J.Vet. Med, A 44, 399-406 (1997)© 1997 Blackwell Wissenschafts-Verlag, BerlinISSN 0931-184X

Dpto. Patologla Animalu Famltad de Veterinaria, CleM Madrid, Spain

Immunohistochemical Study of a Non-invasive CanineThymoma: A Case Report

M. GONzALEZ, A. RODRiGUEZ, M. PIZARRO and P. LLORENS

Address of authors: Dpto. Patologia Animal II Facultad de Veterinaria, UCM Madrid, Spain

ty-1th 5 figures

(Receivedforpublication November 14, 1996)

SummaryWe describe a case of canine thymoma in an l1-year-old pure-bred female Spanish Mastiff. On post­

mortem examination a big mass was found in the thoracic cavity. Histologically the neoplastic parenchymashowed great structural variability; the principal findings included the presence of tumour cells formingcords or sheets, cystic formations, Hassall-corpuscle like squamous formations, and small or intermediatecalibre vessels encircled by areas with eosinophilic material and lymphocytes. In order to determine thehistogenesis of the thymic neoplasia we used immunohistochemical techniques specific for differentmarkers: cytokeratins, vimentin, desmin and neuron specific enolase. A strong positive immunoreactionof tumour cells to wide spectrum cytokerarins confirmed the epithelial origin of the neoplasia.

IntroductionThymomas are defined as primary neoplasms of thymic epithelial cells with varying

proportions of non-neoplastic lymphocytes. The incidence of this tumour is low in domesticanimals (MOULToN, 1990;)UBB et al., 1993; BALAGUER et al., 1995), and particularly in dogs(KLEBANOW, 1992;)OOB et al., 1993; ATWATER et al., 1994; SORDE et al., 1994). Accordingto HADLOW (1978) and MOULTON (1990), however, goats display a high incidence ofthymoma.

As a result of extensive studies of thymomas undertaken in human pathology, mostauthors now associate this tumour with the development of paraneoplastic syndromes. Clinicalpathological conditions similar to human paraneoplastic syndromes including myasthenia gravis,cytopenias polymyositis, systemic lupus erythematous, rheumatoid arthritis, thyroiditis, non­thymic cancer, megaoesophagus and atrio-ventricular block, among others were described(ARONSOHN et al., 1984; HARRIS et al., 1991; KLEBAN OW, 1992; ATWATER et al., 1994HACKETT et al., 1995). However, in numerous cases affected animals have not shown anyclinical signs (BELLAH et al., 1983).

Regarding malignant thymomas, specific clinical signs have been reported in relation tothe mediastinal invaded structures. This tumour has frequently caused pleural and peritonealeffusions (BELLAH et al., 1983; ARONSOHN et al., 1984; SORDE er al., 1994). Previous reportsmention the low rate of neoplastic growth and the scarciry of metastasis (MOULTON, 1990;JUBB et al., 1993; SORDE et al., 1994).

It seems that, among dogs, the German Shepherd breed displays the greatest incidence ofthymoma (PARKER and CASEY, 1976; ARONSOHN et al., 1984; SIMPSON et aL, 1992; SORDEet al., 1994), and large breeds in general are most affected (KLEBANOW, 1992). The mean ageof dogs presenting this neoplasm is 8 years (ARONSOHN et al., 1984; PARKER and CASEY,1976; KLEBANOW, 1992; SORDE et al., 1994). The tumour is not sex-associated, but itsincidence appears to be slightly higher in male dogs (PARKER and CASEY 1976; ARONSOHN

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400 GONzALEZ et al,

et al. 1984; SORDE et al. 1994), Conversely, KLEBANOW (1992) reports a greater incidence inbitches.

Our study describes the pathological and immunohistochemical features of a caninethymoma associated with an osteoblastic osteosarcoma of the right shoulder joint, To charac­terize the tumour cell population and histogenesis, immuno-histochemical techniques usingspecific antisera against intermediate filaments (IF) and neurospecific enolase (NSE) wereapplied.

