Expression of Proliferating Cell Nuclear Antigen (PCNA) in Adrenal Glands from Domestic Ferrets (Mustela putorius furo) with Spontaneous Cortical Hyperplasia and Neoplasia

Ashley L. Ayoob, Laura A. Adams, Raymond P. Campagnoli, and Kenneth S. Latimer

Honors Biology Program, Biological Sciences (Ayoob, Adams) and Department of Pathology, College of Veterinary Medicine (Campagnoli, Latimer), The University of Georgia, Athens, GA 30602

Abstract: The goal of this pilot study was to evaluate the use of proliferating cell nuclear antigen (PCNA) immunoreactivity as a diagnostic technique to histologically differentiate adrenocortical hyperplasia, adrenocortical adenoma, and adrenocortical carcinoma in spontaneous adrenal disease of ferrets. The percentage of nuclei that expressed PCNA reactivity overlapped somewhat for the various diagnostic categories (normal adrenal gland, adrenocortical hyperplasia, adrenocortical adenoma, and adrenocortical carcinoma). Immunoreactivity was observed in <1% of cortical cell nuclei in tissue sections from normal adrenal glands. In contrast, PCNA expression for adrenocortical hyperplasia and adrenocortical adenomas ranged from <1% to 24.8% and from 1.8% to 54%, respectively. PCNA immunoreactivity in adrenocortical carcinomas was present in 4.5% to 78.5% of cortical cell nuclei. Although PCNA expression alone may not reliably distinguish adrenocortical cell hyperplasia from adrenocortical adenoma or carcinoma, a greater percentage of cells from the carcinomas exhibited PCNA reactivity, suggesting that PCNA immunohistochemistry may be a useful prognostic indicator of malignancy.

Key Words: Ferret, Mustela putorius furo, Adrenal gland, Endocrinopathy, Hyperplasia, Adenoma, Carcinoma, Proliferating cell nuclear antigen, PCNA, Immunohistochemistry

Introduction

In recent years, special emphasis has been placed on improved diagnosis of functional adrenocortical hyperplasia and neoplasia in domestic ferrets (Mustela putorius furo). The presence of adrenal neoplasia and adrenal-associated endocrinopathy has been reported frequently in domestic ferrets with an overall incidence of up to 25% in some studies.¹ The presence of adrenal neoplasms is observed equally in males ("hobs") and females ("jills"); however, tumors occur primarily among middle age to older ferrets. Most ferrets that develop adrenal-associated endocrinopathy range from 28 to 84 months of age at diagnosis (the average life span of the domestic ferret ranges from 109 to 120 months of age).² Neoplasms can arise in either adrenal gland; however, approximately 75 to 90% of tumors have been reported to originate in the left adrenal gland.²

Although adrenal tumors are common in ferrets, little is known about their pathogenesis. In addition, improved diagnosis of adrenal neoplasms and differentiation from nodular adrenocortical hyperplasia would be beneficial in a clinical setting. Diagnosis of adrenal neoplasms in ferrets is based upon clinical signs, palpation, ultrasonography, and radiography. These diagnostic options are somewhat limited in scope. Palpation may reveal enlarged adrenal glands in approximately 33% of ferrets with neoplasms.³ Ultrasonography is considered to be diagnostic in only 50% of cases because normal adrenal glands often are too small for consistent detection.¹ Visualization of adrenal neoplasms is only possible when the tumor is unusually large. In contrast, small adrenal gland tumors cannot be identified radiographically because they do not calcify to promote early detection. Currently, adrenolectomy is the preferred treatment for adrenal neoplasms in ferrets.

