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Brief Communication |
Correspondence: 1Corresponding Author: Dr Newman, Department of Pathology, College of Veterinary Medicine, University of Tennessee, Knoxville, TN, 37996-4542
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Abstract |
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Key Words: Carcinoma • cyclooxygenase • feline • neoplasia • pancreas
Cyclooxygenase-2 (COX-II) is an inducible enzyme that is responsible for the production of prostaglandin E2 (PGE2) from arachidonic acid. COX-II expression is often upregulated in neoplastic conditions in humans, where it plays a role in tumorigenesis and also serves as a target for therapeutic intervention. Of veterinary species, dogs are the most common species in which this marker has been evaluated.3,4,7–10,14,15,17,18,20 Canine neoplasms with overexpression include squamous-cell carcinoma,17 renal-cell carcinoma,9 transitional-cell carcinoma,8 prostatic adenocarcinoma,20 nasal carcinoma,3,10 intestinal neoplasia,14 and mammary carcinoma.4,7,18 Apparently, expression occurs more consistently in canine epithelial neoplasms than in sarcomas.1,6,16 Few reports, however, detail the expression of COX-II in feline epithelial neoplasms.1 Nevertheless, in 1 large study of 58 feline carcinomas, 7/19 (37%) of feline transitional-cell carcinomas and 2/21 (9%) of feline oral squamous-cell carcinomas were found to express COX-II.1 In contrast, all examined feline cutaneous squamous-cell carcinomas (6), mammary adenocarcinomas (9), pulmonary adenocarcinomas (8), intestinal adenocarcinomas (7), nasal lymphosarcomas (6), intestinal lymphosarcomas (6), and vaccine-associated sarcomas (10) failed to express COX-II.1 These results discourage the potential for therapeutic success with COX-II inhibitors in this species.1
Upregulation of COX-II is documented in many human epithelial neoplasms, including pancreatic adenocarcinomas.5,11,12,21,22 However, it appears that no specific studies that determine COX-II expression in pancreatic adenocarcinomas in animals have been published. Therefore, the purpose of this study was to assess a series of feline pancreatic adenocarcinomas for the expression of COX-II.
Eight feline pancreatic adenocarcinomas were submitted to the Gastrointestinal Laboratory at Texas A&M University after an e-mail call for such cases to the Comparative Gastroenterology Society listserv, the American College of Veterinary Internal Medicine listserv, and the Veterinary Cancer Society listserv. Five poorly differentiated ductular variants and 3 well-differentiated acinar variants were reviewed and included in the study. The cases were submitted either as unstained slides or as paraffin-embedded tissues that were subsequently sectioned at 3 µm and placed on positively charged slides. Immunohistochemistry (IHC) was performed at the University of Tennessee College of Veterinary Medicine IHC laboratory. All slides were deparaffinized and rehydrated through alcohols to water. Antigen retrieval was performed for 20 minutes at 95°C in a pH 6.0 citrate buffer.a All slides were soaked in Tris-buffered saline solution (pH 7.6) for 5 minutes before being loaded into the computer-controlled automated stainer.b A 3% hydrogen peroxide block was applied for 5 minutes, and this was followed by a 5-minute nonserum protein block.c The polyclonal anti-COX-II antibodyd at a 1 : 1000 dilution was applied and incubated for 30 minutes. A horse radish peroxidase labeled polymer systeme was applied to all slides, which were then incubated for 30 minutes. Slides were then rinsed in Tris-buffered saline solution, and 3,3-diaminobenzidine chromagenf was applied for 10 minutes. After rinsing, all slides were counterstained with hematoxylin,g were dehydrated, and then were coverslipped. Appropriate positive control tissue included feline kidney-macula densa (Fig. 1), feline pancreas obtained at necropsy from a young adult cat without concurrent disease, and feline pancreas as part of the master control block. Negative control slides included each neoplasm, feline pancreatic tissue and the master control block, and each was processed without primary antibody and with substitution of Universal Negative Control + rabbit serum.h
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As in humans, most feline adenocarcinomas of the pancreas are ductular in origin.13 K-ras point mutations in codon 12 are identified in pancreatic adenocarcinomas in both cats and humans.13 Specifically, of 3 examined feline pancreatic adenocarcinomas in 1 study, 2 had K-ras point mutations.13 Undeniably, obtaining large numbers of cases for immunohistochemical assessment is difficult because of the overall rarity of this neoplasm in cats. Therefore, this study relied on referral submissions that were accumulated over a 2.5-year period. As a result, clinical data were not available for these cases. Furthermore, the disease is typically noted late in its course, and a diagnosis is often made at necropsy.19 Metastatic disease is usually noted at the time of diagnosis, and, in 1 study, the survival time to death or euthanasia was 7 days.19
This study appears to be the first attempt to determine COX-II expression in a series of feline pancreatic adenocarcinomas. Typically, COX-II is expressed strongly within pancreatic adenocarcinomas in humans, and several studies have determined expression to range from a low of 41.7% of cases11 to 55% of cases22 to a high of 56%–90% of cases.5,12 The degree of expression in human cases increases during the progression from normal pancreatic ducts to preinvasive ductular lesions to pancreatic adenocarcinomas.12 Upregulation in benign proliferations and preneoplastic cells is seen, but normal pancreatic tissues surrounding neoplastic foci do not usually express COX-II.5,21 Interpretation of the lack of expression in normal tissues is problematic. The lack of expression may reflect the absence of a stimulus to induce the enzyme or possibly the contribution of a negative feedback mechanism related to regionally high expression of the enzyme. In one of the cases in this study, nodular regions of well-differentiated adenocarcinoma were identified, and, in these areas, there was minimally intense cytoplasmic expression in many cells. However, there was more intense expression within fewer cells in the more poorly differentiated regions of the same malignancy.
