PARALLEL MANIFESTATION OF VARIOUS TYPES OF TUMOURS IN CANINE TESTES

Renata Nowaczyk¹, Marcin Nowak¹, Jan P. Madej^2
1 Department of Pathological Anatomy, Pathophysiology, Microbiology and Forensic Veterinary Medicine, Wrocław University of Environmental and Life Sciences, Poland
² Department of Veterinary Prevention and Immunology, Department and Clinic of Obsterics, Ruminant Diseases and Animal Health Care, Wroclaw University of Environmental Sciences, Poland

ABSTRACT

The study aimed at describing a rare in veterinary practice case of parallel manifestation of various tumour types in canine testes. Samples of the altered testes were fixed in 7% buffered formalin, dehydrated and embedded in paraffin blocks. Mouse monoclonal antibodies were used to detect expression of proliferation antigen, Ki-67 in paraffin sections of the tumours. Microscopical examination of the right testicle detected neoplastic proliferation of seminoma type while in the left testicle of the same dog sertolioma was disclosed. In the former tumour expression of proliferation associated antigen, Ki-67 was appraised at (+++) while in sertolioma it was appraised at (-).

Key words: dog, tumours in testes, seminoma, sertolioma.

INTRODUCTION

Tumours in testes develop in dogs much more frequently than in other species of household animals [1,19,25,32,38,44]. Classification of this tumours was presented by World Health Organization (WHO) already in 1974. The accepted classification was based on multi year studies conducted by Nielsen and collaborators [27]. The authors performed histopathological evaluation of 344 tumours of dog testes, including 109 of interstitial cells tumours, 110 of Sertoli cells tumours and 125 seminomas. The studies and analogous investigations performed by many other authors proved that frequencies of the above mentioned tumours are similar in dogs population [18,22,24,31,42].

Similarly to many other tumours, the reasons of neoplasia in testes remains unknown. Frequency of tumour development in the organ evidently increases in undesceded testes [36,37]. In cases of abdominal cryptorchismus the risk of neoplasia exceeds that in inguinal cryptorchismus, which reflects first of all the difference in environmental temperature [17]. Abdominal cryptorchismus predisposes the host to tumours of Sertoli cells since the temperature higher than that in scrotal sac kills most of other cells. Inguinal cryptorchismus exposes testis to temperatures higher than those in scrotal sac but lower than those in abdominal cavity what, according to many authors, predisposes to seminoma development. Environmental temperature was found to exert no significant effect on development of tumours of interstitial cells. Cryptorchismus used to be unilateral and the right testis is more frequently undescended than the left one. If cryptorchismus affects a single testis, the tumour may develop in the other, properly situated testis [8,43]. Cryptorchismus is more frequent among related dogs, which points to genetic type disturbances [7,14].

It should be added that according to many authors orchitis has only a restricted significance for development of neoplasia in the organ. On the other hand, an enhanced risk of testicular tumours is observed in morbid syndromes linked to testicular dysgenesia, such as testicular feminization syndrome or Klinefelter syndrome in males [7,31,36]. The risk of testicular tumour increases also in cases of their insensitivity to androgens, due to mutation in the gene responsible for androgen receptor structure [12].

Using cytogenetical techniques, presence of isochromosome of chromosome 12 short arms, 12 [i(12p)] was disclosed in several testicular tumours. Significance of the disturbances for neoplasia of testicular tumours has not been fully clarified but the aberration represents a chromosomal marker typical for several histological types of testicular tumours of extensive diagnostic significance [7,16,28,36].

Embryonal tumours of testes develop from primitive cells which may further differentiate along the gonadal axis (seminomas) or may transform into populations of pluripotential cells (nonseminomas). The pluripotential cells may remain at the stage of nondifferentiated cells (carcinoma embryonale), differentiated toward extraembryonal tissues (yolk sac tumour, choriocarcinoma) somatic tissues (teratoma) [7,11,20].

The embryonal testicular tumours develop most frequently from preinvasive lesions, termed the intratubular germ cell neoplasia – IGCN [36]. They form large, intratubularly proliferating gonocyte-like cells with a clear cut nucleus and marked amount of glycogen in cytoplasm [2,7,9].

