Diagnosis and Treatment of a Primary Splenic Lymphoma in a Mule M

606 EQUINE VETERINARY EDUCATION / AE / DECEMBER 2011

Case Reporteve_250 606..611 Diagnosis and treatment of a primary splenic lymphoma in a mule M. S. Madron, S. S. Caston*, E. L. Reinertson, A. K. Tracey and J. M. Hostetter† Departments of Veterinary Clinical Sciences; and †Veterinary Pathology, College of Veterinary Medicine, Iowa State University, Iowa, USA.

Keywords: horse; lymphoma; spleen; transthoracic; mule; haemoabdomen

Summary multicentric form is the most common with involvement extending to lymph nodes and lymphoid organs with a The case reported here describes a transthoracic approach high metastatic potential for the abdominal and thoracic for removal of a splenic lymphoma from a 5-year-old mule. organs. Solitary tumours have been reported to occur at Nine months prior to presentation at Iowa State University extranodal locations but are less common than the other College of Veterinary Medicine (ISUCVM), abdominal forms (Tanimoto et al. 1994; Rhind and Dixon 1999; ultrasound was performed as part of a work-up to Montgomery et al. 2009; Gerard et al. 2010). investigate the cause of lethargy, anorexia and anaemia. Grade of lymphoma can be classified as low or Ultrasound examination identified a haemoabdomen high based on morphology of neoplastic lymphocytes and multiple splenic masses. Transabdominal biopsies (Kelley and Mahaffey 1998). A low grade malignancy is were collected for histopathological evaluation and associated with small mature type lymphocytes. A high lymphoma diagnosed. Based on a number of clinical and grade malignancy is associated with large blast type laboratory examinations there was a lack of support lymphoblasts with abundant cytoplasm and large nuclei for multicentric involvement and all results suggested with prominent nucleoli. The most significant and widely solitary splenic involvement. Surgical removal of the spleen reported classification of lymphoma is based on the was accomplished via a transthoracic approach without phenotype of the lymphoid cell type from which the complications. Histopathology of the splenic masses neoplasm was derived. The 2 forms are B cell and T cell revealed a B cell lymphoma. Bone marrow was collected lymphoma. In addition, a T cell rich B cell lymphoma can during surgery and submitted for cytology and was found to occur in horses where the infiltrating cells are reactive T contain a normal lymphoid population with no evidence of cells and B cells are the neoplastic cell population (Kelley neoplastic cells. The mule was discharged from the hospital and Mahaffey 1998). 11 days after surgery. History and case details Introduction

A 5-year-old mule weighing 473 kg presented to ISUCVM for Lymphoma accounts for 0.2–3.0% of all equine neoplasms surgical evaluation of multiple splenic masses. The masses and is the most common neoplasm of the equine had been discovered 9 months prior to presentation. An haematopoietic system (Jacobs et al. 2002; Taintor and abdominal ultrasound to investigate the cause of lethargy, Schleis 2011). Equine lymphoma as a disease is not age, anorexia and anaemia identified a haemoabdomen gender or breed discriminate (Haley and Spraker 1983; van associated with multiple splenic masses. Transabdominal den Hoven and Franken 1983). The clinical presentation, biopsies were collected for histopathology and the results laboratory data, progression of the disease and were not definitive but lymphoma was highly suspected. histopathology findings can be quite different from patient Medical management with dexamethasone (0.05 mg/kg to patient (Meyer et al. 2006). The most commonly bwt per os every other day) was initiated at this time. recognised forms of equine lymphoma have been divided Management of the haemoabdomen was conservative into 4 anatomical forms: multicentric, alimentary, and no treatments were administered as the anaemia mediastinal and cutaneous (Carlson 1996). The never became severe enough to require a transfusion. *Corresponding author email: [email protected] Serial blood work, including complete blood counts (CBC)

