Bull Vet Inst Pulawy 56, 361-367, 2012 DOI: 10.2478/v10213-012-0063-8
SEMINOMA, SERTOLIOMA, AND LEYDIGOMA IN DOGS: CLINICAL AND MORPHOLOGICAL CORRELATIONS
RAFAŁ CIAPUTA, MARCIN NOWAK, MACIEJ KIEŁBOWICZ¹, AGNIESZKA ANTOŃCZYK², KAROLINA BŁASIAK², AND JANUSZ A. MADEJ
Department of Pathology, ¹Department of Surgery, ²Department of Reproduction and Clinic of Farm Animals, Faculty of Veterinary Medicine, Wroclaw University of Environmental and Life Sciences, 50-375 Wroclaw, Poland [email protected]
Received: April 27, 2012 Accepted: September 7, 2012
Abstract
The study aimed at presenting the most frequent male gonadal tumours in dogs, their clinical and histopathological aspects, at outlining aetiopathogenesis and differential diagnosis of the tumours. As examples of the most frequently manifested testicular tumours, three clinical cases were presented, involving tumour of interstitial (Leydig) cells, tumour of Sertoli cells, and seminoma. Respective clinical diagnosis employed USG, X-ray patterns, and morphological and biochemical tests. The surgically sampled material was stained with H+E and an attempt was made to establish expression of E-cadherin, calretinin, and Ki-67. It was shown that histopathological diagnosis of testicular tumours in dogs is frequently very difficult and complex and requires multidirectional studies.
Key words: dogs, seminoma, sertolioma, leydigoma, calretinin, E-cadherin.
Testicular tumours used to be encountered significant effect on the development of leydigomas (5, relatively frequently in household animals and the 34). lesions are most frequently noted in dogs (14, 19, 22, 23, An increased risk of testicular tumour 25, 35). On grounds of multi-year studies of Nielsen et development can be noted in morbid syndromes linked al. (21), involving over 340 cases of testicular tumours, to testicular dysgenesis, such as testicular feminisation the World Health Organisation presented classification syndrome of Klinefelter syndrome in men (4, 27). The of such lesions. In line with results of the studies, it was risk of a testicular tumour is also clearly augmented in accepted that the most common tumour type in testes cases of their insensitivity to androgens, due to mutation involved tumour of interstitial cells (leydigoma), in the gene responsible for structure of androgen followed by tumours of supportive cells (sertolioma) and receptor (7). seminoma. Moreover, prevalence of such tumours in Embryonal tumours of the testes develop from canine population was similar, although seminomas primitive cells, which may continue their differentiation manifested a slightly higher frequency, which was later along the gonadal line (to seminomas) or they may confirmed on several occasions (13, 16, 18, 33). One of transform into a population of pluripotential cells (to principal factors, which increase frequency of non-seminomas). The pluripotential cells may remain at manifestation in testicular tumours is cryptorchidism the stage of non-differentiated cells (carcinoma (27, 28). Most commonly, cryptorchidism is manifested embryonale), they may differentiate into extraembryonal unilaterally and the right testis used to be stopped in tissues (yolk sac tumour, choriocarcinoma), or toward descent more frequently than the left one. If somatic cells (teratoma) (4, 6). cryptorchidism affects only one testis, it may be Evaluation of malignancy grade in canine accompanied by development of a tumour in the other, testicular tumours is based on analysis of morphology normally located testis. Abdominal cryptorchidism manifested by the primary tumour, condition of draining predisposes to the development of sertolioma since the lymph nodes, and presence of distant metastases. The temperature higher than that in the scrotal sac results in first grade encompasses tumours