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 DOI: 10.1177/104063870902100419

2009 21: 535J VET Diagn InvestNuria García, Guillermo Ramis, Francisco J. Pallarés, Juan I. Seva, Carlos M. Martínez, Juan J. Quereda and Antonio Muñoz

(Papio anubis)a Juvenile Olive Baboon Clinical, Histologic, and Immunohistochemical Features of an Undifferentiated Renal Tubular Carcinoma in

  

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J Vet Diagn Invest 21:535–539 (2009)

Clinical, histologic, and immunohistochemical features of an undifferentiated renal tubularcarcinoma in a juvenile olive baboon (Papio anubis)

Nuria Garcıa, Guillermo Ramis,1 Francisco J. Pallares, Juan I. Seva, Carlos M. Martınez,Juan J. Quereda, Antonio Munoz

Abstract. An undifferentiated renal tubular carcinoma was diagnosed in a juvenile male olive baboon (Papioanubis). The animal suddenly appeared depressed and refused to eat. During physical examination, a firm,palpable mass in the left abdominal area and flank pain were detected. Clinical pathology findings included mildanemia, hypoalbuminemia, hyponatremia, and mildly increased serum creatinine and urea concentrations.Radiographs revealed a large mass in the left abdominal area. Exploratory laparotomy disclosed a 10 cm 3 15 cmmultilobulated mass involving the left kidney and adjacent organs. Because of a poor prognosis, the animal washumanely euthanized, and necropsy was performed. Tissue samples of the neoplasm were taken forhistopathological examination. Immunohistochemical staining was done using vimentin, cytokeratin, S-100protein, Ki-67, a-actin, and desmin-specific primary antibodies. Microscopically, elongated and irregular tubuleswere lined by 2 or more layers of atypical epithelial cells. Anisocytosis, anisokaryosis, and frequent mitotic figureswere also observed. Following immunohistochemical staining, the cytoplasm of neoplastic cells was positive forcytokeratin, vimentin, and S-100 protein and negative for a-actin and desmin. Positive nuclear staining for Ki-67was observed. The neoplasm was diagnosed as an undifferentiated renal tubular carcinoma.

Key words: Baboons; immunohistochemistry; juvenile; primate; renal carcinoma.

<!?show "fnote_aff1"$^!"content-markup(./author-grp[1]/aff|./author-grp[1]/dept-list)>The baboon has become an increasingly important

animal model for biomedical research, especially in areassuch as xenotransplantation. In humans, renal cell carci-noma is the third most common urologic malignancy andthe seventh most common neoplasm overall.17 Spontane-ous benign and malignant neoplasms have been reported innonhuman primates, but the overall frequency of neo-plasms in captive primates has been low.1,2,9,14,15,18 Theurogenital tract is the third most affected system, but only 9cases of spontaneous renal neoplasia have been reported inthe baboon.3 A positive correlation between advancing age

and incidence of cancer has been well documented inseveral nonhuman primate species including baboons.3,19

The current study describes an undifferentiated renaltubular carcinoma that originated spontaneously in ajuvenile olive baboon (Papio anubis). This species reachespuberty at 6 years of age.13 The clinical, hematological, andpathological findings in this juvenile baboon are described.

An abdominal mass was detected in a 4-year-old maleolive baboon (weight 9 kg). The animal was part of acolony of 40 breeding baboons maintained in the Prima-tology Unit of the University of Murcia (Murcia, Spain).The colony provided animals for a xenotransplantationresearch program, and the baboons were not subjected toany experimental procedure up to the moment of tissuetransplantation. The colony was founded with animalsfrom Kenya and South Africa, but the animal in the currentreport was born at the University of Murcia facilities. Thecolony was housed in 2 parks containing 20 animals ineach. Husbandry and maintenance were provided in

From the Departments of Animal Production (Garcıa, Ramis,Quereda, Munoz) and Comparative and Pathological Anatomy(Pallares, Seva, Martınez), University of Murcia, Murcia, Spain.

