Diagnosis and Treatment of Chronic T-Lymphocytic Leukemia in a ...

Diagnosis and Treatment of Chronic T-Lymphocytic Leukemia in a Green Tree Monitor (Varanus prasinus) Timothy A. Georoff 1, VMD, Nicole I. Stacy2, DrMedVet, Alisa N. Newton1, VMD, DACVP, Denise McAloose1, VMD, DACVP, Gerald S. Post3, DVM, DACVIM (Oncology), Rose E. Raskin4, DVM, PhD, DACVP 1. Global Health Program, Wildlife Conservation Society, 2300 Southern Boulevard, Bronx, NY 10460, USA Current address for T. A. Georoff: Roger Williams Park Zoo, 1000 Elmwood Avenue, Providence, RI 02907, USA 2. Department of Large Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, FL 32601, USA 3. Veterinary Oncology and Hematology Center, 178 Connecticut Avenue, Norwalk, CT 06854, USA 4. School of Veterinary Medicine, Purdue University, West Lafayette, IN 47907, USA ABSTRACT: A 13-year-old male green tree monitor (Varanus prasinus) presented with lethargy and chronic weight loss. Complete blood count (CBC) revealed marked leukocytosis (total leukocyte count = 278.1 × 103/µl) and peripheral blood films were characterized by marked lymphocytosis comprised of small, welldifferentiated lymphocytes. Bone marrow aspiration revealed increased cellularity with a predominance of well-differentiated lymphocytes. Using immunocytochemistry, neoplastic lymphocytes in peripheral blood films were positive for CD3 epsilon and negative for CD79a and BLA.36, consistent with T-cell chronic lymphocytic leukemia (CLL). Distal tail necrosis was noted 17 days post-presentation and treated with partial caudectomy. Treatment was initiated for CLL with 0.8 mg/kg prednisone q 48 h 22 days post-presentation. CBC on days 94 to 151 post-presentation showed marked reduction in leukocytosis (counts remaining below 40.0 × 103/µL). The animal displayed improvement in activity, appetite, and gained weight during the initial treatment period. Marked leukocytosis (253.4 × 103/µL) returned on day 203 postpresentation, and on day 206 post-presentation the monitor received 0.08 mg/kg chlorambucil PO once in addition to ongoing prednisone administration. The animal died 210 days post-presentation. Histopathologic examination revealed neoplastic lymphocyte infiltration in the bone marrow at multiple sites as well as in liver, spleen, gall bladder, pancreas, kidneys, esophagus, and the oral cavity. Lymphocyte infiltration in multiple organs was primarily associated with areas of dense lymphocytic infiltrates surrounding blood vessels. This report describes the first published attempt of CLL treatment in a saurian species. KEY WORDS: chemotherapy, CLL, leukemia, monitor lizard, neoplasia, Varanus prasinus.

INTRODUCTION Hematopoietic lymphoid tissue neoplasia is the most commonly reported group of tumors in reptiles (Garner et al., 2004). Lymphoid neoplasia with and without leukemia has been reported in the orders Chelonia (Orós et al., 2001; Silverstone et al., 2007), Sauria (Zwart and Harshbarger, 1972; Goldberg and Holshuh, 1991; Suedmeyer and Turk, 1996; Schultze et al., 1999; Hernandez-Divers and Garner, 2003; Gyimesi et al., 2005), and Serpentes (Finnie, 1972; Effron et al., 1977; Catao-Dias and Nichols, 1999; Walker et al., 1999; Lock et al., 2001; Raiti et al., 2002). The occurrence of these tumors was originally reported at 25% of all reptile neoplasms (Effron et al., 1977), but recent studies show a much lower prevalence ranging from 4.6–7.2% (Garner et al., 2004; Sykes and Trupkiewicz, 2006). Reptilian lymphoid neoplasia frequently presents with multicentric organ involvement and blast morphology 106