and serum chemistry panels remained within normal limits, Anaesthesia was maintained with isoflurane in oxygen in a with the exception of persistent anaemia, in the 9 months closed-circle system. preceding presentation to ISUCVM. Transabdominal splenic A transthoracic approach to the spleen utilising a 16th biopsies were repeated 9 months after the initial biopsies rib resection was chosen (Rigg et al. 1987). Once and splenic B cell lymphoma was diagnosed. Because of anaesthetised, the patient was placed in right lateral continued lethargy and anorexia, together with the lack of recumbency and the surgical site prepared in a sterile evidence for multicentric involvement, the owner elected environment, draped and an incise drape (Ioban)1 to pursue a surgical consultation. placed. A 30 cm skin incision centred over the 16th rib was At presentation the mule had a physical examination made at the ventral margin of the epaxial musculature that was within normal limits. There were no signs of malaise and continued to the costochondral junction. The and the mule was in good body condition. The PCV at subcutaneous tissues and muscles were sharply dissected presentation was 27% (reference range [rr] 34–45%) with a to reach the lateral periosteum of the rib. The periosteum total plasma protein of 90 g/l (rr 58–80 g/l). A CBC and of the 16th rib was incised on the centre of the rib from just serum chemistry panel had been performed a few days ventral to the epaxial musculature to the costochondral prior to presentation and these were not repeated. junction. The periosteum was elevated circumferentially Abnormalities on CBC included an anaemia (erythrocytes: from the full length of the exposed rib. Gigli wire was used 4.82 ¥ 106/ml; rr 5.63–12.09 ¥ 106/ml). Haemoglobin to transect the rib as far dorsally as possible. The body of (5.7 pmol/l; rr 6.1–10.6 pmol/l) as well as the mean the rib was pulled laterally and ventrally until it was freed corpuscular volume (57.2 fl; rr 33.5–55.8 fl) were also outside from the costochondral junction. The periosteum of the the reference range. Serum chemistry values included medial side of the rib was incised along with the the following abnormalities; albumin (551 mmol/l; rr endothoracic fascia and costal pleura. Both cranial and 319–536 mmol/l), gamma glutamyl transpeptidase (28 m/l; rr caudal edges of the incised periosteum were sutured to 5–24 m/l) and total bilirubin (6.8 mmol/l; rr 8.55–39.33 mmol/l). the muscular part of the underlying diaphragm using No. 0 Additional examinations were performed to survey for poliglecaprone 25 (Monocryl)2 in a simple continuous multicentric involvement. Peripheral lymph nodes were pattern. This suture line serves the dual purpose of reclosing palpated and found to be within normal limits. A rectal the thoracic cavity as well as facilitating the closure of the examination demonstrated an enlarged spleen and the diaphragm following the splenectomy. The abdomen was caudal margin contained multiple masses of various sizes. accessed by incising through the diaphragm between the No other abnormities were noted on rectal examination. 2 suture margins which exposed the lateral aspect of the Thoracic radiographs and ultrasound evaluations were spleen. A second simple continuous suture line of No. 0 within normal limits with no abnormal lymph nodes or poliglecaprone (Monocryl)2 was used to over sew the nodules. Transabdominal as well as transrectal ultrasound cranial periosteum-diaphragm suture line to ensure revealed multiple splenic masses with a mixed adequate closure of the pleural cavity. The edges of the echogenicity. The masses were present on most areas that incision were packed with saline moistened laparotomy were accessible for transabdominal ultrasound and few sponges and distracted with a Finochietto rib spreader. areas contained normal splenic architecture. No evidence The renosplenic ligament was identified and bluntly of haemoabdomen was noted. At this time a splenectomy dissected exposing the splenic vein and artery. During was recommended based on a lack of evidence dissection to isolate the vessels for ligation the splenic vein supporting multicentric involvement. was inadvertently torn. At this time a blood transfusion was initiated and aminocaproic acid (20 g in 1 l fluid bolus, i.v.) was given. Haemostasis was achieved with 3 ligatures of Surgical details No. 1 polyglactin 910 (Vicryl)2 and a polyamide cable tie3 (188 ¥ 4.8 mm) surrounding both the splenic artery and Prophylactic antimicrobials including gentamicin (6.6 mg/kg vein. The availability of cable ties was a point of bwt i.v. q. 24 h) and procaine penicillin (22,000 iu/kg bwt i.m. preoperative planning as they have been useful in q. 12 h) as well as flunixin meglumine (1.1 mg/kg bwt i.v. previous splenectomies for controlling haemorrhage q. 12 h) were administered 1 h before induction. A normal where visualisation is limited. As it was not possible to healthy molly mule which was a herd mate of the patient was remove the spleen through the original 30 cm incision, the provided by the owner to act as a blood donor. The mule had incision was extended ventrally an additional 15 cm which previously been crossmatched and found to be a suitable necessitated incising the costal arch. The spleen was donor. Seven litres of whole blood were collected from the then exteriorised (Fig 1) and the phrenicosplenic and donor molly 1 h prior to surgery. gastrosplenic ligaments transected. Haemostasis of the The patient was premedicated with xylazine (1 mg/kg gastric vessels was accomplished with an electrosurgical bwt i.v.) and butorphanol (0.05 mg/kg bwt i.v.). Anaesthetic device (LigaSure)4. The abdomen was lavaged with5lof induction was accomplished with guaifensesin (50– sterile saline prior to closure. The abdomen was visually 100 mg/kg bwt, i.v. 5% solution given to effect), ketamine examined and palpated before closure and no (2 mg/kg bwt, i.v.) and diazepam (0.08 mg/kg bwt, i.v.). abnormalities noted.