Diagnosis of testicular tumours is significantly facilitated by their serum markers. Accurate monitoring of alpha-fetoprotein (AFP), gonadotropin (β-HCG) and lactate dehydrogenase (LDH) levels is very important in patients with testicular tumours and particular in patients with embryonal type tumours, for check of their response to treatment and in early detection of their relapse [11]. Serum level of HCG, produced by syncytiotrophoblast cells, is always elevated in cases of choriocarcinoma, in 40-60% cases of carcinoma embryonale and 5-10% cases of seminoma [17]. Alpha-fetoprotein represents a glycoprotein synthetised in normal conditions first of all by embryonal yolk sack and, to a lower extent, by other embryonal tissues. Nonseminomas containing elements of yolk sac (endodermal sinus) may produce AFP, in contrast to seminomas [11,17]. Serum activity of lactate dehydrogenase (LDH) provides another generally applied marker but it is less specific than AFP or HCG. LDH level is augmented in 50-60% cases of metastatic nonseminomas and in 80% advanced cases of seminomas [11,17].

It should be added that high diagnostic and prognostic significance is manifested also by determination of a correlation between expression of the adhesive molecule called trophin and levels of β-HCG. In studies performed by Hatakeyama et al. [13] all samples of testicular tumours which yielded metastases to lungs proved to be trophin-positive and concentration of β HCG in serum correlated in a positive way with expression of trophin in the tumour. In immunohistochemical studies on testicular tumours an evident positive relationship was disclosed between expression of trophin and degree of tumour histological malignancy. Expression of trophin was found to clearly increase with a growing tumour malignancy.

Clinical classification of canine testicular tumours is based on evaluation of the primary tumour, draining lymph nodes and distant metastases. The first degree denotes tumours restricted to testes, the second degree signifies metastases restricted to retroperitoneal lymph nodes and diaphragm and in the third degree tumours distant metastases are present in addition [3,17].

According to most of the authors, metastases of testicular tumours are very infrequent in dogs. In cases of seminomas, spread of the tumours develops mainly through lymphatic vessels and metastases are most frequently located in retroperitoneal lymph nodes [11,35]. Nonseminomas produce metastases both through lymphatic and blood vessels, mainly to lungs, liver, bones and brain [11]. Rare malignant forms of tumours from supporting cells and intraparenchymal cells yield metastases mainly to inguinal and lumbar lymph nodes [3,10,23,24,40]. It should be added that metastases of hormonally active tumours may themselves produce hormones [24].

At the preliminary stage of the disease, testicular tumours manifest most frequently by painful enlargement of the organ, asymmetry or a small tumour. Pain develops usually in cases of a rapid increase in size, bleeding to the tumour or in cases of cryptorchismus. Azoospermy and/or oligospermy accompany the condition [30]. Infertility and dermatoses (akanthosis, seborrhoea) are also observed [30]. Tumours of supporting cells and seminomas may secrete androgens or oestrogens [26,31].

CASE DESCRIPTION

In a mongrel, male dog, 14 years of age, evident discrepancy was noted in size of testes, which suggested development of a tumour. Palpation disclosed a uniform enlargement of the right testes accompanied by a decreased size of the left one. Surface of each testis was smooth and showed no signs of neoplastic proliferation. No adhesions or exudate was noted in the scrotal sac. In line with doctor’s suggestion, both testes were excised and sent for histopathological evaluation.

MATERIAL AND METHODS

Samples of the altered testes were fixed in 7% buffered formalin, dehydrated and embedded in paraffin blocks. Expression of the proliferation-associated antigen Ki-67, was made by using mouse monoclonal antibodies (MIB-1; diluted 1:100). The antigenic determinants were unblocked in the Antigen Retrieval Solution in a microwave oven. The colour reaction was evoked using LSAB2 kit and diaminobenzidine (DAB). All the reactions were accompanied by negative controls using the Primary Negative Control kit. All the reagents and antibodies originated from DakoCytomation, Polska.