Expression of COX-II may be greater in anaplastic neoplasms (as was borne out by detectable staining in our 2 poorly differentiated ductular adenocarcinomas) and thus might be a marker to help determine prognosis of neoplastic histologic subtypes, but studies need to be undertaken to determine disease-free intervals and survival times for various neoplasms in veterinary species.7 However, no apparent correlation exists between COX-II expression and survival in most canine10 or human studies.11,12 Upregulation of COX-II is an early stage in tumorigenesis, and, hence, expression is seen in precancerous lesions with increased expression occurring in malignant forms and metastatic lesions.10
Positive control tissue for COX-II expression has been noted to include the macula densa and thick ascending limb in human and canine kidneys.9 Strong expression is seen in fetal tissue, and this gradually declines as the kidney matures.9 In this study, the feline renal sections exhibited COX-II expression in the macula densa, and this tissue served as a positive control tissue for these cases. Normal COX-II tissue expression is seen only in placenta, brain, and macula densa of the kidney.16 Thus, the lack of expression in our feline pancreatic tissue controls is consistent with previous studies.
The exact role of COX-II in pancreatic neoplasia is only now being determined, but it is thought to be involved in the pathogenesis of the neoplasm.5 Cyclooxygenase-2 is inducible by oncogenes, proinflammatory cytokines, and growth factors.2,10 Upregulation of COX-II results in enhanced production of prostaglandins from arachidonic acid, specifically PGE2, that can, in turn, increase cell proliferation,1,5,8,10,15,17 promote angiogenesis,1,2,6,9,11,16,22 increase metastatic potential,22 alter cell adhesion,1 enhance extracellular matrix degradation,18 activate xenobiotics,1 inhibit apoptosis,1,10,15,20,21 and inhibit immune surveillance.1,5,8,15
Although there is a 93% amino-acid homology between dog and human COX-II,15 this value is not determined in cats. A polyclonal antibody was used in the Immunohistochemistry (IMHC) procedure, but it is possible that it was unable to detect the small level of expression in this feline neoplasm.1 The length of time of fixation and paraffin block storage was also not known for these samples. However, time stored was not thought to influence COX-II staining intensity in a series of canine nasal tumors.10 As in previous canine neoplasms, heterogeneous expression was often more appreciable near regions of necrosis.16 It has been postulated that this may be because COX-II induction is higher in areas of inflammation or that under hypoxic conditions, it is produced to protect cells from apoptosis.16 In addition, the cytoplasmic and perinuclear localization of cellular expression was not unexpected, because it has been previously noted.3,10 While Western blotting can better quantify COX-II expression levels, this procedure is not possible when archived tissues are examined, as was the case in this study.
In humans and dogs, specific COX-II inhibitors are being used as therapeutics, with variable effects. The rarity and the inconsistency of COX-II expression in feline neoplasms suggest that these drugs may have a lower potential as successful anticancer agents in this species.1 Feline pancreatic adenocarcinomas do not appear to consistently express COX-II, and thus COX-II expression may not be a valid prognostic factor for this group of feline neoplasms.
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a. Target Retrieval System, Dako, Carpinteria, CA.
b. Automated Stainer Model S3400, Dako, Carpinteria, CA.
c. Nonserum protein block, Dako, Carpinteria, CA.
d. Polyclonal Rabbit Anti-Cyclo-oxygenase II, Cayman Chemical, Ann Arbor, MI.
e. EnVision+ Anti-rabbit, Dako, Carpinteria, CA.
f. DAB, Dako, Carpinteria, CA.
g. Hematoxylin 2, Richard Allan Scientific, Kalamazoo, MI.
h. Universal negative control rabbit, Dako, Carpinteria, CA.
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  A. R. Tucker and J. R. Smith Prostatic Squamous Metaplasia in a Cat with Interstitial Cell Neoplasia in a Retained Testis Veterinary Pathology, November 1, 2008; 45(6): 905 - 909. [Abstract] [Full Text] [PDF]
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