Molecular Surgical Pathology
Immunohistochemical Expression of Pituitary Tumor Transforming Gene (PTTG) in Pituitary Adenomas: A Correlative Study of Tumor Subtypes
International Journal of Surgical Pathology 18(1) 5–13 © The Author(s) 2010 Reprints and permission: http://www. sagepub.com/journalsPermissions.nav DOI: 10.1177/1066896909356105 http://ijsp.sagepub.com
Fateme Salehi,1 Kalman Kovacs,1 Bernd W. Scheithauer,2 David Cantelmi,1 Eva Horvath,1 Ricardo V. Lloyd,2 and Michael Cusimano2
Abstract Objective. We investigated the correlation between immunohistochemical expression of the pituitary tumor transforming gene (PTTG) and pituitary adenoma subtype. Methods. Pituitary adenomas (n = 89) were stained for PTTG using the streptavidin–biotin–peroxidase complex method and a monoclonal PTTG antibody. Results. PTTG staining was found to be cytoplasmic with a pronounced paranuclear expression pattern. Reactivity was highest in growth hormone (GH) adenomas as compared with other tumors, including prolactin (PRL), follicle-stimulating hormone/luteinizing hormone/α subunit, as well as adrenocorticotrophic hormone–secreting adenomas. PRL adenomas exhibited the lowest expression levels. Among GH adenomas, untreated tumors demonstrated significantly higher PTTG levels than octreotide-treated examples. Although dopamine agonist–treated PRL adenomas tended to show lower expression levels, statistical significance was not reached. Conclusions. Our finding that PTTG was differentially expressed in pituitary adenoma subtypes suggests a cellspecific function for PTTG. Moreover, treatment of GH adenomas with somatostatin analogues lowered PTTG expression. Further investigation into mechanisms mediating cell-specific expression of PTTG is warranted. Keywords GH, PTTG, pituitary adenomas, somatotroph adenoma, lactotroph adenoma
Introduction The pituitary tumor transforming gene (PTTG) was first isolated from the GH3 rat pituitary tumor cell line.1 Subsequently, its function was demonstrated to be that of securin, mediating sister chromatid separation during mitosis.2 PTTG overexpressing cells that are subcutaneously injected into nude mice induce tumor formation, and PTTG elevated expression has been reported in several neoplasms studied, including pituitary tumors as well as in carcinomas of the lung, breast, esophagus, colon, rectum, and ovary. PTTG overexpression correlates with tumor invasiveness and aggressive behavior in thyroid and colorectal carcinomas.3,4 PTTG has been shown to play a role in several cellular processes, including DNA repair, apoptosis, and angiogenesis. PTTG interaction with various cellular factors, including p53, VEGF (vascular endothelial growth factor), and bFGF (basic fibroblast growth factor) has been demonstrated. PTTG also possesses transactivating activity
and induces upregulation of several genes, specifically bFGF and c-myc.5-8 PTTG induces genetic instability and aneuploidy in tumors. Recently, it has been implicated in stem cell proliferation and even in cardiac hypertrophy, thus, expanding the spectrum of processes that it regulates.9,10 Mouse models of PTTG knockout and overexpression have exhibited its roles in tumor initiation and progression, chiefly in the pituitary and thyroid glands. In the retinoblastoma heterozygous deficient (Rb+/) mice, a model of pituitary adenoma development, PTTG ablation (PTTG−/−) results in decreased tumor formation, whereas PTTG overexpression results in an increased incidence of 1
St Michael’s Hospital, University of Toronto, Toronto, ON, Canada Mayo Clinic, Rochester, MN, USA
2
Corresponding Author: Fateme Salehi, Department of Laboratory Medicine, St Michael’s Hospital, Toronto, ON M5B 1W8, Canada Email:
[email protected]
6 pituitary tumors.11,12 Studies have investigated PTTG expression in pituitary adenomas; although there is consensus regarding overexpression of PTTG in pituitary adenomas, it remains unclear whether PTTG is differentially expressed in the various adenoma subtypes. Therefore, we investigated the issue. In addition, the cellular localization of PTTG in pituitary adenomas was investigated.