restricted to the testes; wasting of most cells except of Sertoli cells. In cases of tumours of the second grade produce metastases inguinal cryptorchidism, the temperature is higher than restricted to retroperitoneal lymph nodes and to the that in the scrotal sac but lower than that in abdominal diaphragm, while tumours of the third grade are cavity, which, according to several authors, predisposes additionally accompanied by distant metastases (1, 11). to the development of seminomas (4, 9). On the other In cases of seminomas, the tumours spread mainly by hand, it was demonstrated that temperature exerts no lymphatic vessels and the metastases are located most 362 frequently in retroperitoneal lymph nodes (6, 26). Non- seminomas yield metastases both through lymphatic and blood vessels, mainly to the lungs, liver, bones, and brain (6). Malignant forms of sertoliomas and leydigomas develop metastases mainly to inguinal and sublumbar lymph nodes (1, 17, 18, 31). At the preliminary phase of development, testicular tumours are most frequently manifested by painless augmentation of the organ, asymmetry or a small nodule. Pain develops in cases of a rapid growth, bleeding to the tumour or upon cryptorchism. Azoospermia and/or oligospermia are frequently present (24). The tumour may be accompanied by infertility and dermatoses (acanthosis, seborrhoea) (24). Moreover, sertoliomas and seminomas may be hormonally active (secreting androgens or oestrogens) (20). The study aimed at presenting the most Fig. 1. Ultrasonographic pattern of canine testis in common male gonadal tumours in dogs, their clinical sagittal projection. Note a non-homogenous structure of and histopathological aspects, at outlining parenchyma. aetiopathogenesis and differential diagnosis of the tumours.
Material and Methods
Case 1 – seminoma. The case involved a dog of golden retriever breed with 40 kg of body weight, 10 years of age. The visit was provoked by an evident asymmetry and altered consistency of the testes. Palpation permitted to detect a soft lesion in the left testis. Rectal control of the prostate gland demonstrated its augmentation. Ultrasonography demonstrated increased volume of the prostate and a tumour in the testis. In the USG examination, the structure of the affected testis was non-homogenous, pointing to possible haemorrhagic or necrotic lesions (Fig. 1). The prostate gland manifested a heterogeneous echo-structure, an increased echogenicity, and presence of hypoechogenic cysts. Chest radiogram demonstrated Fig. 2. Hyperechogenic lesion, 21.0x12.7 mm in size, no metastases. Blood morphology and biochemistry within a normal parenchyma in left testis. fitted reference values. Case 2 – sertolioma. Another case involved an eight-year-old dog of the small bulldog breed, weighing 37 kg. The dog was referred to the Department due to markedly enlarged left testis. Upon palpation a tough, compact lesion was detected, with an irregular, nodular surface. Ultrasonography detected a hyperechogenic tumour of a non-uniform structure, 21x12.7 mm in size (Fig. 2). The right testis preserved a normal outlook. Chest radiogram detected no metastatic lesions. Blood morphology and blood biochemistry fitted the reference values. Case 3 – leydigoma. The subsequent case involved a Dalmatian dog of 13 years of age and body mass 26 kg. Clinical examination of the dog detected a clear disproportion in consistency of the testes. Ultrasonography permitted to detect numerous small hypoechogenic cysts in both testes. In addition, in the right testis, a tumour was detected of 45.9x31.9 mm in Fig. 3. Ultrasonographic pattern of a canine testis with a size, containing a hypoechogenic structure of 16 mm in visible, demarcated lesion of 45.9x31.9 mm in size and a size (Fig. 3). hypoechogenic structure of 16 mm in length.