Journal of Herpetological Medicine and Surgery

(Schultze et al., 1999; Lock et al., 2001; Garner et al., 2004). The majority of these tumors are lymphomas with diffuse visceral involvement (Hernandez-Divers and Garner, 2003; Hernandez-Divers et al., 2003; Garner et al., 2004; Sykes and Trupkiewicz, 2006). Lymphoid leukemia without associated solid tumors appears to be more rare (Garner et al., 2004). Most of the reported cases of lymphoid neoplasia in reptiles have been post-mortem diagnoses, and only limited descriptions of treatment regimes and outcomes exist in the published literature. In most cases, this appears to be caused by the advanced stage of disease at the time of diagnosis. In part because of this, lymphoid neoplasia has been considered historically to carry a poor prognosis in reptiles (Suedmeyer and Turk, 1996). The majority of reptilian oncologic treatments are described in snakes, including two reports of attempted treatment of lymphoid neoplasia (Jacobson et al., 1981; Willette et al., 2001). Very little Volume 19, No. 4, 2009

information exists for chemotherapeutic treatment in saurians. One report describes the successful treatment of an acute lymphoblastic leukemia (ALL) in a sungazer lizard (Cordylus giganteus) (Martin et al., 2003). This case report describes the antemortem diagnosis and first report of attempted chronic lymphocytic leukemia (CLL) treatment in a green tree monitor, Varanus prasinus.

CASE REPORT A 13-year-old captive-born, male green tree monitor from a zoological collection presented with lethargy and chronic weight loss. The animal was housed (alone) indoors in a semiarboreal enclosure with an average temperature range between 23.9°C and 29.4°C (75°F and 85°F) and a relative humidity >70%. The monitor lizard was provided fullspectrum ultraviolet light and access to a basking spot. The monitor was offered a diet of insects and dead pinkie mice. This animal was obtained from another zoo four years prior to presentation and had no previous medical problems during its time in either collection. All other green tree monitors housed at the zoo remained clinically healthy. On initial physical examination, the monitor had pale pink oral mucous membranes and was in thin body condition (261 g). Blood was collected from the ventral coccygeal vein under manual restraint and placed in lithium heparincontaining microcollection tubes (BD Microtainer, Becton, Dickinson and Co., Franklin Lakes, NJ). A complete blood count (CBC) was performed, including hemoglobin, manual white blood cell (WBC) count, packed cell volume (PCV), and WBC differential (based on 100 cells). Hemoglobin was determined using an optical absorption photometric analyzer (HemoPoint H2 Photometer, Stanbio Laboratory, Boerne, TX). The WBC count was performed using a semidirect method with a phloxine B solution (Unopette Test 5877, Becton, Dickinson and Co.) in a hemocytometer (Sykes and Klaphake, 2008). The WBC count obtained from the hemocytometer was compared with a visual smear estimate to ensure WBC count accuracy. Whole body radiographs were obtained, and transcoelomic ultrasonography was performed with a 7.5-megahertz (mHz) transducer probe. The animal was treated supportively at this time, receiving 24 ml/kg 2.5% dextrose/0.45% NaCl (Braun Medical, Inc., Irvine, CA) SC once. Blood work results were compared to reference intervals from the International Species Identification System (2002). Complete blood count revealed a marked leukocytosis (278.1 × 103/µL; reference interval: 9.7 ± 5.9 × 103/µL), characterized morphologicaly as a population consisting of entirely well-differentiated, mature lymphocytes. Significant biochemical abnormalities included hypernatremia (180 mmol/L; reference interval: 154 ± 11 mmol/L), hyperchloremia (132 mmol/L; reference interval: 112 ± 16 mmol/ L), increased aspartate aminotransferase (AST) (72 U/L; reference interval: 26 ± 17), increased creatine phosphokinase (CPK) (5892 U/L; reference interval: 1,303 ± 996 U/ L), increased lactate dehydrogenase (LDH) (735 U/L; reference interval: 296 ± 234 U/L), and hypoalbuminemia (18 g/ L; reference interval: 29 ± 8 g/L). Whole body radiographs and transcoelomic ultrasonography were unremarkable. Because of the significant lymphocytosis and concern for potential lymphoid neoplasia, the animal was anesthetized Volume 19, No. 4, 2009