Fig 2: Incision 11 days after surgery. Fig 1: Image of the spleen after being exteriorised. Multiple raised, necrotic nodules are visible. Gross pathology, histopathology The incision was closed in 4 layers. Three interrupted and cytology circumcostal sutures using 16 gauge stainless steel wire were placed around the 15th and 17th ribs to The spleen was severely and diffusely enlarged. It weighed reapproximate them. The periosteum/diaphragm layer 21 kg and had numerous tan to red nodules that bulged was closed in a simple continuous pattern with No. 1 from the capsular surface (Fig 3). The entire spleen was polyglactin 910 (Vicryl)2. The external abdominal oblique submitted for histopathology. Multiple sections were muscle was closed in a simple continuous pattern using evaluated which had a similar microscopic appearance. No. 0 poliglecaprone 25 (Monocryl)2. The subcutaneous The splenic architecture was nearly obliterated by an tissues were closed in a simple continuous pattern using infiltration of neoplastic lymphocytes arranged in densely No. 2–0 poliglecaprone 25 (Monocryl)2. The skin was packed sheets. Neoplastic lymphocytes had moderate to opposed with surgical staples and sterile gauze 4 ¥ 4 scant amounts of eosinophilic cytoplasm and discrete cell sponges placed on the incision and secured with an margins. Neoplastic cells were large and ranged in size adhesive plastic sheet (Ioban)1. An elastic abdominal from 25–35 microns (Fig 4). Nuclei were round, central and bandage5 was placed over the area for additional with prominent nucleoli. There was moderate to mild support and protection during anaesthetic recovery. In anisocytosis and anisokaryosis. The mitotic rate was this case a secure closure of the thoracic cavity was moderate with 1–3 mitotic figures per high power field. achieved and a chest tube was not placed. Recovery There were numerous large foci of necrosis within the from anaesthesia was assisted with a tail rope and was neoplasm. Immunohistochemical stains were used to uneventful. phenotype the lymphoid neoplasm. There was some variation in the staining characteristics for the different lymphocyte markers. Regions within and adjacent to Post operative care necrosis did not stain well for either the T cell marker (CD3) or the B cell marker (CD79). Part of this may be due to less Antibiotics and flunixin meglumine were continued for 7 than optimal cross reactivity for the human antibodies with days. Additional pain management beyond NSAIDs equine tissue. However, multifocally within the neoplastic included butorphanol (0.1 mg/kg bwt i.m.) as needed lymphocytes stained strongly for the B cell marker and for 2 days following surgery. The occlusive wound dressing intermixed with these neoplastic lymphocytes were fewer was replaced with an absorbent cotton pad at 72 h smaller staining T cells. The interpretation was that this after surgery. The abdominal bandage5 was reset as staining pattern was consistent for a B cell lymphoma. The needed. Hand walking for 10–15 min 4 times a day presence of numerous T cells within the neoplasm is was started on Day 3 after surgery and continued suggestive of a T cell rich B cell lymphoma (Kelley and throughout hospitalisation. The abdominal bandaging was Mahaffey 1998). discontinued on Day 7. There was moderate oedema of A bone marrow aspirate was collected during surgery the ventral abdomen and sheath noted after bandaging from the dorsal aspect of the transected 16th rib. The was discontinued but this resolved in 48 h with hand erythroid series was well represented and was found to walking. No other incisional complications were noted have orderly maturation and morphology. Lymphocytes (Fig 2). were 5% of the 300 cell count which is within the reference

Fig 3: Image of the enlarged spleen with multiple nodules.

