Ki-67 and minichromosome maintenance-7 (MCM7) expression in canine pituitary corticotroph adenomas

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Domestic Animal Endocrinology 41 (2011) 207–213 www.domesticanimalendo.com

Ki-67 and minichromosome maintenance-7 (MCM7) expression in canine pituitary corticotroph adenomas H. Ishinoa,*, Y. Haraa, S. Takekoshib, T. Teshimac, A. Teramotod, R.Y. Osamurab, M. Tagawaa a

Division of Veterinary Surgery, Department of Veterinary Science, Faculty of Veterinary Medicine, Nippon Veterinary and Life Science University, Musashino-shi, Tokyo 180-8602, Japan b Department of Pathology, Tokai University School of Medicine, Boseidai, Isehara-shi, Kanagawa 259-1193, Japan c Division of Veterinary Internal Medicine, Department of Veterinary Science, Faculty of Veterinary Medicine, Nippon Veterinary and Life Science University, Musashino-shi, Tokyo 180-8602, Japan d Department of Neurosurgery, Nippon Medical School, Bunkyo-ku, Tokyo 113-8603, Japan Received 30 April 2011; received in revised form 11 July 2011; accepted 24 July 2011

Abstract Pituitary-dependent hyperadrenocorticism (PDH) caused by pituitary corticotroph adenoma is a common endocrine disorder in dogs. The ratio between pituitary height and the area of the brain (P/B) has been used to evaluate the pituitary size. A P/B ratio ⬎ 0.31 indicates an enlarged pituitary, whereas a P/B ratio ⱕ 0.31 indicates a nonenlarged pituitary. The aim of this study was to investigate the expression of proliferation markers Ki-67 and minichromosome maintenance-7 (MCM7) in canine corticotroph adenomas in enlarged and in nonenlarged pituitaries and to evaluate their relation with the size of canine pituitary corticotroph adenomas. Ki-67 and MCM7 expression in ACTH-positive tumor cells was determined by dual-labeling immunohistochemistry in resected corticotroph adenomas from 15 dogs with PDH. The mean ⫾ SD Ki-67 labeling index (LI) was 0.55% ⫾ 0.59% in corticotroph adenomas with nonenlarged pituitaries and 1.6% ⫾ 0.6% in adenomas with enlarged pituitaries. The MCM7 LI in corticotroph adenomas with nonenlarged pituitaries and in adenomas with enlarged pituitaries was 2.9% ⫾ 2.2% and 10.9% ⫾ 3.7%, respectively. The Ki-67 LI and MCM7 LI were significantly greater in the adenomas with enlarged pituitaries than in the adenomas with nonenlarged pituitaries (P ⬍ 0.01 and P ⬍ 0.01, respectively). The MCM7 LI was significantly greater than the Ki-67 LI in adenomas (P ⬍ 0.01). The Ki-67 LI was positively correlated with the MCM7 LI (r ⫽ 0.820, P ⬍ 0.01), and the P/B ratio was positively correlated with the Ki-67 LI (r ⫽ 0.560, P ⫽ 0.03) and the MCM7 LI (r ⫽ 0.854, P ⬍ 0.01). In conclusion, canine corticotroph adenomas in enlarged pituitaries show greater proliferation potential than do adenomas in nonenlarged pituitaries. MCM7 expression was significantly greater than Ki-67 expression in canine pituitary corticotroph adenomas. Thus, MCM7 may be superior to Ki-67 as a proliferation marker in pituitary tumors. © 2011 Elsevier Inc. All rights reserved. Keywords: Corticotroph adenoma; Dog; Pituitary; Proliferation marker; Minichromosome maintenance protein

* Corresponding author. Division of Veterinary Surgery, Department of Veterinary Science, Faculty of Veterinary Medicine, Nippon Veterinary and Life Science University, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo 180-8602, Japan. Tel.: ⫹81 422-31-4151; fax: ⫹81 422-33-8836. E-mail address: [email protected] (H. Ishino). 0739-7240/11/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.domaniend.2011.07.002