for bone marrow aspiration and repeat phlebotomy. The monitor lizard was sedated with 0.08 mg/kg medetomidine (Domitor, Pfizer Animal Health, New York, NY) and 0.4 mg/kg butorphanol (Torbugesic, 2 mg/ml, Fort Dodge Laboratories, Fort Dodge, IA) both IM at 29°C (85°F), intubated using a 16-gauge IV-catheter (Abbocath, Abbott Laboratories, Abbott Park, IL) as an endotracheal tube, and administered isoflurane (AErrane, Baxter Healthcare Corporation, Deerfield, IL) to achieve anesthesia. The proximal left tibia was prepared for sterile surgery and a 23-gauge, 2.54-cm hypodermic needle attached to a 1-mL syringe (without anticoagulant) was inserted into the tibial crest for bone marrow aspiration. Marrow samples were smeared as a thin film onto glass slides immediately following sample collection for cytologic examination. Bone marrow was also submitted for aerobic and anaerobic bacterial culture to rule out bacterial septicemia. Additionally, blood was collected from the ventral coccygeal vein as previously described for repeat CBC and serum biochemical analysis. Antagonism of medetomidine and butorphanol was achieved with 0.4 mg/kg atipamezole (Antisedan, Pfizer Animal Health) and 4 mg/kg naloxone (Naloxone hydrochloride, International Medication Systems, Ltd., South El Monte, CA) IM, respectively. The monitor recovered uneventfully from these procedures and treatment was initiated with 20 mg/kg ceftazidime (Fortaz, GlaxoSmithKline, Research Triangle Park, NC) q 72 h IM pending bone marrow culture results and 24 mL/kg reptile Ringer’s solution (2 parts 2.5% dextrose in 0.45% NaCl, 1 part lactated Ringer’s solution) q 72 h SC, both for 11 treatments. Results of the CBC included a total leukocyte count of 171.6 × 103/µL, with a lymphocytosis of 168.2 × 103/µL (Fig. 1). The bone marrow aspirate contained an adequate number of spicules; however, each was hypercellular. Also, although mildly reduced in numbers, the thrombocyte, granulocyte, and erythroid series were complete and showed orderly maturation. A normal amount of polychromasia was seen in the erythroid series. The number of small welldifferentiated lymphocytes was markedly increased (92%). No infectious agents were identified. Aerobic and anaerobic culture of the bone marrow yielded no growth. Based on these findings, a preliminary diagnosis of chronic lymphocytic leukemia (CLL) was made. Flow cytometric immunophenotyping was performed using whole blood anticoagulated with lithium heparin (BD Microtainer). Three populations of leukocytes were observed based on size and granularity. All three populations were tested for cross-reactivity using canine, human, and bovine species-specific monoclonal antibodies to granulocytic, monocytic, and lymphocytic surface antigens including CD172a (granulocytic) (DH59B, VMRD, Pullman, WA), CD14 (monocytic) (CAM36A, VMRD, Pullman, WA), T lymphocyte molecules CD3 (CA17.2A12, AbD Serotec, Raleigh, NC, and MM1A, VMRD), CD4 (CA13.1E4, Leukocyte Antigen Biology Laboratory, University of California-Davis, Davis, CA), CD8 (CA9.JD3 and CA15.4G2, Leukocyte Antigen Biology Laboratory, University of California-Davis), and B lymphocyte molecules CD21 (CA2.1D6, Leukocyte Antigen Biology Laboratory, University of California-Davis), CD79a (HM57, Dako, Carpinteria, CA), and MHC II (H42A, VMRD). None of the antibodies reacted with any of the three populations of blood leukocytes. Immunocytochemical staining of peripheral blood films using human monoclonal antibodies Journal of Herpetological Medicine and Surgery

107

Figure 1. Photomicrograph of a peripheral blood film from a green tree monitor with chronic T-cell lymphocytic leukemia one day after initial presentation. Numerous small well-differentiated lymphocytes are present among mature erythrocytes. Wright-Giemsa stain.

to lymphocytic surface antigens showed lymphocytes were positive for CD3 epsilon (CD3-12 at 1:25, AbD Serotec) expression and negative for CD79a (HM57 at 1:25, Dako) and BLA.36 (A27.42 at 1:25, Novocastra, Newcastle Upon Tyne, UK) expression (Fig. 2). Positive and negative controls for the described antibodies were routinely performed. Results of immunocytochemical staining were consistent with CLL of T-cell origin. On day 17 post-presentation, the monitor developed distal tail necrosis of 1 cm in length. Two days later, this lesion extended 5 cm in length and exhibited prominent dark purple discoloration. The animal was anesthetized the same day (day 19) using 0.2 mg/kg medetomidine, 12.7 mg/kg ketamine (Ketaset, 100 mg/ml, Fort Dodge Animal Health), and 1.3 mg/kg butorphanol all IM at 29°C (85°F) followed by isoflurane via endotracheal tube for surgical partial caudectomy. Histopathologic evaluation of the amputated tail revealed multifocal acute to subacute severe coagulative necrosis with surface-associated and intravascular bacterial colonies. Aerobic and anaerobic bacteria cultures yielded numerous Bacillus sp. and alpha-hemolytic Streptococcus sp. Ceftazidime at the previous dosage was continued postoperatively (through day 38), and 2.5 mg/kg (5 mg/kg loading dose) amikacin q 72 h IM was added for seven treatments. Based on concerns regarding the animal’s healing ability after surgery and the risk of further infection, initial treatment for CLL with prednisone was delayed until after the immediate postoperative period. 108


