Case Report Ultrasonographic Appearance and Abdominal Haemorrhage Associated with a Juvenile Granulosa Cell Tumour in a Foal J

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Case Report Ultrasonographic appearance and abdominal haemorrhage associated with a juvenile granulosa cell tumour in a foal J. Harper*, A. J. Stewart, L. Kuhnt†, R. W. Waguespack, M. Holland and C. Downs Department of Clinical Sciences; and †Department of Pathobiology, J.T. Vaughan Large Animal Teaching Hospital,Collegeof VeterinaryMedicine,Auburn University,Auburn, Alabama 36849-5522,USA.

Keywords: horse; granulosa thecal cell tumour; neonate; haemoperitoneum; colic; ultrasonography

Summary ultrasonographic appearance, successful surgical removal and histopathological description of a juvenile granulosa Granulosa cell tumours are commonly identified in mares, cell tumour in a neonatal filly foal. but have rarely been identified in equine neonates. This report describes a septic neonatal foal that presented Case summary with haemoabdomen secondary to a ruptured ovarian granulosa cell tumour. The ultrasonographic appearance, successful surgical removal and histopathological History appearance of the tumour is described. Juvenile ovarian granulosa cell tumours differ histologically from adult An 18 h old Arabian filly (weight 42 kg) was referred to the granulosa cell tumours. Ultrasound is a useful way to John Thomas Vaughan Large Animal Teaching Hospital at identify haemoabdomen and abdominal masses in foals. Auburn University for weakness and possible neonatal Juvenile granulosa cell tumour should be considered as a septicaemia. The foal had a normal gestational length

differential for this combination in neonatal foals.eve_20 115..120 (330 days), but had appeared weak since birth. Failure of the foal to stand by 4 h of age had prompted the owner Introduction to assist the foal to stand and nurse from the mare. At 12 h of age the foal still appeared weak. As umbilical Granulosa cell tumour is the most common ovarian tumour haemorrhage had persisted since birth, the owner sought diagnosed in mares (Westermann et al. 2003; McCue et al. veterinary attention for the foal. The referring veterinarian 2006). While routinely diagnosed in adult horses with the placed a suture ligature around the umbilical stump to aid of clinical history, diagnostic ultrasonography and prevent further haemorrhage, administered one litre of i.v. endocrine testing, there have been limited reports of fluids containing 5% dextrose, and approximately 480 ml juvenile granulosa thecal cell tumour in foals (Green et al. of colostrum via nasogastric intubation. The foal was then 1988). Diagnosis in the neonate or weanling patient has referred to Auburn University. been limited to identification during an exploratory celiotomy to investigate the cause of haemoperitoneum Physical examination findings or as a finding on necropsy evaluation (Green et al. 1988). Abdominal ultrasonography is useful for the evaluation of At initial examination, the filly was standing but appeared the abdomen in neonatal foals (McAuliffe 2004; Porter and extremely weak. She appeared disoriented and was Ramirez 2005). It is especially useful for identification of unable to nurse from the dam. The abdomen appeared haemoabdomen and localisation of abdominal masses distended and ventral oedema was noted around the and can aid in the decision to perform an exploratory umbilicus. No joint effusion was palpable, but hindlimb laparotomy (Reef 1998). In this article, we describe the flexor tendon laxity was noted. The sclera were bright red and haemorrhagic foci were observed at the dorsal sclerolimbal junction bilaterally. Mandibular prognasthism *Author to whom correspondence should be addressed. Current address: Texas Specialty Veterinary Services, PLLC, Boerne, Texas was also present. Temperature, pulse and respiratory rates 78006, USA. were within normal limits.

Major differential diagnoses for the filly following initial examination included septicaemia, hypoxic ischaemic encephalopathy, and failure of passive transfer. The owner elected to pursue medical therapy, and diagnostic and treatment plans were initiated.