Materials and MethodsA SO kg l l-year-old female, pure-bred Spanish Mastiff was referred to the Veterinary School of

Madrid after displaying marked lameness of the right forelimb for 3 days. Due to poor body condition thedog was euthanized at the owner's decision. Upon necropsy, we took representative samples from wholebody, including the masses observed in right forelimb and thorax cavity. Samples were fixed in 10 %neutral buffered formalin and 5 usx: sections were cut and stained with haematoxylin and eosin, toluidineblue and Congo red for histopathological diagnosis,

In order to determine the origin of the tumour cells, we used four markers. We developed theperoxidase-antiperoxidase (PAP) immunohistochcmical technique with the following primary antisera:mouse monoclonal anti-bovine vimentin (Sigma, St Louis, MO, USA), mouse monoclonal anti-chickendesmin (Dako, Glostrup, Denmark), rabbit polyclonal anti-neuron specific enolase (Zymed, San Francisco,CA, USA) and rabbit polyclonal anti-keratin wide-spectrum (Dako, Glostrup, Denmark). The sectionswere then incubated overnight at 4°C with the primary antibodies. After washing with PBS, the sectionswere incubated with swine antiserum against rabbit immunoglobulins (Dako-901, Glostrup, Denmark) orrabbit antiserum against mouse immunoglobulins (Miles, Elkhart, USA), depending on whether the firstantibody was polydonal or monoclonal, for 30 min at room temperature with a 11100 dilution, in bothcases, Following a rinse with PBS, the slides wcre incubated with horseradish peroxidase-antiperoxidasecomplex (PAP) produced in rabbit (Dako-Z.96, Glostrup, Denmark), a mouse (Dako, Glostrup, Denamrk),for polyclonal or monoclonal antibody respectively, at a dilution of I!64, for 30 min at room temperature.After the sections were washed twice in PBS, the antigens were visualized with a freshly prepared 0.025 %solution of DAB (3,3' -diaminobenzidine tetrahydrochloride, Sigma Irnmunochemicals. 0-5905. Tablets,St Louis, MO, USA) and 0.1 % hydrogen peroxide (30 %) in PBS for 2 to 10 min. The slides were washedwith distilled water, counrerstained with hacmaroxylin solution (Gill no 2) for 40 seconds, dehydrated andmounted. For negative controls, thc primary antisera were replaced by preimmune rabbit or mouse serum,Samples of normal canine skin and thymus were included in the same slide, which served as an internalpositive control.

Results

ClinicaljindifW

Physical examination revealed weight loss, dehydration, pale mucous membranes, mildtachypnoea and lethargy, and evident deformation of the right shoulder joint, which presentedpain on palpation. Both laterornedial and craneocaudal radiographs displayed a substantialosteolytic process with loss of the outline of humeral diafisis; this image suggested a bonetumour, and the presence of pulmonary metastasis was therefore investigated in order to pursuethe evaluation of the patient,

Thoracic radiographs showed a craneoventral mediastinal mass of homogeneous densitywhich displaced the trachea and oesophagus and masked the cardiac profile. As the animal wasreferred, no previous clinical history was available, however, the radiographic findings revealedthe presence of a possible bone tumour and its pulmonary metastasis.

Because of poor body condition and the severity and extent of the lesions, the dog waseuthanized. For this reason, the realization of complementary probes was not possible,

Macroscopi: .rtu4y

The necropsy revealed an osteolytic process accompanied by the fracture of the head ofthe humerus and exuberant proliferation of connective tissue. After opening the thoracic cavity,we observed the presence of a large (30 X 14 X 20 em), whitish-grey mass of approximately 4.5

Immunohistochemical Study of a Non-invasive Canine Thymoma 401

kg which collapsed the lungs (Fig. 1.) The mass was enveloped by a thick, connective tissuecapsule. No other pathological findings were observed in the remaining organic cavities andviscera.

Histologicalfindings

The histological study focused on the analysis of theoseous and mediastinal masses. Thesubsequent histological diagnosis of the right forelimb lesion was osteoblastic osteosarcoma.The mediastinal mass was enveloped by a connective tissue capsule and did not invadeneighbouring regions. In some subcapsular areas, cystic formations, which presented areas ofpseudostratification and which were covered by a ciliated columnar epithelium, were seen (Fig.2.). The cysts 'Were mainly filled with erythrocytes and macrophages with wide cytoplasms ofvacuolated appearance. Connective tissue trabeculae originating in the capsule penetrated theinterior of the neoplasm.