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1. Introduction Pituitary-dependent hyperadrenocorticism (PDH) caused by an ACTH-secreting corticotrophic adenoma is a common endocrine disorder in dogs [1]. Polydipsia, polyuria, abdominal distention, and skin lesions such as alopecia are common symptoms of hyperadrenocorticism, which results from a chronic overproduction of cortisol. However, in PDH, expansion of the pituitary tumor may lead to neurologic signs because of the intracranial mass effect [2,3]. Because a previous report suggested that there was no clear correlation between the onset of neurologic signs and tumor size in dogs with PDH [4], the neurologic signs may be related to how rapidly the pituitary tumor is growing. In humans, pituitary tumors are classified by size, as either macroadenomas (ⱖ10 mm in size) or microadenomas (⬍10 mm in size). In dogs, the ratio between pituitary height and the area of the brain (P/B) has been used to evaluate pituitary size [5]. This P/B ratio was developed to discriminate enlarged pituitaries from nonenlarged pituitaries with a threshold of 0.31 [5]. Notably, the size of the pituitary tumor is associated with poor prognosis after pituitary surgery in dogs [6], which may be a result of the greater technical difficulty in removing larger tumors. In many neoplasms, Ki-67 is widely used as a marker for tumor cell proliferation. This protein is expressed in the nuclei of cells in the G1, S, G2, and M phases of the cell cycle but not in the G0 phase [7]. In human pituitary tumors, including corticotroph adenomas, the Ki-67 labeling index (LI) has been used to assess tumor proliferation. However, its precise function in cell proliferation is still unknown [8], and its use has been described both positively and negatively [9 – 12]. Therefore, the significance of Ki-67 is strongly debated, and other proliferation markers are needed for pituitary adenomas. Minichromosome maintenance (MCM) proteins, particularly MCM2, MCM5, and MCM7, have recently been described as potential markers of cell proliferation in neoplasms [13]. The MCM proteins are part of the replication system complex that licenses DNA replication, and, once DNA replication is completed, they are displaced from chromatin [14]. Furthermore, MCM proteins have been characterized as showing higher specificity and sensitivity than other conventional proliferation markers, such as Ki-67 [13,15,16]. However, to our knowledge, only one report has described the expression of any of the MCMs in pituitary tumors, in this case MCM2 [17].

Although PDH is a common endocrine disorder in dogs, only one report has investigated tumor proliferation markers in this setting [18]. Therefore, the aim of the present study was to investigate the expression of the newly described proliferation marker MCM7 and a conventional proliferation marker Ki-67 and to evaluate their relation with tumor size in canine pituitary corticotroph adenomas. 2. Materials and methods 2.1. Animals All experimental procedures were approved by the Bioethics Committee at Nippon Veterinary and Life Science University. Fifteen dogs [5 females (2 spayed) and 10 males (3 neutered)] with PDH treated by transsphenoidal hypophysectomy as described by Meij et al [19,20] were included in this study. The dogs included various breeds, and the median age and body weight at surgery were 7 yr (range, 5 to 10 yr) and 11.2 kg (range, 5.8 to 21.9 kg), respectively (Table 1). PDH was diagnosed by clinical signs, routine laboratory examinations, endocrine examinations (plasma ACTH concentrations in 7 dogs and ACTH-stimulation test in 15 dogs), abdominal ultrasonography, and magnetic resonance imaging of the pituitary. Abdominal ultrasonography confirmed that all cases had bilateral enlargement of the adrenal grand (⬎7.5 mm wide). The resected pituitary tissues were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. One section was stained with hematoxylin and eosin, and adjacent sections of the pituitary were subjected to immunohistochemical staining with the use of standard enzyme antibody techniques with a monoclonal mouse antibody to synthetic ACTH1–39 (Dako Japan, Tokyo, Japan) [21]. From the histopathologic examination, a veterinary pathologist diagnosed each case as having a corticotroph adenoma in the anterior pituitary. The dogs were grouped according to the P/B ratio, which was calculated with AZE Virtual Place software (AZE, Tokyo, Japan), on gadolinium-enhanced T1 transverse magnetic resonance images, as previously described [5]. A P/B ratio ⬎ 0.31 was classified as an enlarged pituitary, whereas a P/B ratio ⱕ 0.31 was classified as a nonenlarged pituitary according to the previous report [5]. Nine dogs had enlarged pituitaries with a median P/B ratio of 0.407 (range, 0.32 to 0.793), and six dogs had nonenlarged pituitaries (median, 0.266; range, 0.23 to 0.306). Normal pituitary and tonsil tissues were obtained from a clinically and neurologically normal adult male