Materials and Methods Materials We selected 89 pituitary adenomas randomly for PTTG examination. All had been obtained at surgery of patients with a clinical or pathological diagnosis of pituitary adenomas. Each tumor was classified based on the criteria of the 2004 World Health Organization (WHO) Classification of Endocrine Tumors.13 Specimens included 12 growth hormone (GH), 9 prolactin (PRL), 10 adrenocorticotrophic hormone (ACTH), 11 follicle stimulating hormone (FSH)/ luteinizing hormone (LH)/α-subunit (α-SU), and 9 thyroid stimulating hormone (TSH) adenomas as well as 14 null cell adenomas. In addition, 13 bromocriptine-treated PRL adenomas and 11 octreotide-treated GH adenomas were studied.
Methods All specimens were fixed in 10% buffered formalin, routinely processed, paraffin embedded, cut at 4 to 6 µm, and stained with hematoxylin and eosin. The streptavidin–biotin– peroxidase complex method was used, and antisera were directed against GH PRL, PRL, ACTH, TSH, LH, FSH, and the α-SU of glycoprotein hormones. Methods of immunostaining, antibody sources, and dilutions as well as control procedures have been described previously.14 In many cases, electron microscopy had also been performed for diagnostic purposes. Immunostaining for PTTG used a mouse monoclonal antibody (1:75, Abcam, Cambridge, UK). Routine deparaffinization, rehydration, and blockade of endogenous peroxidase activity were carried out. Sections were then microwaved in 0.1 mM sodium citrate buffer (pH 6.0), incubated with goat antiserum, and exposed to the streptavidin–biotin–peroxidase complex. Diaminobenzidine served as the chromogen. Positive controls of PTTG immunoreactivity used formalin-fixed, paraffin-embedded, mouse testicular tissue. Replacement of the primary antibody with PBS served as a negative control. Immunopositivity for PTTG was evaluated at high magnification (×400). Both the intensity and percentage of positive cells were studied blindly by one of the authors (FS), who repeated the assessments at 2 time points 4 to 6 weeks apart to determine intraobserver reliability. Yet
International Journal of Surgical Pathology 18(1) another observer (DC) independently assessed 43 of the specimens to determine interobserver variability calculations (Cohen’s κ sample size; see below). Both PTTG intensity and percentage positivity were evaluated. The former was assessed on a scale of 0 to 3 (0 = none; 1 = mild; 2 = moderate; 3 = strong). The percentage of immunopositive cells was assessed as the percentage of cells exhibiting reactivity in each field. Areas demonstrating necrosis, fibrosis, and artifacts were excluded. Average immunopositivity intensity and percentage reactivity were recorded as a mean observed in 10 high-power fields. For each specimen, a histological score was calculated by multiplying the intensity score by percentage positivity (1 × percentage of positive cells, 2 × percentage of positive cells, 3 × percentage of positive cells), 300 being the maximal attainable histoscore.
Statistical Analysis ANOVA (SPSS Statistical Program) was used to find significant differences between pituitary adenoma subtypes. The κ statistic was used to determine interobserver and intraobserver variability. The κ sample size calculation was performed using the R statistical program to determine the minimum sample size requiring assessment by 2 observers. Using this program, sample size calculation for κ was determined to be 43. The program required the following parameters to be estimated: κ to detect = 0.90; power = 0.70; proportion of positive ratings = 0.50. For κ value calculation, the percentage positivity was categorized as follows: 0 = 0%; 1 = 25%; 2 = 26% to 50%; 3 = 51% to 75%; 4 = 76% to 100%.
Results PTTG Cellular Localization PTTG immunoexpression was found to be cytoplasmic in all adenomas studied (Figure 1). A predominantly paranuclear pattern of staining was evident in most adenomas, regardless of subtype, with PTTG preferentially marking the Golgi zone (Figure 1). No nuclear PTTG expression was observed (Figure 2).
PTTG Immunoexpression: Percentage of Immunopositive Cells The mean percentage of PTTG immunopositivity across all pituitary adenoma subtypes was 52% ± 16% (range, 21%94%). The percentage of PTTG immunopositive cells among specimens ranged from 0% to 100% of cells. Mean percentages were highest in GH adenomas (94% ± 27%) followed by TSH and null cell adenomas (65% ± 91% and
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Figure 1. PTTG expression in a GH (A), PRL (B), ACTH (C), and null cell adenoma (D); original magnification ×400
Abbreviations: PTTG, pituitary tumor transforming gene; GH, growth hormone; PRL, prolactin; ACTH, adrenocorticotrophic hormone.