1 Corresponding Author: Guillermo Ramis, Departamento deProduccion Animal, Facultad de Veterinaria, Universidad deMurcia, Campus de Espinardo, 30.071 Murcia, Spain. guiramis@um.es

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Figure 1. Multilobulated, white to yellow mass (*) effacingapproximately 80% of the left kidney and involving the adjacentintestine and spleen.

Figure 2. Irregular tubules lined by neoplastic cells thatexhibit anisocytosis and anisokaryosis and contain prominenteuchromatic nuclei, eccentric nucleoli, and finely granulareosinophilic cytoplasm. Occasional clear cells also are present.Numerous apoptotic cells are scattered within the neoplastic cellpopulation. Hematoxylin and eosin. Bar 5 45 mm.

Figure 3. Detail of neoplastic cells. A, hematoxylin and eosin stain. B, positive cytoplasmic staining for cytokeratin within theneoplastic cells. C, positive cytoplasmic staining for S-100 protein within the neoplastic cells. D, positive nuclear staining for Ki-67protein within the neoplastic cells. Bar 5 30 mm (applies to all figure panels).

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accordance with the recommendations listed in the Guide forthe Care and Use of Laboratory Animals.20 The baboonswere fed with simian commercial feed and supplemented withfresh fruit, dry seeds, and vegetables, which varied dependingon the growing season. Routine serological monitoring wasnegative for Hepatitis A, B, and C virus; Simian alphaher-pesvirus 8; Primate T-lymphotropic virus 1 isolate Simian T-lymphotropic virus 1; and Simian immunodeficiency virus.Testing for the latter virus was done by enzyme-linkedimmunosorbent assay. The health protocol included semian-nual treatment of external and internal parasites andtuberculin testing as well as a yearly complete blood cellcount and biochemical profile for each animal.

The juvenile baboon of the current report was homereared because of early maternal rejection and was added tothe colony when he was 1 year old. The animal’s medicalhistory was devoid of any clinical, biochemical, orhematological abnormalities. The clinician first noted thatthe baboon was depressed and refused to eat. A physicalexamination revealed a firm palpable mass in the leftabdominal area and flank pain. Biochemical and hemato-logical evaluations were relatively nonspecific and includedmild anemia (3.4 3 1,012 RBC/l, reference [ref.] interval:4.8–6.3 3 1,012 RBC/l),11 mild hypoalbuminemia (2.7 g/dl,ref. interval: 2.9–4.2 g/dl),11 and mild hyponatremia(135 mg/dl, ref. interval: 143–158 mg/dl).11 Serum ureanitrogen (57 mg/dl, ref. interval: 9–25 mg/dl) and creatinine(1.6 mg/dl, ref, interval: 0.8–1.4 mg/dl) concentrations weremildly increased.11 The low reticulocyte percentage (0.5%)indicated nonregenerative anemia. A urine specimen wasnot available for laboratory analysis. The laboratoryfindings were suggestive of renal disease, but this isgenerally rare in most juvenile animals. Prerenal azotemiawas also a considered because the baboon had inappetenceand was not drinking water. Survey abdominal radiographswere suggestive of a large mass in the left abdominal cavity.Exploratory laparotomy, performed through a midlineincision under isoflurane anesthesia, disclosed a large renal

mass involving the inferior vena cava and the adjacentintestine and spleen. Firm adhesions were present betweencapsular surface of the kidney and adjacent organs (Fig. 1).Because of a poor clinical prognosis, the animal waseuthanized, and a complete necropsy was performed.