In various human neoplasms, immunohistochemical markers that measure the kinetic parameters of the cell have been used successfully as prognostic indicators. One such marker is proliferating cell nuclear antigen (PCNA). PCNA is a 36kD nuclear polypeptide that functions as a co-factor for DNA polymerase d , which participates in DNA synthesis and repair.⁴ PCNA is expressed mainly in the late G₁ and S phases of the mitotic cycle. However, due to its long half-life, immunohistochemical localization of PCNA can be used as a reliable marker of cells undergoing active proliferation.⁵ In human tumors, a correlation has been found between PCNA immunoreactivity and patient prognosis. Tumors with a high proliferation rate (a high PCNA score) have been associated with poorer prognosis than tumors with a low proliferation rate.^4,6 Although PCNA immunoreactivity has improved the diagnosis and prognosis of some human neoplasms, this technique has rarely been used for these purposes in domestic animals. The objectives of this study were to determine if a commercial anti-PCNA antibody was reactive with ferret adrenal tissues, to evaluate the prognostic value of PCNA immunoreactivity in distinguishing adrenocortical hyperplasia from neoplasia, and to determine whether PCNA immunoreactivity could reliably distinguish malignant from benign adrenal neoplasms.

Materials and Methods

Tissues: Case accessions from the Department of Pathology and the Athens Diagnostic Assistance Laboratory, College of Veterinary Medicine, The University of Georgia were reviewed for ferret adrenal glands submitted for histologic examination from 1992 to 1999. During this time period, 269 adrenal gland biopsies were submitted for histologic evaluation. Biopsies with a diagnosis of normal adrenal gland, adrenocortical hyperplasia, adrenocortical adenoma, and adrenocortical carcinoma were retrieved for microscopic review. Selection of eligible case material was based upon the presence of a complete medical records, an intact adrenal gland obtained by excisional biopsy, a short formalin fixation time (< 24 hr), presence of an archived paraffin embedded tissue block, and adequate paraffin embedded tissue for extensive replicate sectioning. After the histologic slides were reviewed for accuracy of diagnosis, 20 tissue sections were selected randomly from this pooled diagnostic material for a pilot project on PCNA immunohistochemical staining. The diagnoses for the 20 randomly chosen biopsies of adrenal gland were: normal adrenal gland (n=5), adrenocortical hyperplasia (n=3), adrenocortical adenoma (n=4), and adrenocortical carcinoma (n=8).

Anti-PCNA antibody: For immunohistochemistry, the primary antibody was a commercially available monoclonal mouse anti-PCNA antibody (DAKO M-0879).^a The antibody was reported to react with all vertebrate species tested and was specifically designed for use on formalin-fixed, paraffin embedded and frozen tissue sections. A Universal Vectastain Elite ABC Kit,^b also purchased commercially, was used to detect and visualize sites of primary antibody binding.

Immunohistochemistry: Formalin-fixed, paraffin embedded tissues were sectioned at 3 to 4 µm and mounted on ProbeOn Plus glass slides.^c The tissue sections were deparaffinized in HemoDe^c (limonene) and rehydrated in graded ethanol to Autobuffer.^d Endogenous peroxidase activity was quenched by immersing the tissue sections in 3% hydrogen peroxide for 15 minutes. The sections subsequently were rinsed in distilled water and placed in autobuffer. To reduce background staining, the tissue sections were blocked for 5 minutes at 30° C with 1.5% normal horse serum in 0.3% Triton X-100 and PBS. The anti-PCNA antibody (primary antibody) was diluted to a 1:50 ratio in PBS with 1.5% horse serum and 0.3 % Triton, placed on the tissues, and incubated at 30° C for 30 minutes. Subsequently, the primary antibody was blotted and the tissues were rinsed with Autobuffer.

The secondary biotinylated horse anti-mouse antibody was diluted to a 1:200 ratio, applied to the tissue sections, and incubated at 30° C for 30 minutes. Subsequently, the tissues were blotted and rinsed in Autobuffer. Avidin-biotin complex was applied to the tissues, incubated at 30° C for 30 minutes, and rinsed with autobuffer. Next, a diaminobenzidine (DAB) chromagen solution (20 m l of 3% hydrogen peroxide in 2.5 ml DAB at 1 mg/ml) was added to the tissue sections, which were incubated at room temperature for 6 minutes. Following a brief rinse in distilled water, the tissue sections were counterstained in Gill’s II hematoxylin for 45 seconds, rinsed in distilled water, dehydrated through a series of graded ethanol solutions to xylene, coverslipped, and examined by light microscopy.