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abnormalities were detected in biochemical or morphological tests. Section of the excised testes detected: - case 1: grey-yellowish-creamy parenchyma, occasionally divided by delicate lamellae of connective tissue, slightly bulging above the plane of cross-section (Fig. 4); - case 2: parenchyma or a nodular structure, grey-beige in colour, with fine light-yellowish spots, also bulging above the plane of the cross-section (Fig. 5); - case 3: a round, well enveloped tumour, with grey- brownish parenchyma of a soft consistency, with evident cysts (Fig. 6) The isolated fragments of altered testes were fixed for 24 h in 7% buffered formalin, routinely Fig. 4. Cross-section of canine testis containing processed to paraffin blocks, and cut to 4 µm thick seminoma. sections. In histopathological examination of haematoxylin and eosin stained microscope preparations, WHO classification of canine testicular tumours was applied. Immunohistochemical investigations were conducted on 4 µm paraffin sections, placed on silanised microscope slides (DAKO). Subsequently, the sections were deparaffinised in xylene and passed through decreasing concentrations of alcohol to water. Calretinin and E-cadherin antigens were detected using EnVision™ FLEX Target Retrieval Solution, High pH (50x) (DAKO), heating the preparations in a water bath at 96ºC for 20 min, while Ki-67 antigens were detected in EnVision™ FLEX Target Retrieval Solution, Low pH (50x) (DAKO), heating the preparations in a water bath at 96ºC for 20 min. Endogenous peroxidase was blocked in Fig. 5. Cross-section of canine testis containing EnVision™ FLEX Peroxidase-Blocking Reagent for 10 sertolioma. min. Then, the sections were overlaid with solutions of primary antibodies: Monoclonal Mouse Anti-Human Calretinin Clone DAK-Calret 1 (DAKO), dilution of 1/100, Monoclonal Mouse Anti-Human E-Cadherin, Clone NCH-38 (DAKO), dilution of 1/50, Monoclonal Mouse Anti-Human Ki-67 Antigen Clone MIB-1 (DAKO), dilution of 1/100. All sections were incubated for 20 min at room temperature. Subsequently, the preparations were washed in EnVision™ FLEX Wash Buffer (20x), overlaid with EnVision™ FLEX /HR SM802 visualisation system, and incubated at room temperature for 20 min. The immunohistochemical reaction was developed with solution of 3,3 diaminobenzidine tetrahydrochloride (DAB), EnVision™ FLEX DAB+ Chromogen (DAKO). Then, the sections were washed in distilled water, counterstained with haematoxylin, and Fig. 6. Cross-section of canine testis containing dehydrated in a row of alcohols. The material was leydigoma. cleared in xylene and closed under cover slips in a balsam. Microphotographs of all studied tumours Prostate gland showed a uniform echogenicity were subjected to computer-assisted image analysis but was enlarged, around 4 cm in length. Chest using a computer coupled to Olympus BX53 optical radiogram documented an accentuated pattern of microscope (Olympus, Japan). The system was bronchi, most probably due to senile calcification of capable of storing images and performing their digital bronchial walls. A marked dilation of pulmonary blood analysis. The measurements were done using cell^A vessels and an increased shadow of the myocardium was software (Olympus Soft Imaging Solution, Germany). noted. No metastases were detected. Except of a slightly increased level of lymphocytes in blood, no 364
Results and Discussion
In microscope examination of the first case, neoplastic hyperplasia of a diffuse seminoma type was detected. Cells with oval or round nuclei and relatively scanty cytoplasm prevailed. Neoplastic cells tightly filled seminiferous tubules and in several sites disrupted their walls and by infiltrating the sublayer formed a uniform neoplastic texture. Moreover, presence of small and focal lymphocyte infiltrates was demonstrated (Fig. 7). Seminoma represents an embryonal tumour, derived from primary genital cells. It is a relatively common testicular tumour in dogs, particularly in older dogs (mean age of 10 years). Its development is Fig. 7. Histological pattern of seminoma: aggregates of predisposed by cryptorchidism. Seminoma can be uni- embryonal cells with oval nuclei and scanty cytoplasm or bilateral, single or multiple. It develops more and typical focal lymphocyte infiltrate. H.E., 100x. frequently in the right, as compared to the left testis. Its size varies and the increase in size may cause enlargement of the testis (13, 18, 33). Seminomas are relatively soft although not as soft as sertoliomas. On