Evaluation of Proliferation Marker (PCNA, Ki-67, and p53) Expression in Avian Hepatic Tissues
Pamela J. Sullivan, Raymond P. Campagnoli, and Kenneth S. Latimer
Department of Pathology, College of Veterinary Medicine, The University of Georgia, Athens, Georgia 30602-7388 (USA)
Abstract. Expression of various proliferation markers was evaluated by immunohistochemical staining in hepatic tissues from 17 birds. The markers that were evaluated included proliferating cell nuclear antigen (PCNA), Ki-67, and p53. Immunohistochemistry was performed using monoclonal antibodies for each marker, as well as polyclonal anti-Ki-67 antibody. The hepatic tissues evaluated included: normal liver (n = 2), bile duct hyperplasia (n = 3), and cholangiocarcinoma (bile duct carcinoma) (n = 12). Only PCNA immunoreactivity was reliable and reproducible in avian liver sections. PCNA expression was increased in 7 of 12 cholangiocarcinomas, but failed to differentiate 5 cholangiocarcinomas from extreme biliary hyperplasia. The results of this study suggest that excessive staining of biliary epithelial cell nuclei or combined staining of biliary and hepatocellular nuclei increases the suspicion of cholangiocarcinoma. Therefore, extreme bile duct hyperplasia and cholangiocellular carcinoma may still be difficult to differentiate using PCNA immunoreactivity alone. Lastly, Ki-67 (monoclonal and polyclonal antibodies) and p53 immunoperoxidase staining is unreliable in avian tissues using the mammalian reagents specified in this manuscript.
Key Words: Avian, Bile duct hyperplasia, Bile duct carcinoma, Cholangiocellular carcinoma, Immunohistochemistry, Ki-67, Liver, Neoplasia, PCNA, p53
Introduction
Common causes of bile duct hyperplasia in poultry include ingestion of mycotoxins (aflatoxin, T-2 toxin), poisonous plants (Crotolaria spectabilis), phenol derivatives (chlorodioxin, pentachlorophenol, coal tar derivatives, and disinfectants), and various other compounds (lithocholic acid, D,L-ethionine, thioacetamide, dimethylnitrosamine, and methimazole). 1 Bile duct hyperplasia also is observed frequently in psittacine and free-ranging birds with liver disease. Cholangiocarcinoma is the most commonly reported hepatic neoplasm in both captive and free-ranging birds. 2-9 These neoplasms may be accompanied by invasion of the hepatic parenchyma, necrosis, and desmoplasia. Metastasis to the lungs, brain, kidney, pleura or serosa may occur late in the disease process. 2
In human and comparative mammalian research, proliferation markers have been used to evaluate cell replication in various tissue lesions, including neoplasia. Proliferating cell nuclear antigen (PCNA) is a highly conserved 36 kD acidic nuclear protein that is expressed during cell replication and DNA repair. 10,11 PCNA interacts with DNA polymerase d and with RF-C protein to bind at DNA primer-template junctions. Immunostaining of S-phase nuclei will detect PCNA in sites of DNA synthesis. Ki-67 antigen is expressed during the G1, S, G2, and M phases of the cell cycle, but is not expressed during the G0 (resting) phase. Because Ki-67 antigen has a short half-life, it can be used as a marker of actively proliferating cells. 12,13 The protein p53 serves as a tumor suppressor gene product for all normal cells. This protein inhibits cell proliferation in the G1 and G2/M phases of the cell cycle. More recently, p53 is thought to either promote cell survival by facilitating DNA repair or promote cell death via apotosis. 14 Expression of mutant p53 is associated with decreased DNA repair, a mutated cell genome, lack of cell proliferation control, and progression to neoplasia. 15,16
Avian pathologists occasionally have difficulty in distinguishing extreme biliary hyperplasia from well differentiated cholangiocellular carcinoma, based upon the examination of H&E-stained liver sections. 2 In such instances, evaluation of proliferation marker immunoreactivity might reliably distinguish hyperplasia from neoplasia. The objectives of this study, therefore, were to determine if mammalian reagents could be used to detect selected proliferation markers (PCNA, Ki-67, and p53) in formalin fixed, paraffin-embedded avian liver tissue. In addition, we wished to determine if such markers could reliably distinguish biliary hyperplasia from cholangiocellular carcinoma.