Image acquisition: During microscopic examination of the immunostained tissue sections, five random images of each biopsy specimen were captured via digital camera for subsequent analysis. These microscopic images were captured at a magnification of 40x by the digital camera and were then saved by computer as TIFF files. The images were analyzed using Image Pro Plus Version 3.0 for Windows.^e The intensity of color analyzed was adjusted to select for stained nuclei and then for normal (unstained) nuclei. The program was also used to obtain counts of positively-stained nuclei and normal nuclei. The data subsequently were analyzed for statistical significance based upon the ratio of positively-stained nuclei to normal nuclei.

Statistical analyses: The four groups were designated as normal adrenal gland, nodular adrenocortical hyperplasia, adrenocortical adenoma, and adrenocortical carcinoma based upon evaluation of H&E stained tissue sections. An analysis of variance was performed to determine significant differences in the percentages of cell nuclei that expressed PCNA immunoreactivity. Significant differences between group means were detected by Fisher's (protected) least significant difference test. The level of significance tested was p=0.05.

Results

The DAKO primary antibody was immunoreactive with ferret tissues, as has been reported for tissues from other vertebrate species. PCNA reactivity was readily discerned during microscopic examination of the tissue sections.

Data related to image analysis parameters and histological diagnoses are presented in Table 1. In general, normal adrenal gland tissue sections had the least PCNA immunoreactivity, while adrenocortical carcinomas had the greatest PCNA immunoreactivity. Considerable overlap in immunostaining occurred within adrenal sections that were diagnosed as nodular adrenocortical hyperplasia and adrenocortical adenoma. This observation is presented in condensed form in Table 2. Although the difference in PCNA immunoreactivity between the groups was highly significant (p=0.01, Table 2), the samples within the various groups overlapped each other somewhat.

Table 1. Histologic diagnosis and PCNA immunoreactivity of adrenal gland tissue sections from 20 ferrets.

Table 2. Ferret adrenal gland biopsies: Histologic diagnoses and percentage of nuclei staining for proliferating cell nuclear antigen (PCNA).

Figure 1. Ferret, normal adrenal gland, case 1, PCNA immunostaining with hematoxylin  counterstain. Stained nuclei are not observed, indicating a lack of cell proliferation.	Figure 2. Ferret, nodular adrenocortical hyperplasia, case 12, PCNA immunostaining with hematoxylin counterstain. Widely scattered cell nuclei are stained, indicating limited cortical cell proliferation.

Figure 3. Ferret, adrenocortical adenoma, case 11, PCNA immunostaining with hematoxylin counterstain. A moderate number of stained nuclei indicate a greater degree of cellular proliferation.	Figure 4. Ferret, adrenocortical carcinoma, case 19, PCNA immunostaining with hematoxylin counterstain. Numerous nuclei are stained indicating marked cellular proliferation.

Discussion

The results of this pilot study indicate that PCNA immunoreactivity may be useful as a prognostic indicator of adrenal malignancy in ferrets as it is in human beings. All of the normal adrenal glands had less than 1% PCNA immunoreactivity in the nuclei of cortical cells. In the normal adrenal gland, cellular proliferation should be minimal. Since PCNA is merely a proliferation marker, both normal cells and neoplastic cells will express this antigen when they are actively engaged in mitosis. Wolkersdorfer, et al.⁷ point out that the adrenal gland has the capability to adapt to the changing needs of the body. Thus, a high rate of normal cell turnover may be found within the adrenal cortex.

