Comp Haematol Int (1996) 6:111-114 ~) 1996 Springer-Veflag London Limited

COMPARATIVE HAEMATOLOGY INTERNATIONAL

Case Report Acute Monocytic Leukaemia (M5a) in a Horse K. S. Latimer 1 and S. L. White 2 1Department of Veterinary Pathology and ZDepartment of Large Animal Medicine, College of Veterinary Medicine, The University of Georgia, Athens, Georgia, USA

Abstract. Acute monocytic leukaemia (M5a) was diagnosed in a 17-year-old Standardbred gelding with lethargy, intermittent pyrexia, oedema of the limbs, harsh lung sounds and submandibular lymphadeopathy. Haematological findings included moderately severe anaemia, thrombocytopenia and a leucocyte count within the reference interval, but characterised by neutropenia and numerous blast cells. Monocytic lineage of the cell population was suggested by examination of Wright-Leishman-stained blood and bone marrow smears. A panel of cytochemical stains disclosed diffuse cytoplasmic c~-naphthyl-acetate esterase activity which could be markedly inhibited or abolished in all leukaemic cells by pretreatment with sodium fluoride. In ultrastructural preparations of bully coat, neoplastic monoblasts had one to two nucleoli, dispersed chromatin, elongated mitochondria, scattered profiles of rough endoplasmic reticulum, bundles of microfilaments and pseudopodia. More differentiated monocytoid cells had infrequent lysosomal granules. Keywords: Alpha naphthyl acetate esterase; Cytochemistry; Horse; Monocyte; Monocytic leukaemia; Ultrastructure

Introduction Non-lymphoid leukaemia and myeloproliferative diseases are reported infrequently in horses. Of these diseases, myelomonocytic leukaemia is the most commonly observed form of haematologic neoplasia in Correspondence and offprint requests to: K. S. Latimer, Department of Veterinary Pathology, College of Veterinary Medicine, The University of Georgia, Athens, GA 30602-7388, USA.

horses (Boudreaux et al. 1984; Spier et al. 1986; Blue et al. 1987; Mori et al. 1991; Buechner-Maxwell et al. 1994). The remaining types of non-lymphoid haematologic neoplasia reported include chronic granulocytic leukaemia (Searcy and Orr 1981), eosinophilic leukaemia (Lewis and Leitch 1975), eosinophilic myeloproliferative disease (Morris et al. 1984), myelomonocytic myeloproliferative disease (Brumbaugh et al. 1982), and monocytic leukaemia (Burkhardt et al. 1984; Straub et al. 1987). The purpose of this report is to describe an additional instance of monocytic leukaemia in a horse.

Case History A 17-year-old Standardbred gelding was referred to The University of Georgia Veterinary Medical Teaching Hospital with a history of lethargy, pyrexia (103.5 °F to 104°F (39.7-40 °C)), and oedema of the limbs. Previous haematological studies revealed anaemia, neutropenia and lymphopenia of undocumented severity. A Coggins test, taken within the past 12 months, was negative. The horse had continued to eat and drink during his illness. Physical examination revealed a temperature of 99.8°F (37.7°C), pulse of 60 beats/min, and respiratory rate of 30 breaths/min. The horse appeared in good body condition. Mucous membranes were pink and the capillary refill time was 2 s. Auscultation revealed slightly harsh lung sounds that were more noticeable over the ventral and midthoracic areas, especially the caudal dorsal left lung lobe. The submandibular lymph nodes were greatly enlarged. Thoracic radiographs disclosed a bronchointerstitial pattern that was more prominent in the anterior ventral lung fields. Mild pulmonary vascular hypertrophy was observed in the caudal dorsal lung fields. A trans-

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tracheal wash contained ciliated columnar epithelial cells, macrophages, fungal debris (contaminants), and mucus that occasionally was arranged in Curschmann's spirals. The cytological diagnosis was mild chronic catarrhal inflammation. Rectal palpation was unremarkable. Blood samples were obtained for a complete blood cell count and biochemical profile. The haematological data (Table 1) revealed moderate anaemia, thrombocytopenia and a leucocyte count that was within the reference interval but characterized by neutropenia and many blast cells (Fig. 1). The blasts varied in size from approximately 12.5 to 20/~m in diameter. Nuclei were eccentrically placed and usually round, oval or slightly irregular. The chromatin pattern was moderately grandular with one to two nucleoli or nucleolar rings. The cytoplasm was blue-grey and cytoplasmic granules were not observed. A few neoplastic cells had indented nuclei