The obtained preparations were microphotographed and the photographs were subjected to computer-assisted image analysis in a set consisting of a computer coupled to Axiophot microscope (Carl Zeiss). The measurements took advantage of MultiScaneBase V 8.08 software working in the Windows environment.

Expression of Ki-67 was estimated in a semi-quantitative manner, evaluating proportion of positive cells [0-5%: lack of reaction (-), 6-25%: scanty reaction (+), 26-50%: moderate reaction (++), above 50%: intense reaction (+++)].

RESULTS AND DISCUSSION

After cross-section of the testes, a grey-yellowish creamy parenchyma was noted in the right (enlarged) testis, in some places separated by delicate connective tissue septa, slightly protruding above the cross-section plane. In the left (decreased in size) testis was noted a small tubercle, of around 5 mm in diameter, with nodal structure, grey-whitish in colour and also protruding above the cross-section plane (Fig.1).

Fig. 1. Cross-section of canine testis. Left testis (A): neoplastic tumour of sertolioma type, right testis (B): neoplastic tumour of seminoma type

In microscope examination of the right testis neoplastic proliferation of seminoma type was disclosed. In the histological pattern cells with oval or spherical cell nuclei and relatively scanty cytoplasm prevailed (Fig.2). Mitotic figures, mostly abnormal, and cells with multiple cell nuclei were numerous. Tumour cells tightly filled seminiferous tubuli and in many cases transgressed tubular wall and infiltrated the sublayer forming a uniform neoplastic layer. Moreover, presence of small lymphocytic infiltrates was observed (Fig.3).

Fig. 2. Histological pattern of diffuse type seminoma with numerous mitotic figures (arrows). HE staining. 400x

Fig. 3. Histological pattern of diffuse type seminoma: infiltrate of lymphoid cells (arrow). HE staining. 400x

In microscope examination the left testis manifested neoplastic proliferation of the supporting cells of Sertoli (sertolioma) of a diffuse character. Spindle shaped cells with oval and, less frequently, spherical, hyperchromatic cell nuclei and clearly vacuoles-filled cytoplasm were noted (Fig.4). Mitotic figures were infrequent.

Expression of the proliferation associated antigen, Ki-67, in the seminoma was appraised at (+++) (Fig. 5), and in the sertolioma at (-). The obtained results of intensity of Ki-67 expression were consistent with the literature data and confirmed the markedly higher mitotic activity in seminoma as compared to sertolioma [4,5,18,21,27].

Fig. 4. Histological pattern of sertolioma with evident vacuolization of cytoplasm (arrows). HE staining. 400x

Fig. 5. High expression of the proliferation-associated antigen, Ki-67 (+++) in seminoma

Seminoma is an embryonal tumour originating from primordial germinal cells. It represents a relatively common testicular tumour in dogs, particularly in older dogs (mean age of manifestation is 10 years). Its development is markedly promoted by cryptorchismus. Seminoma could be unilateral or bilateral, single or multiple. It develops more frequently in the right than left testicle. It forms a tumour of variable size and its growth may markedly enlarge the testis [18,24,42]. Seminomas are relatively soft but not as much as are tumours originating from supporting cells. At a cross section, they show grey or creamy outlook. Large tumours may contain foci of coagulative necrosis, usually free of haemorrhages. In line with the classification accepted by WHO, the intratubular type with or without infiltration, and the diffuse type of seminomas are distinguished. The intratubular type is thought to represent an early developmental stage of seminoma. It consists of embryonal cell clumps which obliterate lumen of seminiferous tubules substituting for their normal epithelial lining. The cells manifest a typical outlook, they are large, of evident borders, they frequently contain a vesicular cell nucleus with noticable nucleolus and scanty, basophilic cytoplasm. Frequently, abnormal figures of cell divisions are seen [18,21,27]. Multiple tumours contained focal accumulations of lymphocytes. In the diffuse, most common type of seminoma the tumours cells are present not only in seminiferous tubules but formed bands or islands of a relative uniform structure. The cells are tightly packed, well outlined, with large cell nucleus and one or two nucleoli [18,27]. The neoplastic structure contained individual vacuolised histiocytes [27]. Normal and abnormal mitoses are frequently present. Focal and/or perivascular accumulations of mature lymphocytes are also present [18,27]. The tumour tended to infiltrate lymphatic vessels and blood vessels of spermatic cord. In dogs metastases of seminoma are rare in contrast to men, in whom the tumours comprise as many as 40-50% embryonal tumours and exhibit higher tendency to develop metastases [15,18]. Malignant forms of the tumour, even if very sporadically, develop also in dogs, including the case described by Takiguchi et al. [41] of seminoma with metastases to scrotal skin, liver, kidney and peritoneum.