The neoplastic parenchyma displayed great structural variability in general, although solidcords or sheets composed of neoplastic cells predominated. These cells contained a largerround or oval-shaped nucleus and granular eosinophilic cytoplasm with indistinct boundaries,and displayed a low mitotic index. Throughout these areas, cells with fusiform nuclei andreticular cytoplasm appeared forming a reticular patrern. The neoplastic tissue exhibited anabundant vascular bed.

Anastomosis occurred between the cords, which enclosed substantial areas containing aneosinophilic and Congo red negative material which was generallyamorphous, but on occasionspresented a filamentous appearance. A small or medium calibre vessel normally occupied thecentre of these areas, which were bounded by an epithelial cell palisade (Fig. 3.) Epithelial cellsdisplayingvarying degrees ofkeratinization, which gave rise to Bassall corpuscle-like squamousformations, were seen (Fig. 4a.)

Likewise, larger numbers of mature lymphocyte cords forming penetrated the tumourparenchyma in some areas. Throughout the whole tumour, mast cells appeared in varyingdegrees of degranulation; the visualization of these cells was greatly facilitated by the use of

Fig. 1. Macroscopic appearance of thymoma with heart and collapsed lungs.

402 GONzALEZ et al.

Eg. 2. Cystic cavity covered by ciliated columnar epithelium. Positive reaction to wide spectrum cytokeratinsin epithelial cells (p.A.P. technique x 94).

Fig. 3. Perivascular space with epithelial cell palisade (H&E. x 188).

Immunohistochemical Study of a Non-invasive Canine Thymoma 403

Fig. 4. (a) Strong positive reaction to wide spectrum eytokeratinsin the Hassall's corpuscle-like structureand in neoplastic cells close to it. (P.A.P. technique x 198); (b) Moderate positive reaction to vimentin in

some neoplastic cells (p.A.P. technique x 94).

toluidine blue. The diagnosis of a noninvasive, mixed thymoma of epithelial and lymphoidorigin was made following the histological examination of the samples.

Immunohistochemistry

In normal canine thymus, cytokeratin marked the reticular network of epithelial cells andcell clusters resembling Hassall's corpuscles. Antiserum against vimentin revealed numerousdendritic and non-dendritic mesenchymal cells, especially in the medulla of normal thymustissue and within the perivascular area. The muscular layer of the blood vessels and large myoidcells displayed desmin-posirive immunoreactivity. NSE immunoreactive cells were occasionallyseen in the medulla of normal thymic tissue.

The immunohistochemical study of the neoplastic tissue revealed a moderate to strongreaction to the wide-spectrum eytokeratins in the tumour cells arranged in cords and in thoseforming; the positive reaction was also intense in the Hassall's corpuscle-like structures and inthe reticular cells near them (Fig. 4a.) Slight cytokeratin irnmunolabelling was likewise seen inthe pseudostratified ciliated columnar epithelium which covered the subcapsular cystic cavities(Fig. 2.) Immunoreactivity to antivimentin serum was intense in the perivascular mes­enchymatous cells (Fig. 5.), and slightly positive in the tumour epithelial cells (Fig. 4b.) Thereaction to the other antibodies used, NSE and desmin, was negative within the neoplasticparenchyma, except for desmin, which was positive in the smooth muscle fibres of the vesselsand myoid cells.

DiscussionUntil recently, veterinary pathologists have used human medical criteria in order to

histologically classify domestic animal thymomas (HARRIS et al., 1991). This was in relation tothe scarcityofcases and the great morphologic variabilitydisplayed, even within a single tumour.

404 GONzALEZ er al.

Fig. 5. Perivascular space: immunoreaction to amivimentin serum in mesenchymatous cells (p.A.P.technique x 94).

We agree with MOCLTON (1990), however, in noting that the use of human medical criteria isnot necessary.