K . S . Latimer and S. L. White

more typical of monocytes (Fig. 2). Pseudopodia and cytoplasmic vacuoles were rare and only observed in more differentiated monocytoid cells. Scattered effete neoplastic cells and mitoses were present within the smear. The appearance of the cells in the WrightLeishman-stained blood smear was suggestive of monocytic or myelomonocytic leukaemia. Mild hyperfibrinogenaemia (700 mg/dl) was also present. The major abnormality in the biochemical profile was hyperproteinaemia (9.4 g/dl). Serum protein electrophoresis characterized the hyperglobulinaemia as a polyclonal gammopathy (Table 2) with beta-gamma bridging. Biochemical changes suggesting major organ system dysfunction were not observed. Sternal bone marrow aspiration biopsy was performed to evaluate the anaemia, thrombocytopenia and abnormal leucocyte population. Marrow particles were hypercellular, but megakaryocytes were not observed. Almost all of the nucleated cells had a similar cytological appearance to the blasts in the peripheral blood. Mitotic figures were numerous. Too few erythroid cells Table 1. Initial haematological findings in a horse with acute monocytic leukemia

Fig. 1. Photomicrograph of a blood smear from a horse with acute monocytic leukaemia (M5a). Monoblasts and promonocytes are present. Wright-Leishman stain, bar = 15/~m.

Parameter

Value

Reference interval

Haematocrit (1/1) R B C ( x 1012/l) H b (g/l) M C V (fl) M C H (pg) M C H C (g/l) Platelets (× 109/1) M P V (fl) Fibrinogen (g/l) W B C ( x 109/1) Neutrophils ( x 109/1) Bands ( × 109/1) Lymphocytes ( x 109/1) Monocytes ( x 109/1) Eosinophils ( x 109/1) Basophils ( x 109/1) Blasts ( x 109/1)

0.26 5.44 104 47.9 19.1 398 71 5.2 7 11.800 0.118 0.000 4.602 0.118 0.000 0.000 6.962

0.27- 0.43 6.0 - 10.43 101 -161 37 - 49 13.7 - 18.2 353 -393 117 -256 4.0 - 6.0 1 - 4 5.6 - 12.1 2.9 - 8.5 0.0 - 0.1 1.16- 5.1 0.0 - 0.7 0.0 - 0.78 0.0 - 0.3 0.0 - 0.0

Table 2. Selected biochemical data from a horse with acute monocytic leukaemia

Fig. 2. Photomicrograph of a blood smear f r o m a horse with monocytic leukaemia (M5a). A differentiated monocyte (left) and monoblast (right) are present. Wright-Leishman stain, bar = 15/tm.

Parameter

Value

Reference interval

Total protein, serum (g/l) Albumin (g/l) Globulin, total (g/l) A l p h a 1 and 2 (g/l) Beta 1 (g/l) Beta 2 (g/l) G a m m a (g/l) A / G ratio

94 26 68 128 5.8 11.1 33.8 038

56-76 26--41 26--40 2-14 4-16 3-9 6-19

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Monocytic Leukaemia in a Horse

were present for detailed cytological evaluation. The diagnosis was haematological malignancy of undetermined cell lineage. Anaemia was attributed to myelophthisis. Additional blood specimens were obtained in heparin for cytochemical staining and ultrastructural examination. Acid ol-naphthyl-acetate esterase staining was performed by published methods (Yam et al. 1971). The remaining cytochemical techniques were performed using commerical kits and following the manufacturers' guidelines (Sigma Chemical Co., St Louis, MO, USA). The blast cell population was strongly positive for cytoplasmic ol-naphthyl-acetate esterase activity, which was inhibited by pretreatment with sodium fluoride. Neither peroxidase nor chloroacetate esterase reactivity was observed. The neoplastic cells also failed to stain following the application of Sudan black B dye. Weak cytoplasmic alkaline phosphatase activity was observed in only a few blast cells. The cytochemical staining pattern was most suggestive of monocyte origin. Transmission electron microscopic examination of thin sections of the buffy coat was performed to further characterize the blast cells. Many cells had round to oval nuclei with irregular indentations or cerebriform nuclear margins (Fig. 3). The chromatin was dispersed, revealing one to two nucleoli. The cytoplasm contained bundles of microfilaments, scattered elongated mitochondria, and occasional profiles of rough endoplasmic reticulum (Fig. 4). The blast cells were devoid of cytoplasmic granules; however, more differentiated monocytoid cells had rare lysosomes. Cytoplasmic projections, typical of pseudopodia, were irregularly spaced around the plasma membrane of some blast cells and more differentiated monocytoid cells. The ultrastructural findings in the leukaemic cells of this horse were similar to those described for monocytic leukaemia in humans and dogs (Glick and Horn 1974; Latimer and Dykstra 1984). On the basis of cytochemi-