On day 22, treatment for CLL was initiated with 0.8 mg/ kg prednisone (Qualitest, Qualitest Pharmaceuticals, Huntsville, AL) q 48 h PO metabolically scaled from the 2 mg/kg PO q 24 h canine dosage previously used for saurian chemotherapy (Martin et al., 2003). This was easily administered by keeper staff in a food item (pinkie mouse) throughout the course of treatment. Ceftazidime and amikacin treatment overlapped the first two weeks of prednisone treatment because of the previous surgery as well as concern for infection secondary to immunosuppression on the scaled prednisone dose. The monitor responded well to initial treatment with improvements in appetite and activity level, as well as an increase in body weight from post-surgery (236 g) to a recheck on day 64 (260 g). The CBC results on day 64 revealed marked leukocytosis (183 × 103/µL) with severe lymphocytosis (181.17 × 103/µL). Significant biochemical abnormalities included moderate hyperuricemia (uric acid = 826.8 µmol/L, 13.9 mg/dL; reference interval: 422.3 ± 392.6 µmol/L [7.1 ± 6.6 mg/dL]), raising concern for renal disease or gout. Given the consistent lymphocyte counts of 100.0 × 103/µL, the addition of chlorambucil to the treatment regimen was initially planned. However, a delay in acquiring the drug and the clinical improvement observed during the prednisone treatment lead to a delay in initiating the chlorambucil until the next scheduled blood collection. The CBC on day 94 revealed a marked reduction in the total leukocyte count to 36.96 × 103/µL and consisted entirely of lymphocytes; the Volume 19, No. 4, 2009

Figure 2. Photomicrograph of a peripheral blood film from a green tree monitor demonstrating positive stain reaction with an antibody to CD3 epsilon antigen in approximately 75% of the lymphocytes. Erythrocytes are negative in contrast. Aminoethylcarbazole and hematoxylin stain, × 100 objective.

reduction in the CBC was attributed to the prednisone monotherapy. Hyperuricemia had further increased to 1,183.7 µmol/L (19.9 mg/dL). The other biochemical abnormalities present on the initial plasma chemistry profile had resolved by this time, with the exception of the hypoalbuminemia. The previous tail amputation site was completely healed by this time, and the animal’s weight had increased to 290 g. Based on continued clinical improvement, weight gain, significant reduction in the blood leukocyte/lymphocyte counts, and hyperuricemia, chlorambucil treatment was not pursued at this time. The monitor lizard continued to show good appetite and activity through day 150. Blood was collected from the ventral coccygeal vein at approximate 30-day intervals for repeat CBC examination as previously described. A summary of serial CBC and chemistry panel data is provided in Tables 1 and 2, respectively. The animal’s condition remained stable, with a maximum weight (310 g) achieved on day 182. On day 203, the total leukocyte count had risen to 253.44 × 103/µL, including 248.37 × 103/µL lymphocytes. The monitor remained active and was eating consistently at this time, but its weight decreased to 260 g. Chlorambucil 0.08 mg/kg (Leukeran, GlaxoSmithKline, Research Triangle Park, NC) PO compounded to 0.2 mg/mL using vegetable oil (Pure Wesson 100% Natural Vegetable Oil, ConAgra Foods, Inc., Omaha, NE) was instituted on day 206. This dose was derived from a metabolic scaling of a feline CLL chemotherapy dose (Workman and Vernau, 2003) and intended for q 7 day dosing. The monitor died on day 210. Post-mortem examination revealed severe diffuse hepatomegaly and diffuse splenomegaly. Histopathology revealed Volume 19, No. 4, 2009

infiltration of leukemic lymphocytes in the liver and in areas immediately surrounding blood vessels in the liver, spleen, gall bladder, pancreas, kidneys, esophagus, and oral cavity. The bone marrow of multiple sites (femur, skull, and coccygeal vertebrae) was heavily infiltrated and replaced by a monomorphic population of small lymphocytes (Fig. 3). The presence of significant bone marrow involvement, association of leukemic lymphocyte infiltration predominantly surrounding blood vessels in multiple organs, and extremely high circulating lymphocyte counts are suggestive of bone marrow serving as the primary tumor site in this case. In addition to the leukemia, gross necropsy and histology confirmed renal interstitial fibrosis and renal gout. Additional samples from the liver were saved in glutaraldehyde and prepared for examination with transmission electron microscopy by routine methods. Small lymphocytes predominated; the cells were characterized by having high nuclear to cytoplasmic ratios and scant cytoplasm. No viral particles were identified on examination of the samples.