Diagnostic procedures

Laboratory evaluation

Blood was collected for a complete blood count (CBC), biochemical profile, fibrinogen and immunoglobulin concentrations, as well as aerobic and anaerobic blood culture. Haemotology revealed a low haematocrit (20%; reference range (rr), 30–40%) indicating anaemia and hyperfibrinogenaemia (7 g/l; rr 1–4). Serum biochemical Fig 2: Doppler ultrasound image of the cranioventral abdomen. evaluation revealed a mild hypernatraemia (152 mmol/l; rr The large ovarian mass is present. Colour doppler shows a large 133–150) and hypoproteinaemia (29 g/l; rr 30–50). Serum vascular structure associated with the mass. The image was IgG concentration indicated a complete failure of transfer obtained with a 2–5 MHz sector scanner transducer and a of passive immunity (<4 g/l; rr > 8). Actinobacillus equuli was displayed depth of 16 cm. subsequently cultured from the blood. Biochemical analysis revealed minimal changes Ultrasonography with the exception of hypoproteinaemia and hypogammaglobuminaemia, both of which were The pulmonary parenchyma appeared ultrasonographically attributable to failure of transfer of passive immunity and normal and no rib fractures were identified. haemorrhage. Anaemia was considered to be most likely Transabodminal ultrasonography revealed a large due to external blood loss secondary to the reported volume of free, echogenic fluid within the abdomen that umbilical haemorrhage; however, haemoabdomen had a characteristic ‘swirl-like’ pattern suggesting a secondary to trauma or congenital vascular rupture was haemoabdomen. A large, complex mass containing also considered possible. An inflammatory process, such as multiple anechoic to hypoechoic, cystic structures was neonatal septicaemia, was also considered due to the identified in the mid-abdominal region. The mass was presence of hyperfibrinogenaemia and inadequate found to extend from the umbilicus to the cranial portion of transfer of maternal antibodies. the abdomen near the liver (Fig 1). The mass had multiple thinned walled circular cystic structures with hypoechoic to anechoic centres suggestive of fluid accumulation with varying echogenicity. Doppler ultrasound showed large blood vessels associated with the mass (Fig 2). The mass occupied approximately one third of the abdomen. The bladder, umbilical vessels, kidneys, liver and spleen all appeared normal.

Abdominocentesis

Abdominocentesis yielded a large amount of free, haemorrhagic fluid with a haematocrit of 20% which was the same as the intravascular haematocrit of 20% and therefore indicative of frank blood from acute haemorrhage.

Treatment Fig 1: Ultrasonogram of the ventral abdomen 5 cm caudal to the xiphoid. The top of the image is ventral. A large mass An i.v. catheter was placed in the jugular vein and the foal (approximately 10 ¥ 15 cm) with variable echogenicity is was administered 2 l of plasma (48 ml/kg bwt i.v.), ceftiofur surrounded by echogenic peritoneal fluid. The liver is visible 1 dorsally. The image was obtained with a 2–5 MHz sector scanner (Naxcel) (5 mg/kg bwt i.v. b.i.d.), amikacin (22 mg/kg bwt transducer and a displayed depth of 16 cm. i.v. s.i.d.), vitamin E (6000 iu per os s.i.d.) and Normosol-R2

be sonographically normal. Congenital lymphosarcoma, teratoma or juvenile granulosa cell tumour were all differentials for this neonate.