Fig. 3. Transmission electron micrograph of a monoblast showing nucleolus, mitochondria, profiles of rough endoplasmic reticulum, rare lysosomes, and pseudopodia. Uranyl acetate and lead citrate stain, bar = 3/~m.

Fig. 4. Transmission electron micrograph of a monoblast showing cytoplasmic details including microfilament bundles, mitochondria, and profilels of rough endoplasmic reticulum. Uranyl acetate and lead citrate staifi, bar = 1.5/~m.

cal staining and ultrastructural examination, a diagnosis of acute monocytic leukaemia was made. Classification of this leukaemia by conventional French-AmericanBritish (FAB) criteria (Jain et al. 1991) resulted in a diagnosis of acute monocytic leukaemia (M5a). Following empirical treatment for presumed infection, the horse was discharged to the owners with a poor prognosis. The horse deteriorated rapidly and was euthanatized within two weeks. Necropsy examination was not allowed.

Discussion The horse of this report was older than previously reported horses with non-lymphoid leukaemia. Other horses with non-lymphoid leukaemia and myeloproliferative disease have ranged in age from 10 months to 16 years, with mean and median ages of 5.8 and 5.0 years, respectively. The two horses previously described with monocytic leukaemia were six and 16 years of age (Burkhardt et al. 1984; Straub et al. 1987). Clinical signs and physical findings in the horse of this report were non-specific and included lethargy, pyrexia, oedema of the limbs, lymphadenopathy and harsh lung sounds. Despite the presence of anaemia, pallor of the mucous membranes was not noticed on initial examination. Clinical signs and physical findings of leukaemia or myeloprofiferative disorders in horses, in descending order of frequency, include pallor of mucous membranes; dependent oedema of the ventrum, limbs; or genitaha; epistaxis, petechiae, or haemorrhage from injection sites; weight loss; dyspnoea or increased lung sounds; lymphadenopathy; depression; fever; weakness; anorexia; and ulcerations of the skin and mucous membranes (Lewis and Leitch 1975; Searcy and Orr 1981; Brumbangh et al. 1982; Boudreaux et al. 1984; Burkhardt et al. 1984; Morris et al. 1984; Spier et al.

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1986; Blue et al. 1987; Straub et al. 1987; Mori et al. 1991; Buechner-Maxwell et al. 1994). Because these signs and findings are non-specific, definitive diagnosis requires complete haematological examination, including examination of blood and bone marrow smears. If the cell lineage is well differentiated, a diagnosis may be made following examination of Romanowskystained blood and bone marrow smears. In contrast, diagnostic difficulties often are experienced if the cell line is poorly differentiated. In such instances, cytochemistry and ultrastructural examination may disclose the identity of the leukaemic cell population (Latimer, 1991; Jain 1986). Ultrastructure and cytochemical staining results in the horse of this report and that reported by Burkhardt et al. (1984) are characteristic of monocytic leukaemia. In contrast, the diagnosis of monocytic leukaemia in the horse described by Straub et al. (1987) is subject to speculation. Specifically, the acetate esterase activity of the leukaemic cells was equivocal and sodium fluoride inhibition was not performed to distinguish monocytes from T-lymphocytes in the few cells that had enzyme activity. Therefore, the cytochemical staining reactions, as presented by Straub et al. (1987), suggest that the blast cell lineage was either lymphocytic or monocytic, but not granulocytic. Common haematological findings in leukaemia include anaemia and thrombocytopenia, secondary to myelophthisis. The total leucocyte count may be elevated, decreased, or within the reference interval; however, blast cells or morphologically abnormal leucocytes may be observed in stained blood films. Neutropenia, if present, predisposes the patient to infection. Hyperfibrinogenaemia is also relatively common and may be related to tissue and organ infiltration or secondary infection. The biochemical profile is most valuable in documenting organ system dysfunction secondary to infiltrative disease, complicating infections, or leukostasis with tissue hypoxia/anoxia (Searcy and Orr 1981; Brumbaugh et al. 1982; Blue et al. 1987; Spier et al. 1987; Mori et al. 1991; Buechner-Maxwell, 1994). Hyperproteinaemia is an indicator of infection and presents as hyperglobulinaemia with a polyclonal gammopathy (Spier et al. 1986; Blue et al. 1987; Mori et al. 1991). This finding is associated with both bacterial (Spier et al. 1986; Mori et al. 1991) or fungal infection (Blue et al. 1987). The intermittent pyrexia, neutropenia and polyclonal gammopathy in the horse of this report suggest occult infection. Too few instances of equine non-lymphocytic leukaemia have been diagnosed to offer a rational treatment regimen. Low-dose cytosine arabinoside was given for 21 days to one horse with acute myelomonocytic leukaemia. This non-cytotoxic treatment was attempted to