At necropsy, a 12 cm 3 15 cm, white to yellow, soft,multilobular mass was observed in the abdominal cavity. Themass appeared heterogeneous on the cut surface and effacedapproximately 80% of the parenchyma of the left kidney.Tissue samples of the mass were taken for histopathologicalexamination. Macroscopic lesions were not observed in otherorgans; however, samples of the lungs, spleen, heart, liver,and brain were also taken for histopathology to exclude thepresence of micrometastasis. The tissue samples were fixed in10% neutral buffered formalin, processed routinely, embed-ded in paraffin, sectioned, stained with hematoxylin andeosin, coverslipped, and examined microscopically. The massinvolving the left kidney consisted of elongated, irregulartubules that were lined with 2 layers of atypical epithelialcells. Individual cells had indistinct cellular borders and weretightly compressed, with nuclear displacement. Tubularepithelial cells also exhibited anisocytosis and anisokaryosis.Individual neoplastic cells had a prominent, euchromaticnucleus with an eccentrically located nucleolus. The cyto-plasm was eosinophilic and finely granular. A few clear cellsalso were observed (Fig. 2). Forty-five mitoses were in ten4003 fields of view and some mitoses appeared atypical.Apoptotic cells and moderate desmoplasia also wereobserved. Scattered mononuclear infiltrates were present inthe interstitium. The differential diagnosis for this renal massincluded renal tubular carcinoma, nephroblastoma, undif-ferentiated sarcoma, and oncocytoma. Based on the histo-logic features of this neoplasm, a tentative diagnosis ofpoorly differentiated renal tubular carcinoma was madebased. Microscopic examination of the lungs, spleen, heart,liver, and brain failed to reveal micrometastases.

Immunohistochemistry was performed to establish adefinitive diagnosis. Replicate 4-mm tissue sections were

Table 1. Characteristics of primary antibodies used for immunohistochemical staining in the current study.

Antigen Clone Dilution Pretreatment

Vimentin V9 1:100 Heat (121uC in citrate buffer, pH 6)Cytokeratin AE1/AE3 1:25 0.1% pronaseCytokeratin MNF-116 1:50 0.1% pronaseS-100 protein Polyclonal 1:400 NoneKi-67 MIB-1 1:200 Heat (121uC in citrate buffer, pH 6)Desmin D33 1:100 Heat (121uC in citrate buffer, pH 6)a-actin 1A4 1:100 Heat (121uC in citrate buffer, pH 6)

Table 2. Anticipated immunohistochemical staining pattern of neoplastic cells in 4 renal neoplasms.*

Tumor/marker Vimentin Cytokeratin a-actin Desmin S-100 Reference no(s).

Undifferentiated renal cell carcinoma + (diffuse) + 2 2 + 19Nephroblastoma + + 2 + 2 6, 8Undifferentiated sarcoma + 2 2 2 2 19Oncocytoma + (only focal areas) + 2 2 +/2 12, 19

* + 5 positive; 2 5 negative.

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stained using the avidin–biotin–peroxidase complex (ABC)technique and several primary antibodies.a The primaryantibody clone, primary antibody dilution, and pretreat-ment regimen for antigen retrieval are presented in Table 1.The anticipated immunohistochemical staining pattern forvarious renal neoplasms considered in the differentialdiagnosis is presented in Table 2. Although mitotic activityhas been used as a prognostic factor for many neoplasms,the mitotic index of renal carcinomas is usually too low toprovide meaningful information about tumor staging andgrading in individual neoplasms.5 Therefore, nuclear Ki-67expression was evaluated to determine the degree of cellularproliferation.5 Evaluation of Ki-67 expression also has beensuggested as a useful marker to differentiate renal tubularcarcinoma from oncocytoma.21 After dewaxing, rehydration,and pretreatment for antigen retrieval, the tissue sectionswere incubated with 0.5% hydrogen peroxide in methanol for30 min, washed in 0.05 M Tris-buffered saline solution(TBS), and blocked for 30 min with normal serumb diluted1:100 in TBS to minimize nonspecific binding of the primaryantibodies. The slides were then incubated with the primaryantibody in a moist chamber for 60 min. After incubation,the slides were washed in TBS, incubated with thebiotinylated antibodyb for 30 min, washed once more inTBS, and incubated with the ABCb reagent for 30 min. Sitesof primary antibody binding were visualized using achromogen solution of 3-39-diaminobenzidine tetrahy-drochloride (0.05%)c and hydrogen peroxide (0.03%) inTBS with a 5-min incubation period. The reaction stoppedafter rinsing the slides in tap water. The slides werecounterstained in Mayer hematoxylin for 3 min, rinsed intap water, dehydrated, and coverslipped. The negative tissuecontrol was normal kidney from an olive baboon. Sections ofa human renal cell carcinoma were used as positive controls.