DISCUSSION Chronic lymphocytic leukemia is a neoplastic clonal proliferation of small, mature-appearing T- or B-lymphocytes manifesting as a persistent and frequently marked peripheral lymphocytosis (Workman and Vernau, 2003). The general distinction between T- and B-cell CLL and corresponding lymphoma of the same cell type in several lymphoid neoplasia classification schemes (Kiel Classification Journal of Herpetological Medicine and Surgery

109

Table 1. Hematologic values for consecutive examinations of a green tree monitor under treatment for chronic lymphocytic leukemia. Previous hematologic data (23Sep04) collected during a routine exam when the animal was clinically normal is provided for comparison. Reference intervals from the International Species Identification System, 2002. Days following initial 23Sep04 presentation: WBC, cells/µL PCV

6,500 38%

Hemoglobin, g/L g/dL Heterophils, % (cells/µL) Lymphocytes, % (cells/µL)

n/a

18Aug08 Day 1

25Aug08 Day 8

278,080

171,600

Monocytes, % (cells/µL)

1% (65)

Azurophilic monocytes, % (cells/µL)

19% (1,235)

183,000

26Jan09 Day 151

23Feb09 Day 179

19Mar09 Day 203

36,960

25,000

79,200

253,440

Reference range 9,717 ± 5,934

23%

19%

19%

26%

22%

22%

30.9 ± 6.7

59 5.9

77 7.7

54 5.4

71 7.1

n/a

75 7.5

77 7.7

90 ± 27 9.0 ± 2.7

8% (2,000)

2% (1,584)

2% (5,069)

3,717 ± 3,313

88% (22,000)

97% (76,824)

98% (248,371)

3,733 ± 3,446

2% (3,432) 100% (278,080)

98% (168,168)

99% (181,170)


100% (36,960)

401 ± 737

1% (1,830)

and World Health Organization) is a circulating lymphocytosis in the absence of a solid tumor (Valli, 2007). The current Revised European/American Lymphoma (REAL) classification scheme recognizes CLL and small cell lymphoma as a continuous disease process (Valli, 2007). In humans, CLL is almost always a proliferation of B lymphocytes, whereas in dogs and cats T-cell CLL is more commonly seen (Vernau and Moore, 1999; Workman and Vernau, 2003). In reptile lymphoid neoplasia cases where cell-type characterization has been pursued, the majority of neoplasias have been of T-cell lineage (Wheeler et al., 2000; Raiti et al., 2002; Hernandez-Divers et al., 2003). Leukemia cell-type characterization was pursued in this case using both flow cytometry and immunocytochemistry. Monoclonal antibodies for determination of cell-lineage used in both flow cytometry and immunocytochemistry typically need to be species specific for reactivity, but crossreactivity is possible. Cross-reactivity did not occur with the antibodies used for flow cytometry in this case. However, CD3 expression was noted in this case with immunocytochemistry using a human monoclonal antibody toward the epsilon chain of the CD3 receptor, which is different from the CD3 canine (CA17.2A12) and bovine (MM1A) antibodies used for flow cytometry. Antibody directed against CD3 epsilon has been found to be more universal as a T-cell marker for a wider variety of species, including several reptile species (Rast et al., 1995; Raiti et al., 2002). The B-cell marker CD79a has not been shown to label reptile B-cells and may not be expressed in reptiles (Gyimesi et al., 2005). The antibodies used for immunocytochemistry in this case were the same as those used by Silverstone et al. (2007) for a diamondback terrapin (Malaclemys terrapin) in which BLA.36 staining was positive for the B-cell neoplastic cells, whereas another B-cell marker, CD79a, was not positive. Sample handling and technical difficulties as well as species 110

19Nov08 Day 94

28%

66% (4,290) 14% (910)

20Oct08 Day 64

4% (1,000)

1% (792)