Fig 4: Section of spleen stained with haematoxylin and eosin. The splenic architecture is lost to a marked infiltration of neoplastic lymphocytes. Multiple foci of necrosis are present within this neoplastic infiltrate. Inset: immunohistochemistry for the B cell marker CD79 is positive in the neoplastic cell population (arrow). Image taken at 200X. range for adult equids (Harvey 2001). The lymphoid Discussion population exhibited no significant atypia. Plasma cells were present but rare and approximately 1% of a 300 cell The timeline of the case presented here from initial count which is within the normal reference range (Harvey diagnosis to surgery is longer than would be expected for 2001). No neoplastic cells were observed in the bone lymphoma. The decision to initially manage the case marrow aspirate. medically was based upon several considerations. The first

being the likelihood that this was the multicentric form of deterioration occurred. A number of clinical and lymphoma and, as such, carried a poor prognosis even laboratory examinations preformed prior to surgery did not with a splenectomy. A second consideration included the yield any evidence of additional organ involvement inherent risks that accompany a surgery of this type. beyond the spleen. Treatment with dexamethasone was initially selected as The surgical procedure utilised provided adequate glucocorticoids are thought to cause lymphoid tumour cell accessibility to the spleen for dissection and ligation of the death by way of endonuclease activation leading to vasculature. Haemorrhage of the splenic vein occurred destruction of chromatin and loss of tumour cell integrity during dissection but was not unforeseen and was able to (Wielckens et al. 1987). However, other than episodes be controlled. The polyamide cable tie3 worked well for of lethargy and anorexia, the overall health of the mule managing the haemorrhage as it could be manipulated did not deteriorate as was expected. Repeated clinical and secured blindly in the abdomen. Laparoscopic- examinations together with haematology were not assisted splenectomy has been reported and benefits supportive of multiorgan involvement and primary splenic include improved visualisation of the visceral aspect of the lymphoma was suspected. In retrospect, additional tests spleen for dissection and transection of vasculature as well for multiorgan involvement could have been utilised. as the phrenicosplenic and gastrosplenic ligaments Abdominocentesis has been shown to be of diagnostic (Ortved et al. 2008). Considerations during surgery for value as peritoneal fluid from horses with alimentary enlarging the incision included removal of the 17th rib or lymphoma sometimes contain neoplastic lymphocytes closure of the incision after splenic vessel ligation and (Zicker et al. 1990; Taylor et al. 2006). In addition, cytology transection followed by a ventral midline approach to of peripheral lymph nodes could have been used to remove the spleen. However, extending the incision screen for additional organ involvement. allowed for removal of the spleen through a single surgical While the spleen is a fairly common site for infiltration of approach. No incisional complications occurred and the equine lymphomas the literature contains very few reports abdominal bandage5 was effective in preventing all but of lymphoma isolated solely to the spleen. Browning (1986) minimal incisional oedema. reported on a 7-year-old Thoroughbred stallion with a large At the time of this report, the case described here splenic mass discovered during an exploratory celiotomy. appears to be a primary splenic lymphoma. It has been 7 The horse was subjected to euthanasia and no gross or months since surgery and all clinical, laboratory and histopathological findings beyond splenic lymphoma were operative findings support this diagnosis. However, made. Marr et al. (1989) reported on a 5-year-old involvement of additional organs with a lack of clinical Thoroughbred gelding that presented for polydipsia and manifestations at this time remains possible. In this case a polyuria. Lymphoma was suspected following clinical definitive diagnosis will likely be supported by continued and laboratory examinations and necropsy identified health or will be re-evaluated if other clinical signs develop. neoplastic cells confined to the spleen. Paraneoplastic In this particular case, surgical intervention has improved syndrome resulting in pseudohyperparathyroidism was the health and well being of this patient. suspected as cardiovascular and renal mineralisation was present. Another case of primary splenic lymphoma Manufacturers’ addresses involved a 4-year-old Thoroughbred stallion racehorse that was subjected to euthanasia after fracturing a forelimb 13M, St. Paul, Minnesota, USA. (Tanimoto et al. 1994). Routine necropsy revealed a solitary 2Ethicon Inc., Somerville, New Jersey, USA. 3 splenic mass composed of neoplastic lymphocytes. No Nelco Products, Pembroke, Massachusetts, USA. 4LigaSure, Covidien, Mansfield, Massachusetts, USA. other gross abnormalities were identified in other organs 5Professional’s Choice, El Cajon, California, USA. and until the time of fracture the animal had been healthy. In contrast to lymphoma isolated solely to the spleen Authors’ declaration of interests the more commonly recognised classifications of equine lymphoma include multicentric, alimentary, mediastinal No conflicts of interest have been declared. and cutaneous. Multicentric involvement is the most common and involves lymph nodes as well as thoracic References and abdominal organs (van den Hoven and Franken 1983; Meyer et al. 2006). Most horses with multicentric lymphoma Browning, A. (1986) Splenic lymphosarcoma in a stallion associated with are subjected to euthanasia because of poor condition an acute abdominal crisis. Vet. Rec. 119, 178-179. and rapid deterioration weeks to months after diagnosis. Carlson, G. (1996) Lymphosarcoma in horses. In: Large Animal Internal Necropsy often reveals a larger tumour load than originally Medicine, 2nd edn., Ed: P. Smith Bradford, Mosby-Year Book, St. suspected. In the case reported here, multicentric Louis. pp 1242-1244. lymphoma with clinical lesions present in the spleen was a Gerard, M., Pruitt, A. and Thrall, D. (2010) Radiation therapy communication: nasal passage and paranasal sinus lymphoma in a strong differential diagnosis early in the disease. However, pony. Vet. Radiol. Ultrasound 51, 97-101. in the 9 months prior to presentation for consultation with Haley, P.J. and Spraker, T. (1983) Lymphosarcoma in an aborted equine the ISUCVM surgery service, no significant clinical fetus. Vet. Pathol. 20, 647-649.