Materials and Methods
Avian tissues: Laboratory reports from the Department of Pathology and the Athens Diagnostic Laboratory were reviewed to identify recent surgical biopsy submissions diagnosed as normal liver, bile duct hyperplasia, and cholangiocellular carcinoma. The H&E-stained tissue sections were reviewed for the presence of adequate hepatic tissue and absence of necrosis, marked inflammation, and severe congestion. Paraffin-embedded tissue blocks subsequently were obtained from archival storage for further study. Seventeen hepatic tissue specimens were selected for study. The initial histologic diagnoses were normal liver (n = 2), bile duct hyperplasia (n = 3), and cholangiocellular carcinoma (n = 12). The study population included: 7 chickens (Gallus gallus domesticus), 3 Amazon Parrots (1 Yellow-headed, Amazona ochracephala; 2 unspecified, Amazona sp. ), 3 Conures (1 Mitered, Aratinga mitrata; 1 Maroon-bellied, Pyrrhura frontalis; 1 Blue-crowned, Aratinga acuticaudata), 1 Green-winged Macaw (Ara chloroptera), 1 Eclectus Parrot (Eclectus roratus), 1 Caique (Pionites sp. ), and 1 Bali Mynah (Leucopsar rothchildi).
Tissue preparation and processing: Formalin fixed, paraffin- embedded tissue blocks were sectioned at 3 µm. Replicate tissue sections were placed on ProbeOna slides and deparaffinized in two washes of Hemo-Dea xylene substitute (5 minutes each), followed by one wash with xylene (5 minutes). The slides were then rehydrated in a series of graded ethanols to distilled water.
Immunoperoxidase staining: After a brief rinse in distilled water, the tissue sections were placed in 3.0% hydrogen peroxide for 15 minutes to quench endogenous peroxidase activity. For antigen retrieval evaluation, slides were placed in 10 mM citrate buffer (pH = 6.0) and subjected to microwave treatment, steaming, or autoclaving. Additional tissue sections also were processed without attempted antigen retrieval. The tissue sections subsequently were loaded into a slide holder. A MicroProbea work station was used to facilitate immunohistochemistry and minimize reagent consumption.
The slides were incubated for 10 minutes at 30°C in normal horse serum diluted in phosphate buffered saline (PBS) and triton bufferb to block nonspecific antibody staining. Subsequently, the primary antibody was applied at a predetermined dilution (PCNA, 1:50; polyclonal Ki-67, 1:100; monoclonal Ki-67, 1:30; and p53, 1:50). c,d The slides were incubated with the primary antibodies for 2 hours at 30°C. After rinsing the slides with Automation buffer,d the secondary antibody (biotinylated anti-mouse IgG)e was applied and the slides were incubated for 1 hour at 30°C. Following rinsing, the avidin-biotin complex (ABC) was applied and the tissue sections were incubated for an additional hour at 30°C. The slides were rinsed again, diaminobenzidine (DAB, 1 mg/ml)b in 0.024% hydrogen peroxide was applied, and the slides were allowed to sit for 5 minutes or until brown chromagen deposition was observed. Finally, the tissue sections were rinsed in deionized water, counterstained with hematoxylin,g coverslipped, and examined microscopically.
Microscopic evaluation of immunoreactivity: Immunoreactivity was graded on a scale of 0 to 4+. A score of 0 was given if chromagen deposition was not observed. Cytoplasmic staining was scored as 1+. Hepatocellular nuclear staining was scored as 2+. Nuclear staining in biliary epithelium was scored as 3+ or 4+, depending upon the number of cells stained. A score of 3+ indicated that <50% of biliary epithelial cells had nuclear staining. The tissue specimen was scored as 4+ if >50% of bile duct epithelial cell nuclei were stained. Background staining, if present, also was recorded.
Results
Of the four antibodies evaluated, only the monoclonal anti-PCNA antibody provided reliable immunoreactivity in avian liver sections (Table 1).
Both the anti-Ki-67 polyclonal and monoclonal antibodies resulted in extensive, generalized background staining that could not be eliminated by blocking or antibody dilution (including staining overnight at 4°C). Immunoreactivity usually was not observed with anti-p53 monoclonal antibody; however, a few tissue sections had faint chromagen deposition within hepatocellular and biliary epithelial cell nuclei.