2,335 ± 1,973

differences can impact negative results; thus, it is best to interpret positive results rather than negative results in this case. It is suspected that the overwhelming numbers of neoplastic cells in this case crowded out many of the normal cells, leaving too few normal cells for comparison purposes. If possible, validation using lymphoid tissue is recommended; unfortunately, these tissues are often difficult to obtain and mammalian positive control tissues are used instead. The control samples used in this case were positive and verified that the antibodies were functional. Traditionally, CLL is thought to originate in the bone marrow in people and dogs (Dameshek, 1967; Vernau and Moore, 1999; Chiorazzi et al., 2005). This is still thought to be the case with B-cell CLL in dogs; however, canine large granular cell (LGL) T-cell CLL is now thought to be a disease of splenic origin with bone marrow involvement occurring with advanced disease progression (Vernau and Moore, 1999). It is unknown whether the T-cell CLL in this monitor followed a similar progression to canine LGL T-cell CLL. Morphologic distribution of lesions in this case was suggestive of bone marrow as the primary organ. In dogs and cats, CLL typically presents in middle-aged or older animals, with animals generally not exhibiting clinical signs at the time of diagnosis (Workman and Vernau, 2003). The disease is slowly progressive, and clinical signs are related to the degree of bone marrow and additional organ involvement. Animals most commonly exhibit lethargy, hypophagia, and progressive weight loss (Workman and Vernau, 2003) similar to the signs initially observed in the monitor lizard in this report. Diagnosis is made based on CBC and cytology or histology of bone marrow or other affected organs. The primary finding on CBC with CLL is a leukocytosis with absolute lymphocytosis. In this monitor, it is unknown how long a peripheral lymphocytosis was present prior to the development of the animal’s clinical signs and CLL diagnosis. Volume 19, No. 4, 2009

Table 2. Plasma chemistry panels for consecutive examinations of a green tree monitor under treatment for chronic lymphocytic leukemia. Previous plasma chemistry data (23Sep04) collected during a routine exam when the animal was clinically normal is provided for comparison. Reference intervals from the International Species Identification System, 2002. Days following initial presentation: Glucose, mmol/L mg/dL Blood urea nitrogen, mmol/L mg/dL

23Sep04

18Aug08 Day 1

25Aug08 Day 8

20Oct08 Day 64

19Nov08 Day 94

26Jan09 Day 151

23Feb09 Day 179

Reference range

9.84 179

7.42 135

0.88 16

5.99 109

6.43 117

5.39 98

6.98 ± 2.75 127 ± 50

0.71 2

1.07 3

2.14 6

2.14 6

1.07 3

0.35 1

1.07 ± 0.71 3±2

0.0 0.0

17.68 0.2

26.52 0.3

17.68 0.2

17.68 0.2

26.52 ± 17.68 0.3 ± 0.2

Creatinine, mmol/L mg/dL Uric acid, mmol/L mg/dL

731.6 12.3

660.2 11.1

452.0 7.6

826.8 13.9

1183.7 19.9

Total protein, g/L g/dL

97 9.7

75 7.5

54 5.4

53 5.3

64 6.4

66 66

Albumin, g/L

39

18

16

18

19

20

g/dL

19Mar09 Day 203

1522.7 25.6

1005.2 16.9

3307.1 55.6

422.3 ± 392.6 7.1 ± 6.6 74 ± 24 7.4 ± 2.4 29 ± 8

3.9

1.8

1.6

1.8

1.9

2.0

2.9 ± 0.8

58 5.8

57 5.7

38 3.8

35 3.5

45 4.5

46 4.6

50 ± 14 5.0 ± 1.4

264

84

88

38

47

65

169 ± 81

Alanine aminotransferase (ALT), U/L

17

3

6

11

9

4

10 ± 3

Aspartate aminotransferase (AST), U/L

45

72

372

80

38

48

26 ± 17

Lactate dehydrogenase (LDH), U/L

228

735

1,582

259

211

266

296 ± 234

Creatine phosphokinase (CPK), U/L

446

5,892

3480

1,198

718

1,595

1,303 ± 996

Cholesterol, mmol/L mg/dL

2.93 113

2.57 99

2.70 104

2.26 87

2.39 92

0.96 37

8.68 ± 4.31 334 ± 166

3.70 14.8

2.62 10.5

2.50 10.0

2.37 9.5

2.70 10.8

2.42 9.7

2.92 ± 0.52 11.7 ± 2.1

1.97 6.1

2.32 7.2

2.00 6.2

2.19 6.8

1.67 5.2

1.55 4.8

1.71 ± 1.38 5.3 ± 4.3

Globulins, g/L g/dL Alkaline phosphatase (ALP), U/L

Calcium, mmol/L mg/dL Phosphorus, mmol/L mg/dL Sodium, mmol/L Potassium, mmol/L Chloride, mmol/L