In general, canine thymomas are rare and benign tumours. On many occasions, affectedanimals display no clinical signs, except when neighbouring structures are invaded (BELLAH etaI., 1983; ARONS( )HN et aI., 1984; KLEBANOW, 1992; SORDE et al., 1994). Such is our case, inwhich the dog did not present any clinical signs until the humeral fracture occurred. Accordingto the owner, the dog had a quiet nature and exercised very little. In our opinion, this couldhave masked the typical signs resulting from the compression and displacement of the thoracicorgans by the neoplastic mass, as well as the typical musculoskeletal complications resultingfrom the paraneoplastic thymic syndromes.

On the other hand, we think that terms more commonly used, benign and malignant, toassign thymomas are inappropriate, such as BELLAH et al. (1983) have named. Since the clinicalbehaviour of this tumour as in human medicine as veterinary medicine is not consistent withthe histological appearance. Therefore, we consider more proper the use of the alternativeterms, noninvasive and invasive.

The age (5-13 years old) and the breed (large) correspond with the parameters establishedby numerous authors (PARKER and CASEY, 1976; ARONSOHN et aI., 1984; KLEBANOW, 1992;SORDEet al., 1994) for the presentation of this type of tumour in dogs. In addition, the majorityof the authors consulted indicate that canine thymomas usually appear together with malignantneoplasms. Despite the fact that the studies consulted mention different tumours which havecoexisted with thymomas in dogs, the occurrence of associated osteosarcoma and thymoma isreported only by ATWATER et al. (1994). According to ARONSOHN ct al. (1984) and ATWATERet al, (1994), the high incidence of non-thymic tumours in dogs with thymomas should be inrelation to deficiencies in the thymus-mediated immunosurveillance system.

The tumour described did not present the common histological pattern displayed by othercanine thymomas, described by veterinary pathologists (MOULTON, 1990; JUBB et al., 1993).Nevertheless, it did share some histological features with other thymomas. Thus, in some areasunder the fibrous connective tissue capsule, we observe cystic formations which presented

Immunohistochemical Study of a Non-invasive Canine Thymoma 405

areas of pseudostratification which were covered by a ciliated columnar epithelium, such asdescribed by PARKER and CASEY (1976) and SIMPSON et al. (1992) in dogs, and VOS et al.(1990) in cats.

One of the most significant thymic findings was the presence of small calibre vesselssurrounded by an ample perivascular area filled with an eosinophilic, amorphous material. Verysimilar figures have been described as angiomatous proliferation by HADLOW (1978) in goats,and by PARKERand CASEY(1976) and MOULTON (1990) in dogs.

The presence ofscattered mast cells in the neoplastic parenchyma is corroborated by someinvestigators (pARKER and CASEY, 1976; JUBB et al., 1993; A1WATER et al., 1994), whoconsider it an almost constant finding in canine thymomas.

Likewise, we observed, as SIMPSON et al. (1992), that in some neoplastic epithelialcords or sheets the cells 'formed groups and underwent different degrees' of keratinization,constituting structures similar to Hassall's corpuscles.

Immunoperoxidase labelling was performed in an attempt to ascertain the nature of thecells. Using intermediate filament (IF) markers, it is possible to distinguish between epithelial,mesenchymal and muscular tissue neoplasms (Vos et al., 1990; PIZARRO et al., 1992; BAL­AGUER et al., 1995). In order to investigate the possible neuroendocrine origin of the tumour,we used also neurospecific enolase (NSE) (TRUE, 1990). The tumour cells were desmin andNSE negative, strongly positive for wide-spectrum keratin, and weakly positive for vimentin.The results indicated that the cells were neither from a muscular nor neuroendocrine origin,but did not help to attribute a mesenchymal or epithelial origin, either. Similar findings havebeen reported in dogs by HARRIS et al. (1991), although in our study the neoplastic cellspresented more intense immunoreactivity to vimentin than to eytokeratins. In our opinion,coinciding with FAWCETT (1995), this different response could be due to the uncertain originof the different types of thymic epithelial cells.