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Table 1 Clinical characteristics, Ki-67 and MCM7 labeling indices in corticotroph adenomas of the dogs included in this study. Case

Breed

Sex

Age (yr)

BW (kg)

ACTH (pmol/L)a

Before Cort (nmol/L)b

After Cort (nmol/L)c

Pituitary height (mm)d

P/Be

Ki-67 LI (%)f

MCM7 LI (%)g

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Yorkshire Terrier Shiba Shetland Sheepdog Mongrel Pembroke Welsh Corgi Miniature Dachshund Beagle French Bulldog Shiba Beagle Shiba Mongrel Mongrel Cavalier KC Spaniel Shiba

MC M M M F M M FS F MC F MC M FS M

6 10 5 8 4 10 6 5 10 6 8 10 8 7 7

5.8 21.9 13.8 10.2 11.2 9.0 12.5 9.0 11.6 13.9 11.2 14.6 16.1 6.8 10.9

19.2 NA 51.1 13.9 NA 11.7 NA 55.5 331.0 20.9 NA NA 18.1 NA NA

232 74 435 235 298 141 171 163 279 320 210 72 199 576 174

1843 737 2185 1644 1371 1371 1123 1070 1264 1280 1468 1093 684 2000 1413

4.6 7.3 10.2 4.9 7 6.2 7 16 7.8 4.4 5 4.6 4.2 5.4 4.2

0.340 0.492 0.714 0.320 0.369 0.344 0.407 0.793 0.551 0.230 0.281 0.251 0.244 0.299 0.306

2.5 2.4 1.6 1.8 1.5 1.1 0.7 1.7 1 0 0 1.4 0.1 1 0.8

11.8 16.7 10.5 9.3 6.9 7.3 7.2 16.1 12.2 1.3 2.2 6.8 1.2 4.2 1.5

Abbreviations: Cort, cortisol; P/B, pituitary/brain ratio; LI, labeling index; MCM, minichromosome maintenance; F, female intact; FC, female spayed; M, male intact; MC, male castrated; NA, not available. a Preoperative plasma ACTH (reference range, 1.3 to 12.9 pmol/L); values are the mean of two samples taken at an interval of 10 to 15 min. b Preoperative serum basal cort (reference range, 17 to 132 nmol/L); values are 0 min after the intravenous administration of 0.25 mg of synthetic ACTH (Cortrosyn; Daiichi Sankyo, Tokyo, Japan). c Preoperative serum after ACTH cort (reference range, 165 to 480 nmol/L); values are 60 min after the intravenous administration of 0.25 mg of synthetic ACTH. d Pituitary height (mm) measured on a preoperative enhanced T1-weighted transverse image. e P/B area ratio ⫻ 10⫺2 mm⫺1 (P/B ratio ⱕ 0.31 ⫽ nonenlarged pituitary, P/B ratio ⬎ 0.31 ⫽ enlarged pituitary). f Ki-67 LI was defined as the percentage of immunoreactive cells expressing Ki-67 and ACTH of all ACTH cells counted. g MCM7 LI was defined as the percentage of immunoreactive cells expressing MCM7 and ACTH of all ACTH cells counted.

Beagle dog (2 yr, 10.5 kg) euthanized in an experiment for fracture treatment. Rat testis tissue was obtained from an 8-week-old Sprague-Dawley rat euthanized in an experiment for spinal cord injury treatment. These experiments were also approved by the Bioethics Committee at Nippon Veterinary and Life Science University. The tissues were fixed in 4% paraformaldehyde and embedded in paraffin or used for protein extraction. 2.2. Immunohistochemistry To detect Ki-67 and MCM7 in pituitary corticotroph adenomas, 2-␮m-thick consecutive paraffin sections were cut from the tissue blocks and mounted on Matsunami Adhesive Silane-coated slides (Matsunami, Osaka, Japan). The sections were deparaffinized in xylene and rehydrated through an alcohol gradient. Antigen retrieval was performed by autoclaving the sections in citrate buffer (10 mM, pH 6.0) at 121°C for 10 min, followed by cooling at room temperature (RT) for 20 min. The slides were then washed three times for 5 min each in 10 mM PBS. Endogenous peroxidase activity was blocked by immersing the sections in 0.3% hydrogen peroxide in absolute methanol for 30 min. The slides were washed three times for 5 min each in PBS