Exploratory celiotomy

A ventral midline celiotomy was performed under general anaesthesia maintained under isoflurane gas anaesthesia. A small amount of clotted blood in addition to frank blood was apparent within the abdominal cavity. Further exploration of the abdomen revealed a large soft tissue mass attached to a stalk of tissue arising from the left side of the dorsal abdomen, most likely the mesovarium. The mass (Fig 3) measured 10 ¥ 15 cm and had a serosal tear which appeared to have haemorrhagic, fresh tissue Fig 3: Exploratory celiotomy revealed an approximately 10 ¥ 15 cm margins and appeared to be the source of internal soft tissue mass involving the left ovary. haemorrhage. The mass appeared to involve the entire left ovary due to its anatomical location and surrounding structures. Using a bipolar electrothermal device (24 ml/kg bwt i.v. q.i.d.). Although the foal was weak and (LigaSure)3, the pedicle was ligated and incised in order to unable to stand without assistance, it had a strong suckle allow complete removal of the mass (Figs 4 and 5). reflex, therefore the foal was offered and nursed 8 ml/kg but An umbilical herniorraphy was performed using sharp (175 ml) of freshly milked mare’s milk by bottle every 2 h. excision and monopolar cautery allowing removal of Over the next 12 h, the filly’s condition dramatically the umbilical contents (arteries, vein and urachus). The improved and she was then able to ambulate and nurse cystotomy site was closed routinely in 2 layers. No further on her own. Intravenous fluid therapy and offering of haemorrhage was identified during surgery. The abdomen mare’s milk via the bottle were discontinued after 24 h. was closed in a routine fashion, and the foal recovered Haematology was performed the following morning and from anaesthesia without complications. showed an increase in the total white blood cell (19.0 ¥ 109 cells/l; rr 6–12 ¥ 109), and neutrophil counts (16.9 ¥ 109 cells/l; rr 3–6 ¥ 109) and fibrinogen concentration (9 g/l; rr 1–4). Post operative treatment On the basis of serial ultrasonographic findings and the analysis of fluid obtained by abdominocentesis, an The foal recovered uneventfully from surgery. exploratory celiotomy was recommended. The most likely Antimicrobials (ceftiofur and amikacin) and flunixin origin of the mass was considered to be the mesenteric meglumine (1.1 mg/kg bwt i.v. b.i.d.) were administered or reproductive tract as all other organs appeared to for 5 days post operatively. The foal was discharged 7 days after surgery with a prescription for cefpodoxime

Fig 5: Excised ovarian granulosa cell tumour. The yellow arrow Fig 4: Ovarian pedicle proximal to the ovarian mass is being shows the serosal tear and source of the internal haemorrhage. ligated using the Ligasure device. Serosanguinous fluid could be aspirated from cysts within the mass.

considered to be a useful marker for tumours of granulosa cell origin, while S-100 protein is a less specific marker, present in many cell types, including normal ovarian and follicular structures, as well as tissues of neural crest origin, etc. (Lin et al. 1991; Schumer and Cannistra 2003). Immunohistochemical staining for cytokeratin, an intermediate filament within the cytoplasm of many epithelial tissues, was negative. Histological and immunohistological findings support a diagnosis of juvenile granulosa cell tumour in this case. Granulosa cell tumours can occur as 2 types, the adult or juvenile type, and these are differentiated histologically. The more common adult type often occurs in a variety of histological patterns, including diffuse, trabecular, microfollicular, macrofollicular or solid-tubular patterns, while the juvenile type is characterised by solid or Fig 6: Ovarian mass: Sheets and nests of neoplastic granulosa cells macrofollicular structures, without microfollicular patterns are surrounded by thecal cells and separated by fibrovascular or Call-Exner bodies (Piura et al. 2008). Cytological and stroma. The thick outer fibrous capsule is shown at the top. nuclear characteristics also differ in that neoplastic cells in Haematoxylin and eosin (H&E). Bar = 300 mm. the adult type have small amounts of cytoplasm, grooved nuclei and a low mitotic rate. In contrast, the neoplastic (10 mg/kg bwt per os b.i.d.) for 7 days. Follow-up physical cells within juvenile tumours have moderate to abundant examination performed 3 months after surgery, revealed amounts of eosinophilic cytoplasm, increased cytological no abnormalities. atypia, nongrooved nuclei and a relatively higher mitotic rate (Schmidt and Kommoss 2007). Histology