In conclusion, we believe that the use of immunohistochemical techniques is very usefulin establishing a differential diagnosis with other neoplastic or non-neoplastic lesions such asthymic hyperplasia, mediastinal or thymic lymphosarcoma, chemodectoma, parathyroid tumoursand lung metastatic tumours, among others. As the clinical prognosis depends on a correctdiagnosis of the lesion, and as thymomas can be treated surgically (VOS et al., 1995), the valueof these immunohistochemical techniques is unquestionable.

ReferencesARONSOHN, M. G., K. L. SCHUNK,). L. CARPENTER and N. W. KING, 1984:Clinical and pathologic

features of thymoma in 15 dogs.). Am. Vet. Med. Assoc. 184, 1355-1362.AlWATER, S. W., B. A. POWERS, R D. PARK, R. C. STRAW, G. K. OGILVIE, and S.]. WITHROW, 1994:

Thymoma in dogs: 23 cases (1980-1991).J. Am. Vet. Med. Assoc. 205, 1007-1013.BALAGUER, L.,J. ROMANO and A. MoRA, 1995: A poorly-differentiated squamous cell thymoma in a

chickenwith lymphoma.Avian Patho!' 24, 737-741.BELLAH,J. R.,M. E. STIFF, and R G. RUSSELL, 1983:Thymoma in the dog: two case reports and review

of 20 additionalcases.J. Am. Vet. Med.Assoc. 183, 306-311.PAWCEIT, D. W., 1995:Tratado de histologia. 12th edn. Ed. Interamericana New York: McGraw-Hill,

pp. 479-492.HACKETI, T. B., D. R VAN PELT, M. D. WILLAND, L. G. MARTIN, G. D. SHELTON, and W. E.

WINGFIELD, 1995:Third degree atrioventricularblock and acquired myastheniagravis in four dogs.]. Am. Vet. Med. Assoc. 206, 1173-1176.

HADLOW, W.]., 1978:High prevalenceof thymoma in the dairygoat. Vet. Patho!' 15, 153-169.HARRIs, C. L.,). S. KLAUSNER, D. D. CAYWOOD, and). R. LEININGER, 1991:Hypercalcemia in a dog

with thymoma.]. Am. Anim. Hosp. Assoc. 27, 281-284.JUBB, K. V. P., P. C. KENNEDY, and N. PALMER, 1993:Pathology of domestic animals.volume 3, 4th

edn. Orlando: AcademicPress Inc., pp. 218-220.KLEBANOW, E. R., 1992:Thymoma and acquired myasteniagravis in the dog: a case report and review

of 13 additionalcases.J. Am. Auirn. Hosp. Assoc. 28, 63-69.MOULTON,]. E., 1990:Tumors in domestic animals. 3rd edn. Berkeley: University of CaliforniaPress,

pp. 267-268.PARKER, G. A., and H. W. CASEY, 1976:Thymomas in domestic animals. Vet. Pathol. 13,353-364.

406 GONzALEZ et al.

PIZARRO, M., C. BR;\ND;\l:, M. A. SANCHEZ,and). M. FLORES,1992: Immunocytochemical identificationof a bovine peritoneal mesothelioma.). Vet. Med, A 39, 476--480.

SIMPSOI', R. M., D. ). WATERS, D. H. GEBHARD, and H. \'f. C\SEY, 1992: Massive thymoma withmedullary differentiation in a dog. Vet. Pathol. 29, 416--419.

SORDE, A., M. Pl..'MAROL"', M. D. FONDEVII.L'\, and X. MANTEe..., 1994: Psychomotor epilepsyassociated with metastatic thymoma in a dog. J. Small Anirn, Pract. 7, 220-227.

TRl'E, L. D., 1990: Atlas of diagnostic immunohistopathology. Gower Medical Publishing: New York.Vos,). H.,J. STOLWIJK, F. c.S. R,\MAERKERS, 1. A. C. M. VANOOSTERHOl'T, and T. S. G. A. M. V....N

DEN INGH, 1990: The use of keratin antisera in the characterization of a feline thymoma. .J. CompoPathol. 102, 71-77.