a cross-section they manifest a homogenous, grey or creamy outlook. Large tumours may contain foci of coagulative necrosis, usually free of haemorrhages. In line with WHO categories, seminomas include an intratubular type or type free of infiltration and a diffuse type. The intratubular type is thought to represent the early stage of seminoma development. It consists of aggregates of embryonal seminiferous cells, replacing normal lining of seminiferous tubules. Outlook of the cells is typical: they are large, clearly demarcated, they contain large, most frequently vesicular cell nucleus with evident nucleolus and a scanty, basophilic cytoplasm., Abnormal figures of mitotic division are frequently noted (13, 15, 21). Many of the tumours contain focal aggregates of lymphocytes. In the diffuse Fig. 8. Histological pattern of seminoma: abnormal type, the most frequent form of seminoma, tumour cells figures of mitotic division. Note numerous, mostly transgress seminiferous tubuli and form bands or islands abnormal figures of mitoses and cells with a few cell of a relatively uniform texture. The cells are tightly nuclei. H.E., 400x. packed and well outlined. They contain a large cell
nuclei with one or two nucleoli (13, 21). In the structure
of neoplastic cells, individual vacuolised histiocytes are
dispersed (21). Normal and abnormal mitoses are frequent. Focal and/or perivascular aggregates of mature lymphocytes can be noted (13, 21). The tumour manifests a tendency to infiltrate lymphatic and blood vessels of spermatic cord. Metastases of seminomas in dogs are seldom encountered, in contrast to men, in whom the tumours comprise as many as 40%-50% embryonal tumours and show a much more pronounced tendency to develop metastases (10, 13). Even if very sporadic in dogs, a malignant form of the tumour may be encountered, e.g., the described by Takiguchi et al. (32) case of seminoma with metastases to scrotal skin, liver, kidneys, and peritoneum. In the other case, microscope examination disclosed neoplastic proliferation of Sertoli cells (sertolioma) of a diffuse type. The histopathological Fig. 9. Histological pattern of sertolioma: hyperplasia of pattern contained spindle-shaped cells with oval, less spindle shaped cells with hyperchromatic nuclei. H.E., frequently round hyperchromatic nuclei and loci of a 400x. clearly vacuolised cytoplasm. Figures of mitotic division
were infrequent (Fig. 9). 365
Principal function of the cells involves secretion of male hormones of the androgen group. Macroscopically, the tumours used to be described as small, individual or multiple foci of yellow to brown colour, soft in palpation, clearly demarcated from the surrounding tissue, frequently containing haemorrhagic foci (8). They may be located in a single, or both testes (30, 12). The histological pattern includes neoplastic cells of a round or multiangular shape (8), less frequently elongated, spindle-shaped (25). Cytoplasm of neoplastically transformed Leydig cells may contain vacuoles of various size. Cell nuclei are usually small, round, with individual nucleoli (25). Similarly to sertolioma tumours, histopathology of leydigoma may include erythrocytes-filled cysts (8). Fig. 10. Histological pattern of leydigoma: large oval The cases of testicular tumours described in our cells with vast cytoplasm, numerous lipid vacuoles, and paper represent the most frequently encountered focal haemorrhages. H.E., 200x. tumours of male gonads encountered in veterinary practice. This has inspired the authors to describe them in detail both in their histopathological and clinical The tumour develops from Sertoli cells of aspects, attempting to clarify their aetiopathogenesis and seminiferous tubuli. Around half of sertoliomas are to facilitate differential diagnosis of testicular tumours in disclosed in the testes with atrophy of seminiferous dogs. tubules due to cryptorchism (3, 18). The risk of Pathomorphologists describing testicular sertolioma development clearly increases when the tumours frequently encounter difficulties in establishing testes do not descend to the scrotal sac (18). Most the correct diagnosis. This may reflect, on one side, frequently, the tumour is unilateral, solid on its cross- frequently insignificant differences between healthy as section, white or grey, demarcated from the surrounding compared to neoplastically transformed tissue (e.g., tissue. It may be sufficiently large to evidently deform some cases of leydigoma) and, on the other side, the testis. Large malignant tumours infiltrate tunica