did not. To determine the proportion of cells within a specimen that were methylated at the CDKN2A/p16 CpG island we used the quantitative COBRA for simultaneous detection of a methylated and unmethylated CpG island. Fig. 3
shows this analysis in representative specimens. In all 6 of the postmortem pituitary tissues we only detected sequence corresponding to an unmethylated CpG island. From corticotroph adenomas, where MS-PCR gave a positive methylation signal, between 80 and 90% of the specimen was methylated and the remainder unmethylated. None of the adenomas in which MS-PCR predicted an unmethylated CpG island were methylated by the COBRA technique. In apparently normal pituitary and corticotroph hyperplasia the ratio of methylated versus unmethylated CpG island was reversed from that seen in adenomas. In these cases, between 80 and 90% of individual specimens were found to be unmethylated and the remainder methylated. Only those cases found to give a positive display for methylation by MS-PCR were found to be methylated by the COBRA methodology. Thus, in those cases where methylation is not associated with loss of p16 protein a likely explanation is that a significant proportion of cells within the specimen do not harbor this change, at least at the sites investigated by this technique.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This particular tumor suppressor gene was chosen because of its known association with pituitary tumorigenesis (reviewed in 12 ) as well as being a regulatory component of the RB1 G1-S cell cycle pathway. Although RB1 knockout mice develop neurointermediate lobe tumors of the corticotroph lineage, RB1 gene mutations are uncommon in human pituitary tumors. However, loss of pRB has been demonstrated frequently in human somatotrophinomas as a consequence of or associated with methylation of its CpG island (21) . Accordingly, we sought specificity for p16 gene methylation by examining RB1 methylation and found in all of the instances this to be unmethylated. This suggests that methylation did not simply reflect nonspecific methylase activity within a specimen. It also suggests that in corticotroph adenomas, as in somatotrophinomas and nonfunctional tumors, methylation-associated loss of p16 and pRb are mutually exclusive events (30) .
We now show a similar frequency of p16 methylation in corticotrophinomas (55%) to that seen in nonfunctioning tumors (70%) that is also significantly associated with loss of cognate protein by IHC analysis. However, in those patients in whom a discrete adenoma could not be identified, methylation of this CpG island, whilst frequent, was not associated with complete loss of p16 protein. Furthermore, and in contrast to corticotroph adenomas, MS-PCR showed coamplification of sequence that represented an unmethylated CpG island.
Several studies have shown that extinction of gene expression, through CpG island methylation, is density dependent (35) . However, the MS-PCR technique does not reflect methylation density but reports on the status of specific residues within the CpG island (27) . We reasoned that the lack of association between p16 methylation and loss of protein expression in tissue that represented either hyperplasia or apparently normal pituitary that only a proportion of cells within a specimen harbored this epigenetic change. Indeed, quantitative COBRA analysis showed this to be the case, because only 10–20% of the cells within the specimen harbor a methylated p16 CpG island. The reverse was true in adenomas where, and in marked contrast, 80–90% of cells were methylated. Thus, in the adenomas, presumed to reflect a monoclonal expansion, the methylation pattern and density is faithfully replicated in each of the progeny cells. However, in cases of corticotroph hyperplasia and apparently normal pituitary, associated with Cushing’s disease, this reflects a predominantly polyclonal population. Thus, the proportion of cells that harbor this epigenetic change combined with the methylation density within individual cells will impinge on expression status as assessed by immunohistochemistry.
There is increasing evidence that corticotroph hyperplasia and a finding of apparently normal pituitary despite a thorough histological analysis are associated with Cushing’s disease in between 15% and 30% of cases, and removal of apparently normal pituitary tissue results in clinical and biochemical cure of Cushing’s disease (7
, 8
, 36, 37, 38)
. The key issue is, therefore, whether this apparently normal pituitary tissue harbors pathological change, which cannot be revealed by conventional histology and/or immunohistochemistry. Our studies provide strong support for the fact that what is removed in cases deemed to represent nonadenomatous tissue is indeed abnormal. In other tumor types CpG island methylation has been described in preneoplastic tissue (22)
, and our data are compatible with these findings. However, and perhaps more contentious, is the definition of true preadenomatous tissues. Thus, identification of p16 methylation in nonadenomatous pituitary tissue may simply reflect identification of cells that are actually "adenomatous" albeit very few and "scattered" throughout the gland. Our findings are consistent with this explanation because the MS-PCR technique is reported to reliably detect 0.1% methylated DNA present in an otherwise unmethylated sample (27)
. However, and equally plausible, is that we have detected changes in cells that are the ancestral-clonal origins of these tumor cells, where, in this case, p16 methylation does indeed represent one of the earliest identified changes preceding adenoma formation. Which of these two explanations is correct we are at present unable to determine. Regardless of the precise mechanistic interpretation of p16 methylation in the context of Cushing’s disease the molecular pathological approach described herein identifies abnormal cells, irrespective of histological findings. Given the high frequency of this change in Cushing’s associated nonadenomatous pituitaries (70%) this relatively simple technique could provide a useful addition to conventional investigations in circumstances of doubtful pituitary histopathology.
To our knowledge this is the first report that shows a molecular pathological change, namely, methylation of the p16 gene CpG island common to the three histological patterns of corticotrophs in the pituitary in Cushing’s disease. Our data strongly suggest that there is an intrinsic abnormality within the pituitary tissue removed from patients with Cushing’s disease even when no adenoma is identified, as witnessed by the methylation of p16. This does not seem to be a secondary effect of the disease state as it was not identified in association with glucocorticoid therapy or in ectopic ACTH syndrome. Methylation appears to affect only a subpopulation of cells within these glands. This might reflect the presence of a small clone of neoplastic corticotrophs not yet big enough to have the recognized architecture of an adenoma or the ancestor-clonal origins of these tumor cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Requests for reprints: William E. Farrell, Centre for Cell and Molecular Medicine, School of Postgraduate Medicine, Keele University, North Staffordshire Hospital, Stoke-on-Trent ST4 7QB, United Kingdom. Phone: 44-1782-555225; Fax: 44-1782-747-319; E-mail: w.e.farrell{at}keele.ac.uk
Received 7/31/03; revised 11/17/03; accepted 11/19/03.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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