Using the anti-PCNA antibody, visible chromagen deposition was not observed in normal liver. In liver sections with a diagnosis of bile duct hyperplasia, multifocal nuclear staining was observed in <50% of the biliary epithelial cell nuclei (Figs. 1 & 2).
 |
 |
| Fig. 1. Avian liver, bile duct hyperplasia, H&E stain. Cords of well differentiated biliary epithelial cells are present (upper left). Hepatocytes (lower left) have a normal appearance. |
Fig. 2. Avian liver, bile duct hyperplasia, anti-PCNA antibody with hematoxylin counterstain. Scattered biliary epithelial cell nuclei (upper left) stain brown indicating PCNA expression. |
Chromagen deposition was more intense and was observed in hepatocyte and biliary epithelial cell nuclei in tissue sections of cholangiocarcinoma (Figs. 3 & 4).
 |
 |
| Fig. 3. Avian liver, cholangiocellular carcinoma, H&E stain. Proliferating biliary epithelial cells form irregular tubules. Moderate desmoplasia also is present. |
Fig. 4. Avian liver, cholangiocellular carcinoma, anti- PCNA antibody with hematoxylin counterstain. Biliary epithelial cell nuclei stain dark brown indicating marked PCNA expression. |
However, 5 of 12 cholangiocellular carcinomas had staining patterns similar to those with biliary hyperplasia.
Attempted antigen retrieval by steaming, microwaving, and autoclaving avian tissue sections was unsuccessful. Background staining was increased with anti-Ki-67 antibodies, while any slight immunoreactivity to p53 was ablated. Changing primary antibody dilutions and pre-blocking tissue sections for a longer period of time did not alleviate the excessive background staining with the anti-Ki-67 antibodies. Immunoreactivity with anti-PCNA antibody was similar with or without antigen retrieval techniques. Therefore, immunohistochemical detection of PCNA subsequently was performed without antigen retrieval techniques.
Discussion
The results of this study indicate that mammalian anti-PCNA antibodies are cross-reactive in avian tissues. However, the anti-Ki-67 and anti-p53 antibodies cannot reliably detect their associated proliferation markers in avian hepatic tissues.
Proliferating cell nuclear antigen (PCNA) is expressed in cells that are replicating or engaged in DNA repair. 10,11 Therefore, this marker is not a specific indicator of proliferation. PCNA expression in avian biliary epithelium was observed in bile duct hyperplasia and cholangiocellular carcinoma, but was not observed in normal biliary epithelium. In addition, PCNA expression sometimes was present in hepatocyte nuclei surrounding foci of cholangiocellular carcinoma, suggesting a paracrine effect of neoplastic biliary epithelial cells on adjacent normal hepatocytes. In human tissues, minimal PCNA reactivity is observed in normal hepatocytes. While PCNA expression may be increased in lymphoid tissues, it may be downregulated in some carcinomas. Interestingly, normal cells adjacent to carcinoma cells may dramatically increase PCNA expression. 11 A similar scenario also exists in some avian cholangiocarcinomas.
In the avian liver sections examined, PCNA immunoreactivity was used to correctly identify 7 of 12 cholangiocarcinomas. However, the 5 remaining cholangiocarcinomas had a similar staining pattern to that of bile duct hyperplasia. In contrast, PCNA immunoreactivity was not detected in normal liver, indicating low expression of this proliferation marker in health as has been described in human liver tissue. 11
The anti-Ki-67 antibodies failed to stain avian tissues specifically. The excessive background staining prevents the use of these antibodies in detecting Ki-67 expression in avian tissues. Although the anti-P53 monoclonal antibody produced slight immunoreactivity in a few avian tissue sections, the staining was faint and difficult to interpret with certainty. This antibody also appears to be a poor choice for study of p53 production in paraffin-embedded avian tissues.
In summary, PCNA expression can be detected in avian liver sections with bile duct hyperplasia and cholangiocellular carcinoma using commercially available mammalian reagents. Immunostaining for PCNA expression cannot always provide a definitive diagnosis of biliary hyperplasia or cholangiocellular carcinoma, compared to examination of H&E-stained tissue sections. However, increased suspicion of cholangiocellular carcinoma exists when excessive staining of biliary epithelial cells or combined staining of biliary and hepatocellular nuclei is observed.