Harvey, J.W. (2001) Bone marrow examination. In: Atlas of Veterinary Ortved, K.F., Witte, S., Fleming, K., Nash, J., Woolums, A.R. and Peroni, J.F. Hematology, Ed: J.W. Harvey, W.B. Saunders, Philadelphia. pp (2008) Laparoscopic-assisted splenectomy in a horse with 93-123. splenomegaly. Equine vet. Educ. 20, 357-361. van den Hoven, R. and Franken, P. (1983) Clinical aspects of Rhind, S.M. and Dixon, P.M. (1999) T cell-rich B cell lymphosarcoma in the lymphosarcoma in the horse: a clinical report of 16 cases. Equine tongue of a horse. Vet. Rec. 145, 554-555. vet. J. 15, 49-53. Rigg, D.L., Reinertson, E.L. and Buttrick, M.L. (1987) A technique for Jacobs, R., Messick, J. and Valli, V. (2002) Tumors of the hemolymphatic elective splenectomy of equidae using a transthoracic approach. system; Equine. In: Tumors in Domestic Animals, 4th edn., Ed: D.J. Vet. Surg. 16, 389-391. Meuten, Iowa State Press, Ames. pp 157-159. Taintor, J. and Schleis, S. (2011) Equine lymphoma. Equine vet. Educ. 23, Kelley, L.C. and Mahaffey, E.A. (1998) Equine malignant lymphomas: 205-213. morphologic and immunohistochemical classification. Vet. Pathol. Tanimoto, T., Yamasaki, S. and Ohtsuki, Y. (1994) Primary splenic 35, 241-252. lymphoma in a horse. J. vet. med. Sci. 56, 767-769. Marr, C., Love, S. and Pirie, H. (1989) Clinical, ultrasonographic and Taylor, S., Pusterla, N., Vaughan, B., Whitcomb, M. and Wilson, W. (2006) pathological findings in a horse with splenic lymphosarcoma and Intestinal neoplasia in horses. J. vet. int. Med. 20, 1429-1436. pseudohyperparathyroidism. Equine vet. J. 21, 221-226. Wielckens, K., Delfs, T., Muth, A., Freese, V. and Kleeberg, H.J. (1987) Meyer, J., DeLay, J. and Bienzle, D. (2006) Clinical, laboratory, and Glucocorticoid-induced lymphoma cell death: the good and the histopathologic features of equine lymphoma. Vet. Pathol. 43, evil. J. Steroid Biochem. 27, 413-419. 914-924. Zicker, S., Wilson, W. and Medearis, I. (1990) Differentiation between Montgomery, J.B., Duckett, W.M. and Bourque, A.C. (2009) Pelvic intra-abdominal neoplasms and abscesses in horses, using clinical lymphoma as a cause of urethral compression in a mare. Can. vet. and laboratory data: 40 cases (1973–1988). J. Am. vet. med. Ass. J. 50, 751-754. 196, 1130-1134.

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