172 4.5 124

180 5.1 132

150 5.2 112

Additional biochemical abnormalities at presentation in the monitor lizard included hypernatermia, hyperchloremia, increased AST, CPK, and LDH, and hypoalbuminemia. Hypernatremia and hyperchloremia likely were caused by dehydration from excessive water loss or reduced water intake. Underlying renal disease could be considered a possible underlying cause as hyperuricemia developed over the treatment course, and chronic renal disease was Volume 19, No. 4, 2009

137 4 103

147 4.2 111

156 4.6 119

154 ± 11 4.1 ± 0.8 112 ± 16

identified at necropsy. Sodium and chloride values normalized by the day 8 recheck following fluid therapy and supportive care. Plasma AST and LDH are considered to have wide tissue activities, and increases are not highly specific to a particular tissue in reptiles, although a concurrent elevation in CPK suggests that the changes were related to increased muscle catabolism. Hepatopathy caused by neoplastic infiltration should also be considered even though Journal of Herpetological Medicine and Surgery

111

Figure 3. Photomicrograph of a section of bone marrow from a green tree monitor with chronic T-cell lymphocytic leukemia showing heavy infiltration with a predominantly monomorphic population of small lymphocytes. Hematoxylin and eosin.

hepatomegaly was not identified at presentation. The hypoalbuminemia noted may have been caused by renal disease or hepatopathy based on post-mortem findings. Chronic lymphocytic leukemia is currently considered a treatable but incurable disease in veterinary medicine. Because of the slow progressive nature of CLL, chemotherapeutic treatment is frequently not recommended during the initial phase of disease when patients are asymptomatic but may become necessary with disease progression (Workman and Vernau, 2003). The general goal of CLL therapy is not to eliminate all abnormal cell types but rather to manage the disease. Some of the criteria used for initiation of treatment in humans and animals include marrow failure (anemia, thrombocytopenia), weight loss >10% body weight within six months, splenomegaly, and persistent lymphocytosis (guidelines varying between 60,000 and 100,000 lymphocytes/µL) (Workman and Vernau, 2003; Ahmadi et al., 2009). Survival times in dogs with CLL with or without treatment average one to two years (Workman and Vernau, 2003). Treatment was pursued in the monitor’s case because of the development of clinical signs including weight loss, distal tail necrosis, and persistent lymphocytosis >160,000 cells/µL. 112


Chlorambucil is currently considered one of the most effective drugs for CLL treatment in veterinary medicine (Workman and Vernau, 2003). The most common documented side effects of chlorambucil in humans and animals are myelosuppression and gastrointestinal toxicity, but additional adverse effects including increases in serum uric acid and uric acid nephropathy secondary to tumor lysis syndrome have also been reported (Fitzgerald et al., 1981; Plumb, 2005). Prednisone causes lymphocytolysis and is frequently used as adjunctive therapy to chlorambucil. The combination of chlorambucil and prednisone in the treatment of CLL may be superior to chlorambucil monotherapy (Leifer and Matus, 1986). The monitor was initially started on prednisone prior to acquiring the chlorambucil, and the animal showed a positive clinical response to this initial therapy by day 64. Additionally, all biochemical abnormalities with the exception of hypoalbuminemia normalized by the day 94 recheck. Because of the development of distal tail necrosis on day 17, subsequent surgery, and concern for additional tail necrosis with repeated coccygeal venipuncture during the postoperative period, a CBC was not rechecked immediately prior to the initiation of prednisone (on day 22), obscuring Volume 19, No. 4, 2009