63% ± 13%, respectively). The lowest mean percentage of PTTG immunoreactive cells was found in PRL adenomas (21% ± 17%; Figure 2).
Percentage PTTG
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PTTG Immunoexpression: Intensity of Immunopositive Cells
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Figure 2. Percentage of PTTG immunoreactive cells in various pituitary adenoma subtypes: the numbers in parentheses refer to the number of tumors of each cell type tested. Whereas the lowest PTTG immunostaining percentage was exhibited by PRL adenomas, GH adenomas demonstrated the highest percentage of PTTG immunopositive cells Abbreviations: PTTG, pituitary tumor transforming gene; GH, growth hormone; PRL, prolactin; ACTH, adrenocorticotrophic hormone; FSH, follicle stimulating hormone; LH, luteinizing hormone; α-subunit; α-SU.
The mean intensity for PTTG immunostaining across all adenoma subtypes was 1.62 ± 0.66 (range, 0.36 to 2.40; Figure 3). The highest intensity of PTTG immunostaining was observed in GH and TSH adenomas (2.40 ± 0.58 and 2.4 ± 1.03, respectively). The lowest PTTG staining intensity was observed in treated PRL adenomas (0.36 ± 0.79).
PTTG Histoscore The histoscore, obtained by multiplying PTTG intensity and percentage of cells immunopositive for PTTG, was calculated for all tumors. The mean histoscore across all tumor subtypes was 110 ± 25 (range 34-200; Figure 4).
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PTTG Histoscore in Medically Treated and Untreated GH and PRL Adenomas
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The PTTG immunoreactivity histoscore was significantly higher in GH adenomas that were untreated medically as compared with those that were treated (P = .02; Figure 5). Although the PTTG histoscore was lower in treated PRL adenomas as well, this difference was statistically significant (P = .13).
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Figure 3. Intensity of PTTG immunoreactive cells in various pituitary adenoma subtypes: the numbers in parentheses refer to the number of tumors of each cell type tested. Treated and untreated PRL adenomas exhibited the lowest intensity of PTTG immunohistochemical staining, whereas GH adenomas demonstrated the highest intensity scores
Abbreviations: PTTG, pituitary tumor transforming gene; GH, growth hormone; PRL, prolactin; ACTH, adrenocorticotrophic hormone; FSH, follicle stimulating hormone; LH, luteinizing hormone; α-subunit; α-SU.
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Figure 4. Histoscore of pituitary adenoma subtypes: to calculate the histoscore, average intensity and percentage scores were multiplied for each subtype. The lowest histoscore is seen in PRL adenomas and the highest score in GH adenomas Abbreviations: PTTG, pituitary tumor transforming gene; GH, growth hormone; PRL, prolactin; ACTH, adrenocorticotrophic hormone; FSH, follicle stimulating hormone; LH, luteinizing hormone; α-subunit; α-SU.
Whereas null cell, TSH, and GH adenomas demonstrated the highest histoscores (200 ± 2, 159 ± 80, and 154 ± 70, respectively), the lowest histoscore was expressed by PRL adenomas (34 ± 40).
Intraobserver reliability was high—a κ value of 0.93 indicating almost perfect agreement. A high degree of interobserver variability was also noted, as indicated by a κ value of 0.87.