and treated for 30 min with 10% normal goat serum (Dako Japan, Tokyo, Japan) in PBS. The slides were incubated with the primary antibodies overnight at 4°C. The antibodies were anti-MCM7 monoclonal rabbit antibody (Cell Signaling Technology Japan, Tokyo, Japan) diluted 1:400 in PBS and anti-Ki-67 monoclonal rabbit antibody (Lab Vision, Fremont, CA, USA) [18,22] diluted 1:100 in PBS. After being washed three times for 5 min each in PBS, the slides were incubated for 1 h at RT with polymer-horseradish peroxidase– conjugated anti-rabbit IgG antibody (Dako Japan). The slides were washed three times for 5 min each in PBS, and immunoreactivity was visualized with 50 mM TrisHCl buffer (pH 7.6) containing 0.01% 3,3-diaminobenzidine tetrahydrochloride, 0.005% H2O2, and 0.01% sodium nitrate. After the detection of Ki-67 or MCM7, the slides were immunohistochemically stained for ACTH to label corticotrophs. To achieve this, after visualizing the Ki-67 or MCM7, the slides were washed three times for 5 min each in PBS and then incubated with anti-ACTH1–39 monoclonal mouse antibody (Dako Japan) [21] diluted 1:200 in PBS for 1 h at RT. The slides were washed three times for 5 min each in 10 mM Tris-buffered saline (TBS) and incubated with

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alkaline phosphatase-conjugated F(ab’)2 fragments of goat anti-mouse IgG antibody (Abcam, Tokyo, Japan) diluted 1:400 in TBS for 1 h at RT. The slides were washed three times for 5 min each in TBS, and the immunoreaction was visualized with a Vulcan Fast Red Chromogen kit (Biocare Medical, Concord, CA, USA). After being washed in deionized water, the sections were lightly counterstained with hematoxylin, dehydrated through an alcohol gradient and xylene, and coverslipped. Normal canine tonsil tissue sections were used as positive controls for Ki-67. Normal canine pituitary tissue sections were used as positive controls for MCM7 and ACTH. Negative controls were obtained by omitting the primary antibodies. 2.3. Evaluation of Ki-67 and MCM7 LIs Slides were observed under an Olympus BX51 light microscope connected with a DP71 camera (Olympus, Tokyo, Japan) at magnification of ⫻400. The fields were scanned across the slide to avoid overlap. At least five randomly selected fields containing ACTH-positive cells were counted by one of the authors blinded to the source of each slide with the use of Image J software version 1.43 (http://rsb.info.nih.gov/ij/). The Ki-67 and MCM7 LIs were defined as the percentage of immunoreactive cells coexpressing Ki-67 and ACTH or MCM7 and ACTH of all ACTH-positive cells counted. On average, 1,000 ACTH-positive cells were counted per slide. The Ki-67 and MCM7 LIs were each determined on three slides for each case. 2.4. Western blot analysis Samples from canine pituitary tissues (normal pituitary, corticotroph adenoma) and rat testis were transferred to a mortar and crushed in liquid nitrogen with a pestle. The tissue powders were transferred to ProteoJET Mammalian Cell Lysis Reagent (Fermentas, Glen Burnie, MD, USA) and incubated for 10 min at RT. The lysates were then centrifuged (16,000 ⫻ g for 15 min at 4°C), the supernatant fluids were collected, and protein content was measured with the bicinchoninic acid method (DC Protein Assay; Bio-Rad Japan, Tokyo, Japan) according to the manufacturer’s instructions. Then, total protein samples (20 ␮g) were electrophoresed by 7.5% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (GE Healthcare Japan, Tokyo, Japan). The membrane was incubated with a monoclonal anti-MCM7 antibody (Cell Signaling Technology Japan, Tokyo, Japan) diluted 1:1000 in Can Get Signal solution 1 (Toyobo, Tokyo, Japan).