interpretation of the direct affects of prednisone on lymphocyte counts. The lymphocyte count on day 64 remained relatively unchanged from the previous day 8 sample, even though weight gain and increased activity were observed by that time. Lymphocyte counts did eventually decrease on days 94 and 151 relative to the initial three CBCs; however, it is unknown whether the lymphocyte count had changed significantly between day 8 and day 64. Thus, changes in the lymphocyte count mirroring the animal’s clinical change cannot be ruled out. Alternatively, the results may indicate a slower onset of drug action and longer time to reduction of lymphocyte counts relative to clinical changes in reptiles. It is also possible that prednisone monotherapy has a limited effect on lymphocyte counts in the treatment of saurian leukemia. Prednisone when used alone for treatment of ALL in a sungazer lizard appeared to have minimal effect on reducing total leukocyte counts (Martin et al., 2003). In this case, the elevated plasma uric acid noted at days 64 and 94 post-presentation, the potential for tumor lysis syndrome, and the lack of dosing information for chlorambucil in saurians created concern that the addition of chlorambucil could worsen pre-existing renal disease or precipitate renal gout. Increases in uric acid, blood urea nitrogen (BUN), AST, and CPK thought to be caused by tumor lysis were seen in a diamondback terrapin undergoing combination chemotherapy for ALL with chlorambucil, cytosine arabinoside, and prednisone (Silverstone et al., 2007). The authors of this report attributed toxemia resulting from tumor necrosis as a possible contributing factor in this animal’s death 46 days following initiation of chemotherapy. These factors, coupled with the good response to the metabolically scaled prednisone, led to withholding the chlorambucil for most of the monitor lizard’s treatment. Chlorambucil was later administered following a documented significant increase in plasma uric acid (3,307.1 µmol/L, 55.6 mg/dL) when the animal’s condition appeared to decline again. Treatment with allopurinol was also planned following the assessment of the lizard’s response to the chlorambucil; however, the monitor died prior to adding this treatment. Histopathology did not show evidence of apparent complications caused by chlorambucil toxicity, such as sepsis secondary to myelosuppression or gastrointestinal toxicity. However, histopathology did show evidence of interstitial renal fibrosis consistent with chronic renal disease. It is unknown whether initiation of chlorambucil earlier in the treatment plan would have led to a prolonged survival time; thus, its use in this case should be interpreted conservatively. The development of distal tail necrosis was an unusual development in the case. Histopathologic lesions on the tail biopsy were not specific as to a causative etiology. Both venipuncture and hypercoagulability secondary to leukostasis, independently or combined, are considered likely potential causes of the tail necrosis by the authors. Distal tail necrosis developed within two weeks of the second (day 8) blood sample. All coccygeal venipuncture samples obtained from the monitor were obtained without significant trauma to the tail, and no additional tail lesions developed subsequent to venipuncture following the partial caudectomy and initiation of prednisone treatment. Hyperleukocytosis causing leukostasis and hypercoagulability is a well-described phenomena with myeloproliferative neoplasia in humans but rarely associated with CLL. There is Volume 19, No. 4, 2009

one report of major vessel thrombosis caused by stasis from abnormal accumulation of lymphocytes seen with CLL in a person (Cukierman et al., 2002). External trauma or bite wound to the tail are also considerations; however, no external lesions were identified, and the monitor lizard was housed alone and not fed live prey items. There were several additional challenges with the management of this case that factored into the ability to adjust chemotherapy. First, weekly to daily sampling was considered to have potential for deleterious effects to the monitor because of the animal’s small body size and presumptive slow rate of reptile erythrocyte turnover (Rodnan et al., 1957). Second, because of previous distal tail necrosis, there was concern that frequent coccygeal venipuncture for recheck CBC and plasma chemistry would compromise tail healing following partial caudectomy and lead to additional progressive tail necrosis. The distal tail necrosis and subsequent surgery also prohibited recheck of the CBC immediately prior to initiation of prednisone treatment, limiting the ability to assess the direct effect of prednisone on lymphocyte counts. Other sites for phlebotomy were impractical because of the animal’s size. The recheck interval for the CBC and plasma chemistry profiles was arbitrarily defined at approximately 30 days and seemed to provide a reasonable ability to monitor trends in lymphocytosis. The usage of oral prednisone was easy for keeper staff to administer to the monitor and did not subjectively appear to negatively impact the activity or natural behaviors of the animal. Metabolic scaling of prednisone, as done in this case, appeared to provide clinical benefit and may be an appropriate dose schedule for CLL chemotherapy based on reductions in the lymphocyte counts on days 94 and 151. Although data are limited at this time for CLL chemotherapy in saurian species, this report suggests that metabolic scaling may serve as an adequate starting point for future treatment of CLL in other lizard species. Acknowledgments: The authors would like to thank the Wildlife Conservation Society’s veterinary technician staff for their review of peripheral blood films; the Wildlife Conservation Society’s Department of Herpetology for assistance with the long-term management of this case; the Veterinary Oncology and Hematology Center for the donation of chlorambucil; Dr. Melinda Wilkerson from the Department of Diagnostic Medicine/Pathobiology, Kansas State University and the Kansas State University Clinical Immunology/Flow Cytometry Laboratory for performing flow cytometric immuno-phenotyping; and Linda Wright from the Department of Pathology, University of Florida for processing samples for ultrastructural analysis. The authors also thank Drs. Maren Connolly and Bonnie Raphael for their helpful comments in review of the manuscript.

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