K. S. Latimer and S. L. White

promote blast cell maturation; however, treatment did not affect progression of the disease (Spier et al. 1986). In another horse with subleukaemic myelomonocytic leukaemia, combined treatment with cytosine arabinoside and prednisolone also was ineffective (BuechnerMaxwell et al. 1994). Although chemotherapy protocols for other species probably can be adapted for the horse, treatment of non-lymphoid leukaemia in the horse is probably not economically feasible except under rare circumstances. Acknowledgements.We thank Dr Jimmy C. Nash, 200 James Road, Alpharetta, Georgia 30201, USA, for the referral of this horse.

References Blue JT, Perdrizet J, Brown E (1987) Pulmonary aspergillosis in a horse with myelomonocytic leukemia. J Am Vet Med Assoc 190:1562-1564 Boudreaux MK, Blue JT, Durham SK et al. (1984) Intravascular leukostasis in a horse with myelomonocytic leukemia. Vet Pathol 21:544-546 Brumbaugh GW, Stitzel KA, Zinkl JG et al. (1982) Myelomonocytic myeloproliferative disease in a horse. J Am Vet Med Assoc 180:313-316 Buechner-Maxwell V, Zhang C, Robertson J e t al. (1994) Intravascular leukostasis and systemic aspergiUosis in a horse with subleukemic acute myelomonocytic leukemia. J Vet Intern Med 8:258--263 Burkhardt E, v Saldern F, Huskamp B (1984) Monocytic leukemia in a horse. Vet Pathol 21:394--398 Glick AD, Horn RG (1974) Identification of promonocytes and monocytoid precursors in acute leukemia of adults: Ultrastructure and cytochemical observations. Br J Haematol 26:395-403 Jain NC (ed) (1986) Schalm's veterinary hematology, 4th edn. Lea and Febiger, Philadelphia, pp 772, 890-895,909-939 Jain NC, Blue JT, Grindem CB et al. (1991) Proposed criteria for classification of acute myeloid leukemia in dogs and cats. Vet Clin Pathol 20:63-82 Latimer KS (1991) Leukocytic hematopoiesis and Diseases affecting leukocytes. In: Colahan PT, Mayhew IG, Merritt AM, Moore JN (eds) Equine medicine and surgery, 4th edn. American Veterinary Publications, Goleta, pp 1782-1783, 1815--1819 Latimer KS, Dykstra MJ (1984) Acute monocytic leukemia in a dog. J Am Vet Med Assoc 184:852-855 Lewis HB, Leitch M (1975) A case of granulocytic leukemia in the horse. Proc Int Symp Equine Hematol, Am Assoc Equine Pract 1:141-143 Moil T, Ishida T, Washizu T et aL (1991) Acute myelomonocytic leukaemia in a horse. Vet Patho128:344-346 Morris DD, Bloom JC, Roby KAW et al. (1984) Eosinophilic myeloproliferative disorder in a horse. J Am Vet Med Assoc 185:993-996 Searcy GP, Orr JP (1981) Chronic granulocytic leukemia in a horse. Can Vet J 22:148-151 Spier SJ, Madewell BR, Zinkl JG et al. (1986) Acute myelomonocytic leukemia in a horse. J Am Vet Med Assoc 188:861-863 Straub R, Tschidu P, Gerber H (1987) Akute monozyt~re Leukhmie beim Pferd. Pferdeheilkunde 3:251-253 Yam LT, Li CY, Crosby WH (1971) Cytochemical Identification of monocytes and granulocytes. Am J Clin Pathol 55:283-290