alba, diagnostic problems may be posed by certain forms of epididymis, and spermatic cord (18). In view of their leydigoma and sertolioma, which may resemble each histological structure, sertoliomas are categorised into other very much. Finally, the so called mixed tumours their intratubular types: type free of infiltration or should be mentioned, the correct diagnosis of which diffuse type. The former type is formed by neoplastic requires highly experienced histopathologists. In such cells with indistinct outlines, round, or oval cell nucleus cases, the continuously developing immunohisto- and a vacuolised, acidophilic cytoplasm, frequently chemical techniques may be helpful. Using appropriate containing grains of lipochromic pigments (15). The cellular markers, a given type of a tumour may be cells are arranged in a palisade manner on basement confirmed or excluded and in some cases even prognosis membrane of seminiferous tubules and they may can be specified. These methods are routinely used in penetrate the membrane. Mitotic figures are very rare (2, doubtful cases of testicular tumours in men. In 3). The diffuse type cells have an irregular size and veterinary medicine, unfortunately, routine shape. They form large aggregates with no evident histopathological diagnosis does not include canalicular structures, separated by the blood vessel- immunohistopathological methods, which reflects high supplied parenchyma and fibrous septa (18, 21). Spaces prices of the antibodies and much more restricted filled with erythrocytes are also encountered (25). knowledge on behaviour of detected proteins in animals, Around 20%-30% dogs with sertolioma and their significance for carcinogenesis. For the demonstrate signs of hyperoestrogenism, exhibiting a purpose of this study, we have selected such markers as combination of feminisation, gynecomastia, atrophy of calretinin, E-cadherin, and Ki-67 proliferation antigen. It the other testis, metaplasia of prostatic epithelium (with should be added that calretinin belongs to the newest frequent its suppurative inflammation), alopecia, and markers applied in diagnosis of testicular tumours in atrophy of bone marrow (20, 25, 29). dogs. In the subsequent studied case, neoplastic Our attempts to estimate the localisation and proliferation of Leydig cells (leydigoma) was diagnosed. level of chosen proteins brought satisfactory results. The histopathology included large oval or multiangular Estimations of calretinin in leydigioma resulted in a cells, with rich cytoplasm, containing numerous fine strong cytoplasmic and nuclear reaction in vacuoles. The cells manifested a small to an average size neoplastically transformed supportive Leydig cells and hyperchromatic nuclei. Mitotic figures were (Fig. 11). infrequent and tumour structure contained focal In seminoma expression of calretinin was haemorrhages (Fig. 10). scanty and in seminoma no expression of the marker Leydigoma is a tumour originating from an was detected. Possibly, the presence of the marker uncontrolled proliferation of interstitial Leydig cells. positively correlates with hormonal function of the tumour. 366
tumour, and was detected in the spindle-shaped interstitial cells typical for the tumour (Fig. 12). In this case, cellular reaction manifested the typical for the protein membranous character and a less pronounced cytoplasmic character. In seminoma and leydigoma expression of E-cadherin proved to be very weak. In microscope patterns of seminoma, in which we attempted to demonstrate expression of Ki- 67 proliferation antigen, a pronounced nuclear reaction was detected in stimulated cells, ready for a mitotic division. This pointed to a high proliferative potential of the tumour and manifested positive correlation with an unfavourable course of the disease (Fig. 13). In sertolioma and leydigoma expression of Ki-67 was weak (+). Fig. 11. Strong expression of calretinin in Summing up, histopathological diagnosis of neoplastically transformed supportive Leydig cells. testicular tumours in dogs is frequently very difficult About 100x. and complex. Nevertheless, an experienced histopathologist using clinical anamnesis, macroscopic pattern of the tumour, its histological analysis and, at present, also immunohistochemical characteristics can establish a correct diagnosis. This is of a high practical significance since a reliable histopathological diagnosis allows for a precise prognosis and for selection of appropriate methods of therapeutic management.
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