Peroxidase-conjugated F(ab’)2 fragments of donkey anti-rabbit IgG (Amersham Biosciences, Piscataway, NJ, USA) diluted 1:5000 in Can Get Signal solution 2 (Toyobo) were used as the secondary antibody. Binding was visualized with a chemiluminescence detection kit (SuperSignal West Femto Maximum Sensitivity Substrate; Thermo Fisher Scientific, Yokohama, Japan). Total protein extract from rat testis was used as a positive control [23]. 2.5. Statistical analysis Statistical analyses were performed with Statistical Package for Social Sciences software version 16.0J (SPSS, Tokyo, Japan). The Ki-67 LI, MCM7 LI, and the P/B ratio were tested for normality with the Shapiro-Wilks test. Both the Ki-67 LI and the MCM7 LI were normally distributed, whereas the P/B ratio was not normally distributed (P ⬍ 0.05). Student t test was used to compare the Ki-67 LI and the MCM7 LI between adenomas in enlarged and nonenlarged pituitaries. Paired t tests were used to compare Ki-67 LI with MCM7 LI. Correlations among the Ki-67 LI, MCM7 LI, and the P/B ratio were determined with Pearson product-moment correlation test (between Ki-67 LI and MCM7 LI) or Spearman rank correlation coefficient test. Values of P ⬍ 0.05 were considered statistically significant. 3. Results 3.1. Ki-67 and MCM7 LIs Ki-67- and MCM7-positive nuclei were immunohistochemically visualized as brown (Fig. 1). The mean ⫾ SD LI of Ki-67 in corticotroph adenomas was 0.55% ⫾ 0.59% in nonenlarged pituitaries and 1.6% ⫾ 0.6% in enlarged pituitaries (Fig. 2A; Table 1). The mean LI of MCM7 in corticotroph adenomas was 2.9% ⫾ 2.2% in nonenlarged pituitaries and 10.9% ⫾ 3.7% in enlarged pituitaries (Fig. 2B; Table 1). The Ki-67 LI was significantly higher in the adenomas with enlarged pituitaries than in the adenomas with nonenlarged pituitaries (P ⬍ 0.01). Similarly, the MCM7 LI was significantly higher in the adenomas with enlarged pituitaries than in the adenomas with nonenlarged pituitaries (P ⬍ 0.01). The MCM7 LI was significantly higher than the Ki-67 LI in pituitary corticotroph adenomas (P ⬍ 0.01). The Ki-67 LI was positively correlated with the MCM7 LI (r ⫽ 0.820, P ⬍ 0.01). Furthermore, the P/B ratio was positively correlated with the Ki-67 LI (r ⫽

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Fig. 1. Ki-67 and ACTH double-labeled immunohistochemistry (A), MCM7 and ACTH double-labeled immunohistochemistry (B) of pituitary corticotroph adenoma tissue obtained from a dog (Case 8) with an enlarged pituitary. The Ki-67- and MCM7-positive nuclei are visualized as brown, and the ACTH-positive cytoplasm is visualized as red. Bar ⫽ 50 ␮m.

0.560, P ⫽ 0.03) and the MCM7 LI (r ⫽ 0.854, P ⬍ 0.01). 3.2. Determination of MCM7 expression by Western blot analysis Western blot analysis (Fig. 3) showed the presence of canine MCM7 protein (molecular weight, 80 kDa). These results imply that the antibody used had sufficient specificity for canine MCM7. The analysis also showed that MCM7 is expressed in both normal and neoplastic canine pituitary tissues. 4. Discussion Pituitary-dependent hyperadrenocorticism caused by a corticotrophic adenoma is a common endocrine disorder in dogs. Despite its frequency, little is known about the pathogenesis of pituitary tumors in dogs.

Moreover, only one report has examined the proliferation marker Ki-67 in this setting [18]. In the present study, we examined the expression of the proliferation markers Ki-67 and MCM7 in ACTH-positive tumor cells. We demonstrate that the expression of these proliferation markers in corticotroph adenomas with enlarged pituitaries was significantly higher than that in adenomas with nonenlarged pituitaries in PDH dogs. In addition, both the Ki-67 LI and the MCM7 LI were positively correlated with pituitary size determined by the P/B ratio. In human pituitary tumors, Ki-67 has long been used as a proliferation marker, and it is considered to be a main predictive indicator in the recent World Health Organization classification [24]. However, the clinical significance of Ki-67 is strongly debated. Some investigators have found that the Ki-67 LI was higher in ACTH-secreting macroadenomas than in microadeno-

Fig. 2. Boxplots for the Ki-67 (A) and MCM7 (B) labeling indices in pituitary corticotroph adenomas in nonenlarged (NE) and enlarged (E) pituitaries. **P ⬍ 0.01 versus adenomas in nonenlarged pituitaries.