Sources and Manufacturers
a. ProbeOn Plus slides, Hemo-De, and MicroProbe Work Station, Fisher Scientific, Pittsburgh, PA (USA).
b. Triton X-100 and diaminobenzidine (DAB), Sigma Chemical Co. , St. Louis, MO (USA).
c. Monoclonal mouse anti-human Ki-67 (MIB1) antibody, Biogenex, San Ramon, CA, (USA).
d. Polyclonal rabbit anti-human Ki-67 antibody, monoclonal mouse anti-proliferating cell nuclear antigen (PCNA) antibody, and monoclonal mouse anti-human p53 protein antibody, DAKO Corp. , Carpinteria, CA (USA).
e. Automation buffer, Biomeda Corp. , Foster City, CA (USA).
f. Biotinylated anti-mouse IgG and ABC reagent, Vectastain Elite, Burlingame, CA (USA).
g. Hematoxylin, Surgipath, Richmond, IL (USA).
Acknowledgments
This study was supported by the Department of Pathology and the Cowan Avian Health Foundation. Ms. Sullivan completed this research project as a junior veterinary student during a summer clerkship.
References
1. Riddell C: Avian Histopathology, 1st ed. Am Assoc Avian Pathol, Allen Press Inc. , Lawrence, KS, 1987, pp. 57-65.
2. Latimer KS: Oncology. In: Ritchie BW, Harrison GJ, and Harrison LR (eds): Avian Medicine: Principles and Application. Wingers Publishing Inc. , Lake Worth, FL, 1994, pp. 657-658.
3. Anderson WI, Dougherty EP, Steinberg H: Cholangiocarcinoma in a four-month-old double yellow-cheeked Amazon parrot (Amazona autumnalis). Avian Dis 33:827-828, 1989.
4. Elangbam CS, Panciera RJ: Cholangiocarcinoma in a blue-fronted Amazon parrot (Amazona aestiva). Avian Dis 32:594-596, 1988.
5. Potter K, Connor T, Gallina AM: Cholangiocarcinoma in a yellow-faced Amazon parrot (Amazona xanthops). Avian Dis 27:556-558, 1983.
6. Reece RL: Observations on naturally occurring neoplasms in birds in the state of Victoria, Australia. Avian Pathol 21:3-32, 1992.
7. Coleman CW: Bile duct carcinoma and cloacal prolapse in an orange-winged Amazon parrot (Amazona amazonica amazonica). J Assoc Avian Vet5:87-89, 1991.
8. Hillyer EV, Moroff S, Hoefer H, Quesenberry KE: Bile duct carcinomas in two out of ten Amazon parrots with cloacal papillomas. J Assoc Avian Vet 5:91-95, 1991.
9. Latimer KS, Niagro FD, Rakich PM, Campagnoli RP, Ritchie BW, McGee ED: Investigation of parrot papillomavirus in cloacal and oral papillomas of psittacine birds. Vet Clin Pathol 25:158-163, 1997.
10. Shivji MKK, Kenny MK, Wood RD: Proliferating cell nuclear antigen is required for DNA excision repair. Cell 69:367-374, 1992.
11. Hall PA, Levison DA, Woods AL, Yu CC-W, Kellock DB, Watkins JA, Barnes DM, Gillett CE, Camplejohn R, Dover R, Waseem NH, Lane DP: Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: An index of cell proliferation with evidence of deregulated expression in some neoplasms. J Pathol 162:285-294, 1990.
12. McCormick D, Chong H, Hobbs C, Datta C, Hall PA: Detection of the Ki-67 antigen in fixed and wax-embedded sections with the monoclonal antibody MIB1. Pathol 22:355-360, 1993.
13. Pinder SE, Wencyk P, Sibbering DM, Bell JA, Elston CW, Nicholson R, Robertson JFR, Blamey RW, Ellis IO: Assessment of the new proliferation marker MIB1 in breast carcinoma using image analysis: Associations with other prognostic factors and survival. Br J Cancer 71:146-149, 1995.
14. Smith ML, Fornace AJ: The two faces of tumor suppressor p53. Am J Pathol 148:1019-1022, 1996.
15. Smith SJ, Luo J-C, Brandt-Rauf P, Marion M-J: Mutant p53 protein as a biomarker of chemical carcinogenesis in humans. J Occupat Environ Med 38:743, 1996.
16. Hollstein M, Soussi T, Thomas G, von Brevern M-C, Bartsch H: p53 gene alterations in human tumors: Perspectives for cancer control. Recent Results Cancer Res 143:369-389, 1997.
This Page Last Updated May 15, 1998 |
