A 7-year-old castrated male Poodle dog was referred for evaluation of persistent lymphocytosis that had lasted for more than 20 months and gradually worsened. Specific infections were ruled out in that canine SNAP 4Dx (IDEXX Laboratories, Westbrook, ME) for heartworm, Lyme, Ehrlichia canis, Anaplasma phagocytophilum was all negative before the visit. On physical examination, the dog had hyperthermia (rectal temperature: 39.4 C) and tachypnea. The size of superficial lymph nodes was found to be normal on palpation. Hematologic findings included leukocytosis, lymphocytosis, and neutropenia (Table 1, Day -28). On peripheral blood smear, lymphocytosis with well-differentiated small lymphocytes and neutropenia were found (Figure 1A). Results of serum chemistry were within normal ranges. Abdominal ultrasonography showed hepatosplenomegaly. Chronic inflammation and immune mediated diseases, which could primarily induce persistent lymphocytosis were excluded and lymphoproliferative disease was highly suspected. Therefore, bone marrow aspirate was performed to identify marrow infiltration and to evaluate the cytological types and proportions of hematopoietic cells. Bone marrow aspirate cytology showed hypercellularity and infiltration with small-sized mature lymphocytes (Figure 1B); the percentage of lymphoid cells was 45%. Differential diagnosis for the lymphocytosis of bone marrow include ALL, CLL, and stage V lymphoma. Among them, the dog was diagnosed with CLL based on the results of clinical examination mentioned above.
For the evaluation of immunophenotype by flow cytometry, both blood and marrow samples were stained with a panel of antisera specific for the T-lymphocyte markers [CD3 (CA17.2A12; Serotec Inc, Raleigh, NC, USA), CD5 (YKIX322.3; Serotec Inc, Raleigh, NC, USA), CD4 (YKIX302.9; Serotec Inc, Raleigh, NC, USA), and CD8 (YCATE55.9; Serotec Inc, Raleigh, NC, USA)], B-lymphocyte marker CD21 (CA2.1D6; Serotec Inc, Raleigh, NC, USA), and precursor cell marker CD34 (1H6; B-D Biosciences, San Jose, CA, USA) (Colorado state university, Fort Collins, CO, USA). After the exclusion of dead cells by propidium iodide staining, populations of lymphocyte were gated on the forward scatter vs. log side scatter plots. Neoplastic lymphocytes predominantly consisted of CD21+ cells with 66.73% of all nucleated cells in the blood sample and 31.71% of all nucleated cells in the bone marrow sample, respectively. Among lymphocytes, cells with CD21 expression were 87.52% in the blood, and 75.60% in the bone marrow, respectively. These results revealed a B-cell origin, which indicated B-cell CLL (Figures 2A,B). Expression of the remaining markers as mentioned above was unremarkable. Moreover, polymerase chain reaction for antigen receptor rearrangements (PARR) using blood samples was performed to determine clonality by detecting rearranged antigen receptor genes (Colorado state university, Fort Collins, CO, USA). DNA was extracted from blood, and the size of the antigen receptor hypervariable region was detected by PCR with amplification of immunoglobulin for B cells and T-cell receptor gamma sequences for T cells. PARR results of the dog showed a positive reaction to immunoglobulin sequence with prominent bands while a negative reaction was shown to T-cell receptor gamma sequence as no clonal bands, indicating B-cell clonality.
Treatment was initiated with chlorambucil (0.2 mg/kg, PO, QOD; Excella GmbH, Germany) and prednisolone (30 mg/m2 surface area, PO, SID; Daesung, Korea) 28 days after the first admission (Table 1, Day 0). One week later, the chlorambucil dosage was reduced to 0.1 mg/kg (PO, QOD), and prednisolone dosage was reduced to 20 mg/m2 (PO, SID). After another week, the chlorambucil dosage was reduced to 0.1 mg/kg (PO, EOD), and prednisolone dosage was reduced to 10 mg/m2 (PO, EOD). Moreover, additional medication was prescribed, involving the administration of liver supplement medications (Zentonil; 0.1 T/kg, PO, divided; Vetoquinol, USA.) and ursodeoxycholic acid (10 mg/kg, PO, BID; Daewoong, Korea) for the control of elevated liver enzymes.
The dog's leukocyte and lymphocyte counts did not resolve remarkably after 3 weeks of chlorambucil and prednisolone chemotherapy (Figure 1C). Moreover, clinical signs including hyperthermia, tachypnea, and hepatosplenomegaly were not resolved. Therefore, imatinib (10 mg/kg, PO, SID; Novartis, Switzerland) was additionally administrated to the original therapy (Table 1, Day 22). Two weeks after combined treatment, the white blood cell (WBC) counts decreased from 35.46 x 109/L to 21.53 x 109/L, and the imatinib dosage was decreased from once daily to every other day to prevent myelosuppression. Moreover, hyperthermia was resolved, and the dog had normal body temperature (rectal temperature: 38.9 C). However, 9 weeks later, WBC counts increased to 30.4 x 109/L (Table 1, Day 100). Therefore, the imatinib dosage was increased to once daily. After 2 weeks, WBC counts increased from 30.4 x 109/L to 32.32 x 109/L and mild anemia (HCT 36.7%; reference range, 37.3-61.7%) occurred as a side effect of myelosuppression. Considering deterioration of the side effect, the dosage of imatinib was changed from once daily to 2 days taking on and 1 day taking off in a 3-day cycle. Moreover, 18 days after additional administration of imatinib, test for c-kit mutation in the blood sample was performed to check mutations identified in the c-kit gene (Colorado state university, Fort Collins, CO, USA). There was no internal tandem duplication in c-kit exons 8 and 11, indicating no c-kit mutation in the dog. After 2 weeks, WBC counts dramatically decreased to 16.86 x 109/L, and anemia was resolved (Table 1, Day 127). WBC and lymphocyte count of this dog was well-controlled with the current treatment regimen. On day 386, WBC counts were within the normal range (15.41 x 109/L; reference range, 5.05-16.76 x 109/L), and lymphocyte counts were also within the normal range (3.2 x 109/L; reference range, 1.05-5.1 x 109/L; Figure 1D).
Approximately 1 year after starting treatment, flowcytometry was re-assessed for evaluating the current treatment response (Figure 2C). The number of cells expressing the CD21 molecule decreased from 24,678/muL at the first admission date to 704/muL (reference range, 85-350) showing a highly favorable response to medication (Figure 2D). The dog responded well to therapy with chlorambucil, prednisolone, and imatinib for 2 years and remained clinically normal.