Multiple sections of formalin-fixed tissue were stained Discussion with haematoxylin and eosin (H&E) and the immunohistochemical stains anti-vimentin, anti- Granulosa cell tumour is the most common reproductive cytokeratin, anti-S100, and anti-inhibin4. Histologically, tumour diagnosed in the adult mare (Bosu et al. 1982; normal ovarian architecture was effaced by a thickly Westermann et al. 2003; McCue et al. 2006). Although the encapsulated mass of neoplastic granulosa and thecal adult form is more commonly identified, juvenile forms of cells. Solid, cystic, or macrofollicular patterns of granulosa cell tumours have been reported in human proliferating granulosa cells were bordered by infants (Young et al. 1984; Calaminus et al. 1997; Vassal spindle-shaped thecal cells and separated by bands of et al. 1998), a neonatal foal (Green et al. 1988) and a fibrovascular trabeculae (Fig 6). The stroma varied from Holstein heifer (Masseau et al. 2004). Complications collagenous to mucinous. Follicular-like aggregates of associated with juvenile granulosa tumours in foals, heifers polygonal granulosa cells were lined by palisading and human infants include abdominal pain and/or peripheral cells and had indistinct cellular margins, distension due to rupture of the tumour and subsequent moderate amounts of lightly eosinophilic, multivacuolated haemoabdomen (Green et al. 1988; Masseau et al. 2004; cytoplasm and round to ovoid, vesicular nuclei. Some Dechant et al. 2006). Numerous studies have reported macrofollicles were filled with vacuolated fluid. Mitotic rupture of the granulosa cell tumour during or prior to figures were uncommon, but variable, ranging from 0–3 surgery in horses (Green et al. 1988; Alexander et al. 2004), per 400x field. The mass contained many large, central cattle (Masseau et al. 2004), sheep (Gardner et al. 2005) cystic spaces filled with haemorrhage, fibrin, and cell and humans (Sehouli et al. 2004; Givalos et al. 2005). debris. Small foci of well-differentiated and mineralised Clinical signs associated with haemorrhagic shock can bone, with adipose tissue-filled medullary spaces, and manifest as elevation in respiratory and heart rates, cartilage, were rarely present within the fibrous capsule. signs of weakness, and hypovolaemia, while pain Few capsular vessels were occluded by fibrin thrombi. associated with a ruptured tumour can result in signs of Immunohistochemical staining of the neoplastic colic. These signs in the equine neonate should be granulosa cells revealed diffuse and strong cytoplasmic evaluated by haemotology, ultrasonographic evaluation, immunopositivity for vimentin, an intermediate filament abdominocentesis, and/or exploratory celiotomy. found within the cytoplasm of many mesenchymal Granulosa cell tumours are routinely diagnosed based and mesodermal-origin cells, and moderate, irregular on the results of clinical history, endocrine assay evaluation cytoplasmic immunopositivity for inhibin and S-100. Inhibin, and ultrasound examination. The typical sonographic as a hormonal product of normal ovarian granulosa cells, is appearance of an ovarian granulosa cell tumour is a