Sertolioma, originating from Sertoli cells, represented another tumour identified by us in the dog. Around 50% the tumours originating from Sertoli cells are noted in testes with atrophy of seminiferous tubules due to cryptorchismus [5,24]. The risk of sertolioma development increases in cases when testes have not descended to scrotal sac [24]. The tumour used to be unilateral, hard, white or grey at the cross section surface, well separated from the surroundings. It may be sufficiently large to clearly disfigure testis. Large malignant tumours infiltrate tunica albuginea, epididymis and spermatic cord [24]. In respect to histological structure sertoliomas are categorized into intratubular type, with or without infiltration, and the diffuse type. The first type consists of neoplastic cells with unclear borders, round or oval cell nucleus, small nucleolus and vacuolised acidophilic cytoplasm, frequently containing granules of lipochrome stains [21]. The cells are arranged in a palisade manner in relation to basement membrane of seminiferous tubules and may penetrate the membrane. Mitotic figures are very rare [4,5]. In the diffuse type tumours cells are of irregular size and shape. They form large clumps with no evident tubular structures, divided by well blood supplied parenchyma and fibrous septa [24,27].

Around 20-30% dogs with sertolioma manifests signs of hyperoestrogenism, characterized by a combination of feminisation, gynecomastia, atrophy of the other testis, metaplasia of prostatic epithelium (frequently with its suppurative inflammation), alopecia and bone marrow atrophy [26,31,32,39].

Basement membrane (BM) is always present around seminiferous tubules and blood vessels in normal testes and in the intratubular form of sertoliomas and seminomas. Alterations in BM (its fragmentation or absence) develop in the invasive types and in the diffuse type of the tumours and provide an additional prognostic factor [6,29,32,34,40].

Metastases of tumours to testes develop sporadically and they may include lung cancers, prostate carcinoma, cancers of urinary bladder, kidney, stomach and colon. involvement of testes is possible in cases of some systemic neoplasms, e.g., diffuse large B-cell lymphoma (DLBCL)) of extranodular localization [33].

CONCLUSIONS

1. Tumours in testes develop in dogs much more frequently than in other species of household animals. Most often diagnosed are seminomas, Sertoli cells tumours and interstitial cells tumours.

2. Frequency of tumour development in testes evidently increases in cases of cryptorchismus. Enhanced risk of testicular tumours is also observed in morbid sundromes linked to testicular dysgenesia and insensitivity to androgens.

3. Diagnosis of testicular tumours is significantly facilitaded by their serum markers.

4. Seminoma represents a relativly common testicular tumour in dogs, particulary in older dogs. Around 50% tumours originating from Sertoli cells are noted in testes with atrophy of seminiferous tubules due to cryptorchismus.

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Accepted for print: 6.08.2007

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Renata Nowaczyk
Department of Pathological Anatomy, Pathophysiology,
Microbiology and Forensic Veterinary Medicine,
Wrocław University of Environmental and Life Sciences, Poland
C. K. Norwida 31, 50-375 Wrocław, Poland

Marcin Nowak
Department of Pathological Anatomy, Pathophysiology,
Microbiology and Forensic Veterinary Medicine,
Wrocław University of Environmental and Life Sciences, Poland
C. K. Norwida 31, 50-375 Wrocław, Poland

Jan P. Madej
Department of Veterinary Prevention and Immunology,
Department and Clinic of Obsterics, Ruminant Diseases and Animal Health Care,
Wroclaw University of Environmental Sciences, Poland
Norwida 31, 50-375 Wrocław, Poland
email: jan_madej@interia.pl

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