Discussion PTTG Expression in Adenoma Subtypes Abundance of PTTG expression in pituitary adenomas has been documented in several studies using both immunohistochemical and/or polymerase chain reaction techniques. Here, we investigated the quantitative relationship of PTTG expression and pituitary tumor subtype using routine immunohistochemistry. Our results showed that null cell adenomas had the highest PTTG histoscore, followed by TSH and GH adenomas. The lowest levels of expression were encountered in PRL adenomas. Pairwise comparison of adenoma subtypes using ANOVA showed that GH adenomas expressed significantly higher PTTG histoscores as compared with PRL, FSH/LH/α-SU, and ACTH adenomas as well as medically treated GH and PRL adenomas. Moreover, TSH adenomas showed substantially greater PTTG histoscores as compared with PRL adenomas as well as treated GH and PRL adenomas. Our results are in keeping with findings that indicated higher PTTG expression in GH-secreting adenomas as compared with other subtypes. Hunter et al,15 analyzing PTTG mRNA levels in 40 pituitary tumors (12 GH, 5 PRL, 5 ACTH, 18 nonfunctioning) showed that the expression levels in GH-secreting adenomas were 2.7-fold higher as compared with those in nonfunctioning adenomas.15 Furthermore, in vitro GH secretion and PTTG expression were also found to be correlated (R = 0.41; P < .01). Interestingly, targeted PTTG overexpression using the mouse α-SU promoter induces focal PTTG expression not only in LH cells but in GH-producing cells as well.16 The α-SU promoter targets PTTG expression to pituitary stem cells and is unassociated with nonfunctional mature somatotroph cells.16 Thus, the unexpected finding of PTTG expression in GH cells in this mouse model further supports the existence of a strong link between PTTG and GH-producing cells in the normal pituitary gland.
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Treated PRL Untreated PRL
Treated GH Untreated GH
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Figure 5. Treated and untreated GH and PRL adenomas: statistically significant differences are seen between treated and untreated GH adenomas when comparing PTTG intensity, percentage positivity, and histoscore Abbreviations: PTTG, pituitary tumor transforming gene; GH, growth hormone; PRL, prolactin.
Uccella et al17 found that GH as well as PRL cell adenomas showed elevated PTTG expression as well as multiple chromosome gains when compared with other pituitary adenomas. These 2 adenoma types also showed abnormal nuclear shape and increased numbers of centrosomes.17 Previous studies had already shown increased chromosome numbers in both GH and PRL cell adenomas.18-20 Elevated expression of PTTG in these adenomas is in line with the role of PTTG in the inhibition of premature sister chromatid separation and its importance to maintaining euploidy.21 During metaphase, sister chromatids bind via the cohesion molecule, which is degraded by separin, thus, permitting progression of the cell cycle into anaphase and sister chromatid separation. By binding to separin and thereby inhibiting degradation of cohesion, PTTG has been shown to inhibit chromatid separation.2 Not surprisingly therefore, PTTG−/− mouse embryo fibroblasts demonstrate aneuploidy.22 In addition, a strong correlation has been shown between overexpression of PTTG and chromosomal aberrations in colorectal and thyroid neoplasms.23,24 Thus, the higher PTTG histoscore found in GH adenomas may correlate with the higher reported incidence of aneuploidy in these tumors.18-20 Our findings showed the lowest PTTG expression in PRL adenomas, which also have a reported increased incidence of abnormal karyotypes. Given these discrepancies, further research into the correlation between aberrant PTTG expression and ploidy in pituitary adenomas is needed. In vitro studies also support a relationship between PTTG and the function of GH and PRL cells. Horwitz et al,25 studying the effects of PTTG on PRL and GH secretion in vitro, found overexpression of PTTG C terminus to abrogate PRL secretion 10-fold, whereas GH secretion was increased 4-fold.25 Interestingly, subcutaneous injection of transfected cells overexpressing PTTG C terminus showed decreased PRL and elevated GH serum levels.25 These results suggest that PTTG stimulates PRL expression and inhibits GH production—findings that further support the notion that PTTG is intimately associated with GH and PRL cell function in the normal pituitary gland. In contrast to the above-noted studies, some investigators have reported a lack of correlation between PTTG
expression and the function of various pituitary adenoma cell types. Minematsu et al,26 examining 101 pituitary adenomas (29 GH, 12 PRL, 6 ACTH, 1 FSH, 3 TSH, and 50 nonfunctioning) found PTTG mRNA to be elevated in most adenoma types, with levels not being significantly different.