multiloculated (‘honeycombed’) mass, however they can S-100, cytokeratin and calretinin (Schmidt and Kommoss appear as a solid ovarian mass, or as a single fluid-filled 2007). Although immunostaining characteristics of the cyst (Bosu et al. 1982; Hinrichs and Hunt 1990; Reef 1998; adult and juvenile types are similar, this does allow McCue et al. 2006). Equine ovarian teratomas often differentiation from other types of ovarian tumours, such as have a similar sonagraphic appearance as granulosa cell those of epithelial or germ cell origin. Overall clinical tumours (Reef 1998; McCue et al. 2006). Ultrasonography is behaviour of these tumour types in man is similar when a useful diagnostic technique for evaluation of suspected diagnosed at an early stage, but juvenile type tumours causes of haemoperitoneum in the neonatal foal have been noted to be more aggressive, with more rapid (Green et al. 1988; Dechant et al. 2006). Transcutaneous and frequent recurrence, when diagnosed at more abdominal ultrasonography in the equine neonate allows advanced stages (Piura et al. 2008). visualisation of nearly the entire abdominal contents and While granulosa cell tumours have a classical organs due to their smaller size compared to adult horses appearance during surgery and ultrasonographic (Reef 1998; McAuliffe 2004). Potential causes of evaluation, other tumours are possible including haemoperitoneum in the neonatal foal include rupture of teratomas. Although the mass contained small the umbilical vein or arteries, abdominal trauma involving intracapsular foci of bone and cartilage, this was the liver or spleen and abdominal masses or tumours considered to represent chondro-osseous metaplasia, and (Green et al. 1988; Dechant et al. 2006). was not part of the neoplastic population of cells. A Classic behavioural changes associated with ovarian diagnosis of teratoma was excluded in this case because granulosa cell tumours in mares have not been reported in of the relatively small size, uniformity, well-organised and equine neonates with juvenile granulosa cell tumours. well-differentiated nature of the chondro-osseous tissue, Mares with ovarian granulosa cell tumours often have which was present only in the peripheral outer fibrous elevated levels of oestrogen or testosterone and capsule. A teratoma is a neoplasm consisting of 2 or 3 decreased levels of progesterone (Stickle et al. 1975; germ cell layers (ectoderm, endoderm or mesoderm), Stabenfeldt and Hughes 1980; Westermann et al. 2003; often in a haphazard admixture of poorly or widely McCue et al. 2006). Inhibin concentrations are also differentiated elements. It is possible that local trauma elevated in mares with ovarian granulosa cell tumours and or vascular disturbances due to the large size of this mass this is considered the gold standard for diagnostic testing may have contributed to the reactive or metaplastic (Piquette et al. 1990; Bailey et al. 2002; Watson et al. 2002; changes. It is not uncommon in humans for neoplastic Charman and McKinnon 2007; Ellenberger et al. 2007). or non-neoplastic processes to undergo metaplasia in However, hormonally inactive granulosa cell tumours are many tissues, including ovarian tissue (Morizane et al. 2003; present within the equine population and should be Mukonoweshuro and Oriowolo 2005). considered when evaluating a patient for this type of Prognosis for survival and future reproductive tumour (McCue et al. 2006; Zelli et al. 2006). Hormone performance for patients diagnosed with granulosa cell concentrations in the limited number of reported cases of tumours is good (Bosu et al. 1982; McCue et al. 2006). equine neonatal ovarian granulosa cell tumour have not Although bilateral granulosa theca cell tumours have been reported in the literature. Unfortunately, hormone been reported (Frederico et al. 2007), rarely is the opposite concentrations were not evaluated in our patient, and ovary involved and it generally returns to normal function therefore it is still unknown if granulosa cell tumours in after removal of the affected ovary (Bosu et al. 1982; equine neonates are hormonally active. McCue et al. 2006). Approximately 3 months following In man, ovarian juvenile granulosa cell tumours are surgery, follow-up examination of the described patient commonly noted in children, although they may arise at revealed a healthy filly, and no abnormalities were any age, and are usually unilateral and well encapsulated reported by the owner over the following 2 years. (Piura et al. 2008). These lesions are considered benign, but Ultrasonography is a useful tool for diagnosing granulosa recurrence and intraperitoneal seeding following rupture cell tumour in the adult and neonate. It appears particularly has been reported (Gittleman et al. 2003). Juvenile valuable in indentifying complications such as rupture and granulosa cell tumours, as in this case, are noted to differ haemoperitoneum in patients afflicted by the juvenile histologically from adult granulosa cell tumours by the lack form of granulosa cell tumour. Follow-up transabdominal of microfollicular, trabecular or solid-tubular type patterns, and transrectal ultrasound examinations have been and an absence of Call-Exner bodies (Piura et al. 2008). recommended to evaluate the remaining ovary and Juvenile type tumours also differ cytologically, compared monitor for possible, though unlikely, metastatic disease. to adult type tumours, and are composed of polygonal to elongated cells with variable, but often abundant, eosinophilic cytoplasm, nongrooved nuclei and relatively Manufacturers’ addresses increased overall atypia. Both juvenile and adult 1Pharmacia & Upjohn, Kalamazoo, Michigan, USA. granulosa cell tumours are reported to have similar 2Abbott Laboratories, North Chicago, Illinois, USA. immunohistochemical staining characteristics, and are 3Tyco Healthcare, Boulder, Colorado, USA. vimentin and inhibin positive, with variable staining of 4Dako, Carpinteria, California, USA.

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