Following immunohistochemical staining, the neoplasticcells were positive for vimentin, cytokeratin, and S-100protein. In addition, .70% of the neoplastic cells expressedKi-67 protein (Fig. 3). The neoplastic cells were negativefor desmin and a-actin. A definitive diagnosis of undiffer-entiated renal tubular carcinoma was based on thehistologic and immunohistochemical findings.

Spontaneous neoplasia has been documented across allsubspecies and most hybrid combinations of baboon.3,4,15

Neoplasms involving the urogenital tract account for 21% ofall primate tumors, but only 9 spontaneous neoplasms havebeen reported in baboons.3 Renal adenoma, renal carcinoma,and nephroblastoma have been reported previously inbaboons,3,4 but the low incidence of spontaneous neoplasmsis probably because most nonhuman primates are euthanizedat a young age in most research settings.

Previous investigations have reported a positive correla-tion between age and incidence of spontaneous neoplasia inbaboons.3,4,19 The neoplasm in the baboon of this report isunusual in that it occurred in a juvenile animal. To date,only nephroblastoma has been described in young ba-boons.4 Renal adenoma and renal carcinoma have beenreported in baboons only older than 15 years.3,4

In humans, renal cell carcinoma may remain clinicallyoccult for most of its clinical course. The classic triad offlank pain, hematuria, and presence of a flank mass is

uncommon (10%) and usually indicative of advanceddisease. Twenty-five percent to 30% of human patientsare asymptomatic, and the renal cell carcinomas aredetected incidentally during radiographic studies.

In the baboon of the current report, the neoplastic renalcells expressed cytokeratin and vimentin. If the neoplasm ispositive for cytokeratin and vimentin, a diagnosis of renalcarcinoma can be made. In contrast, sarcomas are positivefor vimentin and negative for cytokeratin. Diagnosis ofnephroblastoma is based on the histologic observation ofembryonic glomeruli and blastemal proliferation, oftenwith muscular and osseous metaplasia.10 However, thesehistologic features were not found in the neoplasm of thepresent report. Desmoplasia is a common feature innephroblastomas and undifferentiated carcinomas19 butmay also be observed in renal carcinomas. Immunohisto-chemically, human nephroblastomas are reportedly posi-tive for vimentin,7 cytokeratin,6 and desmin8; however, theneoplasm in this baboon was negative for desmin and othermuscle markers such as a-actin. Therefore, a diagnosis ofnephroblastoma was excluded. Although the S-100 stainingis a useful marker necessary to differentiate some types ofrenal carcinoma from oncocytoma,16 vimentin stain wasuseful for this distinction in the neoplasm reported in thisbaboon. Expression vimentin in oncocytomas has beencontroversial. Recent studies of human oncocytomasdocument focal expression of vimentin in some neoplasticcells,12 but more uniform, diffuse, cytoplasmic expressionof vimentin was observed in the neoplastic cells of thisbaboon. Expression of Ki-67 is much lower in oncocytomasthan in renal cell carcinoma.21 Based on the histologicfeatures and immunohistochemical staining of the neo-plasm in the baboon of the current report, a diagnosis ofoncocytoma also was excluded.

More than 70% of the neoplastic cells in the renal tubularcarcinoma from this juvenile baboon had intense nuclearexpression of Ki-67, indicating marked cellular prolifera-tion. Although this degree of Ki-67 expression suggestsmoderate malignant potential, metastases were not detectedin other tissues and organs that were examined. Only 30%of human renal carcinomas metastasize to other organs.17

Thus, the definitive diagnosis was undifferentiated renaltubular carcinoma.

Sources and manufacturers

a. Dako North America Inc., Carpinteria, CA.

b. Vector Laboratories Inc., Burlingame, CA.

c. Sigma-Aldrich, St. Louis, MO.

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