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Fig. 3. Western blotting for MCM7. Lane 1, normal canine pituitary tissue; lane 2, canine corticotroph adenoma; lane 3, rat testis. The bands were located at the expected molecular weight of MCM7 (80 kDa) recognized with the antibody. Total protein extract from rat testis was used as a positive control.

mas [10], whereas others found no such difference [11,25]. Similar to human pituitary corticotroph adenomas, a previous study reported contrasting results in Ki-67 LI compared with our findings [18]. In that study, the Ki-67 LI did not differ between adenomas in enlarged and nonenlarged pituitaries, and it was not correlated with the P/B ratio [18]. Their results may have been influenced by a single elevated value in the adenomas in nonenlarged pituitaries and the absence of Ki-67 expression in two adenomas in enlarged pituitaries. Moreover, it seems that the nuclear antigen Ki-67 was expressed in the cytoplasm rather than in nuclei in their report. In the present study, we did not find Ki-67 expression in the cytoplasm. Although its precise function in cell proliferation is still unknown [8], its expression in the cytoplasm may not be related to cell proliferation. Therefore, these differing outcomes may have occurred because of differences in Ki-67 localization. However, immunohistochemical analysis of Ki-67 has shown that it has high interlaboratory variability even when using the same tissue and the same antibody [26]. Thus, the different methods used to detect Ki-67 (eg, the use of different fixation and antigen retrieval methods) may have led to these conflicting results. It is considered that some experience is needed for objectively interpreting the results of Ki-67 immunohistochemistry in endocrine tissues. Notably, Ki-67 LIs in pituitary tumors vary considerably among reports [9–12,25]. Therefore, it seems that immunohistochemical detection of Ki-67 in pituitary tumors is inconsistent, and Ki-67 may not be a suitable proliferation marker in such tissues. It has also been suggested that Ki-67 plays a role in ribosome biosynthesis rather

than being directly responsible for cell proliferation [27]. Thus, it seems that proliferation activity determined by the Ki-67 LI alone is questionable. Considering these factors, more appropriate proliferation markers for pituitary tumors have been needed. The MCM proteins are essential components in the control of DNA replication [14,28,29]. Numerous studies have suggested that MCM proteins show greater specificity and sensitivity than other conventional proliferation markers, such as Ki-67 [13,15,16]. Therefore, we assessed MCM7 expression in canine pituitary corticotroph adenomas. In the present study, we found that the MCM7 LI was significantly higher than the Ki-67 LI. However, in human GH-secreting and clinically nonfunctional pituitary adenomas, it has been reported that MCM2 expression did not differ from that of Ki-67 [17]. Some studies reported a higher proliferation index in ACTH-secreting adenomas than in other pituitary hormone-secreting adenomas [11,30]. Thus, these different outcomes may have occurred because the proliferation index in ACTH-secreting adenomas differs from that in other pituitary adenomas, or because canine pituitary tumors show greater proliferation than human pituitary tumors. Otherwise, there may be differences between the individual MCM proteins. Nevertheless, the high sensitivity of the MCM proteins is probably because Ki-67 is required for the initiation of replication, representing the point of convergence of many signaling pathways involved in cell growth. Moreover, in quiescent and resting cells, MCM proteins still maintain replication competence, unlike Ki-67 [14,28,29], supporting the validity of our results. We also found that the MCM7 LI in the adenomas with enlarged pituitaries was significantly higher than that in the adenomas with nonenlarged pituitaries and that the MCM7 LI was positively correlated with the P/B ratio. Because MCM7 is directly responsible for cell proliferation [14, 28,29], these results suggest that tumor cells in an enlarged pituitary show greater proliferation potential than do tumor cells in a nonenlarged pituitary. Furthermore, the positive correlation between the MCM7 LI and the P/B ratio raise the possibility that adenomas with greater proliferation potential may grow more rapidly, resulting in the development of large tumors. However, the P/B ratio was only determined at the time of surgery, and the growth rate of adenomas was not evaluated in the present study. Thus, it is not possible to confirm an association between MCM LI and the growth rate of pituitary corticotroph adenomas. In conclusion, to our knowledge, this is the first study describing MCM expression in canine pituitary tumors. We found that, in dogs with PDH, adenomas in enlarged pituitaries have significantly higher Ki-67 LI and MCM7 LI than do adenomas in nonenlarged pituitaries. This in-

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