Lowest and Highest PTTG Expression in PRL and GH Adenomas, Respectively It is of note that the highest PTTG levels were observed in GH adenomas, with PRL adenomas showing the lowest expression levels. Factors underlying this stark difference may relate to the process of cytodifferentiation that gives rise to PRL- and GH-secreting cells from a common ancestral cell line expressing the transcription factor Pit-1. Indeed, the Pit-1 gene is selectively expressed in pituitary adenomas expressing GH, PRL, and TSH.27-29 Evidence supports the role of ER-α in the development of PRLexpressing cells from somatotrophs expressing Pit-1.30 Interestingly, estrogen is shown to regulate PTTG expression in the context of PRL adenoma development.31,32 Estrogen increases PTTG levels concordant with early lactotrophic hyperplasia. This may relate to the fact that estrogen induces bFGF, which in turn induces PTTG expression. In turn, PTTG mediates the secretion of bFGF, which augments PRL cell proliferation and PRL secretion, as well as angiogenesis. The stimulation of angiogenesis is mediated by VEGF and bFGF. Thus, PTTG is an important regulatory element in PRL adenomagenesis because it is expressed and mediated early in the process.32 Given the association of PTTG with early PRL adenoma development, it is possible that PTTG plays a role in the cytodifferentiation of mammosomatotrophs into lactotrophs. The clear difference in PTTG levels and GH versus PRL levels may stem from this yet unverified role and may indicate that the levels of PTTG expression are either partly responsible for this shift in differentiation or are a by-product of the cellular events mediating it. The differential expression of PTTG in a cell-specific manner warrants further investigation into its role in cellular differentiation. The
10 notably higher expression of PTTG in GH adenomas as compared with most other adenoma types suggests a role for PTTG in GH adenoma development and/or progression.
Medically Treated Versus Nontreated GH and PRL Cell Adenomas Our study found a significantly higher PTTG histoscore in untreated GH adenomas as compared with octreotidetreated ones (P = .005), indicating an effect of treatment on PTTG expression. Bromocriptine-treated PRL adenomas showed somewhat lower PTTG expression histoscores, but the difference did not reach statistical significance (P = .13). The finding of abrogated PTTG expression in treated GH adenomas supports a role for PTTG in the mechanism of adenomagenesis. It appears that following medical treatment, PTTG is downregulated as evidenced by lower percentages of PTTG immunopositive cells as well as lower mean intensities of PTTG immunoexpression. Morphological, including ultrastructural studies of GH adenomas treated with somatostatin analogues (octreotide, lanreotide) demonstrate only mild alterations, including an increase in the number and size of secretory granules, which is an indicator of inhibited protein secretion, but no nuclear changes. This is in contrast to the dramatic changes seen in PRL adenomas treated with dopamine agonists.33 Several explanations may be offered regarding decreased PTTG expression in somatostatin analogue–treated GH adenomas. The first is that decreased PTTG expression in treated GH adenomas is a primary effect of medical treatment. It is known, for instance, that somatostatin analogues inhibit cell proliferation via several mechanisms, including inhibition of DNA synthesis and cell cycle progression, thus, affecting the expression level of many cell cycle regulatory factors.34,35 One such factor, p27, is upregulated in vitro, an observation in keeping with its role in cell cycle arrest.35 It would be valuable to investigate the relationship between expression of the proliferation marker Ki-67 and PTTG in treated GH adenomas. The second possible explanation for decreased PTTG expression in treated GH adenomas is that PTTG reduction is simply the result of cell cycle arrest and decreased cellular proliferation in such tumors.34,36 Reduced cellular proliferation and cell cycle activity does result in a decrease in immunoreactivity of several cell cycle proteins, one of which may be PTTG. One more explanation may relate to the fact that somatostatin analogues inhibit the MAPK (mitogen-activated protein kinase) pathway in GH3 cells factors.34,35 In pituitary folliculostellate cells, this pathway mediates induction of PTTG via EGFR ligands.37 Thus, PTTG reduction in somatostatin analogue–treated GH adenomas may be a result of abrogation of the MAPK pathway. Although in our study, the percentage of PTTG immunopositive cells was found to be higher in dopamine