In contrast to normal adrenal glands, those glands with nodular hyperplasia may exhibit a very wide range of PCNA immunoreactivity. The percentage of stained nuclei ranged from <1% to 24.8%. This variability in immunostaining could be attributed to the length of time that abnormal cellular proliferation has been present. A ferret that has only very recently shown abnormal adrenal function may not have a very high proliferation rate. However, a ferret that has exhibited the clinical signs of disease for an extended period of time may have no density-dependent inhibition properties, thus coming closer to a transition to malignancy. Therefore, an extreme rate of cellular proliferation may be a characteristic of the early stages of cancer. In addition, the variability in cellular proliferation also could be explained by an inaccurate histologic diagnosis, based upon the examination of H&E stained tissue sections alone. When cases were reviewed for accuracy of the histologic diagnosis, several cases diagnosed as adrenocortical hyperplasia cases appeared to actually be carcinomas. An example is case 16. This ferret was originally diagnosed with nodular and capsular accessory cortical nodular hyperplasia. However, review of the H&E stained slide revealed features of a carcinoma wherein cortical cells dissected and infiltrated the capsule, periglandular fat, and blood vessels; changes that are consistent with a carcinoma.

The adrenocortical adenomas, as well as cases of nodular adrenocortical hyperplasia, exhibited a wide range of immunoreactivity. The percentages of stained corticocellular nuclei ranged from 1.8% to 54%. There are several explanations for this observation. First, the lesions may have been misdiagnosed based upon the evaluation of H&E stained tissue sections (case 15 was originally diagnosed as a adenoma). Second, the staining intensities for adenomas and nodular hyperplasias may vary. Third, the degree of cellular proliferation may vary with early or late disease states or may differ when secondary inflammation is present (case 18).

All carcinomas exhibited marked PCNA immunoreactivity. Generally, the degree of immunostaining revealed that >4.5% of the adrenocortical cells were actively proliferating. The greatest degree of PCNA immunoreactivity was observed in the adrenal biopsy from case 20, in which 78.5% of the adrenocortical cells demonstrated nuclear staining. Based on our observations, any adrenal gland that exhibits positive nuclear PCNA immunoreactivity in >25% of the adrenocortical cells staining can be considered likely to be cancerous.

A few conclusions can be drawn from this pilot study. It is obvious that distinguishing adrenocortical hyperplasia and adrenocortical neoplasia in ferrets can be difficult when relying only on examination of H&E stained tissue sections. However, using PCNA immunoreactivity and routine histopathology apparently improves diagnostic accuracy. Furthermore, PCNA reactivity may be a useful prognostic indicator by demonstrating how rapidly abnormal cell populations are proliferating.

Sources and Manufacturers

a. DAKO Corporation, Carpinteria, CA, USA.

b. Vector Laboratories, Burlingame, CA, USA

c. Fisher Scientific, Pittsburgh, PA, USA.

d. Biomeda Corporation, Foster City, CA, USA

e. Media Cybernetics, Silver Spring, MD, USA

References

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2. Neuwirth L, Isaza R, Bellah J, Ackerman N, Collins: 1993, Adrenal neoplasia in seven ferrets. Vet Radiol Ultrasound 34:340-346.

3. Wheler CL, Kamieniecki CL: 1998, Ferret adrenal-associated endocrinopathy. Can Vet J 39:175-176.

4. Roels S, Tilman K, Ducatelle R: 1999, PCNA and Ki-67 proliferation markers as criteria for prediction of clinical behaviour of melanocytic tumours in cats and dogs. J Comp Pathol 121:13-24.1

5. Suzuki T, Sasano H, Nisikawa T, Rhame J Wilkinson DS, Nagura H: 1992, Discerning malignancy in human adrenocortical neoplasms: utility of DNA flow cytometry and immunohistochemistry. Mod Pathol 5:224-236.

6. Bergada I, Venara M, Maglio S, Diez B, Bergada C, Chemes H: 1996, Functional adrenal cortical tumors in pediatric patients: a clinicopathologic and immunohistochemical study of a long-term follow-up series. Cancer 77:771-777.

7. Wolkersdorfer GW, Marx C, Brown JW, Scherbaum WA, Bornstein SR: 1996, Evaluation of apoptotic parameters in normal and neoplastic human adrenal. Endocrine Res 22:411-419.

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