International Journal of Surgical Pathology 18(1) agonist–treated PRL adenomas as compared with untreated ones, the difference did not reach statistical significance. This finding may appear counterintuitive as dopamine agonist– treated PRL adenomas exhibit decreased mitotic activity, lower MIB-1 labeling indices, and p27 expression—all indicators of inhibition of proliferative activity.38 Dopamine agonist treatment of PRL adenomas induces conspicuous morphology changes, including marked reduction in rough endoplasmic reticulum and Golgi complexes as well as nuclear alterations.33 Such nuclear changes suggest that the drug effect is at the transcription level and that the increased PTTG levels observed in treated PRL adenomas may be transcriptionally mediated. Although, as a cell cycle protein, PTTG affects cellular proliferation, antiproliferative effects have also been demonstrated.1,39,40 Several studies have showed a lack of correlation between PTTG and proliferative activity.3,41,42 In one series of 101 pituitary adenomas, no correlation was found between PTTG expression and cell proliferation.43 Indeed, PTTG may in fact suppress proliferation through induction of apoptosis and delay of mitosis, effects seen in treated PRL adenomas.9,33 It has been proposed that PTTG exerts its proliferative effects in a dosedependent manner.3,44,45 Thus, a tendency to see higher PTTG levels in treated PRL adenomas may point to its role in mediating antiproliferative effects in PRL cells. It is of note that some PRL adenomas respond only mildly to antidopaminergic treatment, with corresponding morphological changes being minor. Although such tumors fail to demonstrate alterations in membranous organelles (rough endoplasmic reticulum and Golgi) or in the nucleus, they do exhibit an increase in secretory granule size and number as well as lysosomal activity and crinophagy.33 These changes suggest a modification at the posttranslational level, corresponding to diminished hormone secretion and increased lysosomal degradation.33 A decrease in protein secretion, as observed in PRL adenomas mildly responsive to dopamine agonist therapy, may have led to accumulation of PTTG in some of the treated PRL adenomas in our series because PTTG was recently shown to be a protein product secreted by pituitary cells.43 It remains to be determined, however, whether the decreased PTTG expression seen in treated GH and PRL adenomas is secondary to the cellular changes described or is more directly mediated by the medical therapeutic agents.
Cellular Localization of PTTG The cellular localization of PTTG is unclear. In the nucleus, it acts as a transcriptional activator, in addition to an inhibitor of premature sister chromatid separation.5-8 To date, however, the role of cytoplasmic PTTG remains the subject of study,7,8,46,47 one of which recently demonstrated that pituitary PTTG is a secretory protein in pituitary tumor cells.43
Salehi et al. In our series of 89 pituitary adenomas, only cytoplasmic PTTG expression was noted, the pattern of staining being decidedly paranuclear. No nuclear immunoreactivity was noted. These results are in keeping with those of most other studies showing cytoplasmic PTTG expression48 in pituitary tumors.15,32,49 Recently, a study of 25 PRL-secreting adenomas by Wierinckx et al50 reported nuclear expression of PTTG to be among the prognostic indicators of tumor aggressiveness, other factors being numerous mitoses, high Ki-67 labeling indices, and p53 expression.50 It is possible that nuclear PTTG expression is limited to aggressive adenomas and that our series lacked such tumors. No pituitary carcinomas were examined in our series. The paranuclear pattern of PTTG expression is in keeping with its expression in the Golgi apparatus, which is a paranuclear structure, and in the endoplasmic reticulum, further supporting the recent findings that PTTG is a secretory protein.43 The paranuclear localization of PTTG may also facilitate its nuclear trafficking, a phenomenon observed with respect to other proteins.51 Alternatively, a paranuclear localization may facilitate the association of PTTG with other factors as in the case with cytosolic phospholipase A2α and its coupling with cyclooxygenases.52 More research is clearly needed into the various substances and structures that potentially interact with PTTG.
Conclusion In this study, we demonstrated variable cytoplasmic PTTG expression levels in different pituitary adenoma subtypes, the highest levels of immunoexpression being found in GH adenomas and the lowest in PRL adenomas. Although PTTG overexpression in pituitary tumors is well established, its specific role in pituitary tumor initiation and progression remains to be determined. Our results and discussion focus on the cell-specific nature of PTTG regulation of tumorigenic mechanisms in pituitary tumors and underscore the importance of differential PTTG expression in the spectrum of adenoma subtypes. Declaration of Conflicting Interests The authors declared no conflicts of interest with respect to the authorship and/or publication of this article.
Funding The authors received no financial support for the research and/or authorship of this article.
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