Feline lymphocytic leukemia is a common neoplastic disorder characterized by the proliferation of neoplastic lymphocytes in bone marrow. When lymphoid leukemia is suspected in cats, the diagnostic process often involves peripheral blood assessment, including complete blood count (CBC) with differential count, flow cytometry (FC) analysis, and polymerase chain reaction (PCR) for antigen receptor rearrangement (PARR).

Immunophenotypes of lymphoid leukemia in cats have been reported. For chronic lymphocytic leukemia (CLL), 94% (17/18) exhibited T-cell origin in a study of 18 feline CLL (2). In particular, 16 cats displayed disease originating from T-helper lymphocytes (CD3+/CD4+/CD8–), while one cat demonstrated the double-negative T-cell phenotype (CD3+/CD4–/CD8–). Notably, only one cat (6%) was CD21–positive, indicating B-cell origin CLL. In contrast, 67% (4/6) exhibited B-cell lineage, while one cat (17%) showed T-cell lineage in cats with acute lymphoblastic leukemia (ALL) (12). Interestingly, one cat demonstrated the presence of both the B-cell marker (CD21) and the T-cell marker (CD8), suggesting a potential mixed lineage or aberrant marker expression in that particular case.

Non-B, non-T cell lymphoproliferative diseases are characterized by the absence of B-cell or T-cell-specific antigens and instead express common lymphocyte antigens. Compared to studies of non-B, non-T leukemia in human ALL (6,9), there is limited research available on non-B, non-T cell-origin lymphocytic leukemia in veterinary medicine. To date, two out of five cases of ALL in dogs (14) and only one cat have been reported for non-B, non-T lymphoma/leukemia (5). Here, we report a rare case of non-B, non-T feline ALL.

A 7-year-old neutered male, domestic shorthair cat showed anorexia and lethargy. The CBC indicated that he had severe anemia (hematocrit: 14.5%, reference interval: 30.3-52.3%). The leukogram also showed remarkable lymphocytic leukocytosis, with a lymphocyte count of 41,600 cells/μL (reference interval: 920-6,880 cells/μL), a total white blood cell counts of 45,570 cells/μL (reference interval: 2,870-17,020 cells/μL) and a neutrophil count of 140 cells/μL (reference interval: 2,300-10,290 cells/μL). Additionally, severe thrombocytopenia was observed, with a platelet count of 7,000 cells/μL (reference interval: 151,000-600,000 cells/μL). Hyperglobulinemia with 5.4 g/dL (reference interval: 2.8-5.1 g/dL) was identified in the biochemistry profile (Table 1).

Table 1 . Selected laboratory results of the cat with acute lymphoblastic leukemia.

Parameters	Reference interval	Case
Red blood cells	6.54-12.20 (×1012/L)	2.51
Hematocrit	30.3-52.3 (%)	14.5
Hemoglobin concentration	9.8-16.2 (g/dL)	4.5
Mean corpuscular volume	35.9-53.1 (fL)	57.8
Mean corpuscular hemoglobin concentration	28.1-35.8 (g/dL)	31.0
Red blood cell distribution width	15.0-27.0 (%)	21.0
Reticulocytes	3.0-50.0 (×103/μL)	36.4
White blood cells	2.87-17.02 (×109/L)	45.57
Segmented neutrophils	2.30-10.29 (×109/L)	0.14
Lymphocytes	0.92-6.88 (×109/L)	41.60
Monocytes	0.05-0.67 (×109/L)	3.75
Eosinophils	0.17-1.57 (×109/L)	0.03
Basophils	0.01-0.26 (×109/L)	0.05
Platelets	151-600 (×109/L)	7
Creatinine	0.8-2.4 (mg/dL)	0.9
Blood urea nitrogen	16-36 (mg/dL)	13
Albumin	2.2-4.0 (g/dL)	3.2
Globulin	2.8-5.1 (g/dL)	5.4
Alanine aminotransferase	12-130 (U/L)	27
Alkaline phosphatase	14-111 (U/L)	20
Gamma-glutamyl transferase	0-4 (U/L)	0
Total bilirubin	0.0-0.9 (mg/dL)	0.3

Feline leukemia and feline immunodeficiency viral infections was excluded based on a commercial kit (SNAP^® Feline Triple kit, IDEXX, Westbrook, ME, USA), and blood-borne diseases were ruled out based on a multiplex feline real-time PCR panel (POBGEN^TM Feline anemia pathogens test, Green Vet, Yongin, Republic of Korea), including 13 pathogens (anaplasma, ehrlichia, babesia, leptospira, bartonella, hemoplasma, rickettsia, lyme diseases, hepatocytozoon, theileria, feline leukemia virus, feline immunodeficiency virus, and feline coronavirus).

Diagnostic imaging was not remarkable: splenomegaly and a blunt margin at the caudoventral aspect of the liver were found, but no significant changes were observed in the intra-abdominal lymph nodes during the abdominal ultrasonography.

Blood smear examination indicated severe non-regenerative anemia and thrombocytopenia [<1 polychromatophilic cells, 11 red blood cell (RBC) anisocytosis and 1.3 platelets under ×1,000 microscopic examination]. The majority of white blood cells (WBCs) were medium-to-large round cells with moderate basophilic cytoplasm. When a manual differential count of 500 WBCs was performed, the following were observed: 70% of the WBCs were intermediate-large lymphocytes (immature lymphocytes) characterized by nuclei that were 2-2.5 times the size of an RBC, whereas 15% of the WBCs were small lymphocytes (mature lymphocytes) characterized by nuclei that were 1-1.5 times the size of an RBC (Fig. 1A). The May Grunwald-Giemsa stain used to differentiate granular lymphocytes revealed intermediate-to-large lymphocytes that exhibited intracytoplasmic granules, but these granular lymphocytes accounted for less than 5% of the total lymphocytes (Fig. 1B). Considering the results of bicytopenia, lymphoblastic leukocytosis without lymphadenopathy, lymphoid leukemia was strongly suspected. Confirmative tests, such as a FC analysis and clonality test, were performed.

Figure 1. Peripheral lymphocytosis in blood smear examination. (A) Morphologically, 70% of white blood cells (WBCs) were intermediate-large lymphocytes characterized by nuclei that were 2-2.5 times the size of red blood cells (RBCs), while 15% of the WBCs were small lymphocytes characterized by nuclei that were 1-1.5 times the size of an RBC (×400, Diff-Quik stain). (B) There were few intermediate-to-large lymphocytes with intracytoplasmic granules (5% of total lymphocytes) (×1,000, May Grunwald-Giemsa stain).

Flow cytometric immunophenotyping was performed using antibodies such as rat anti-human CD3 FITC (CD3-12 clone; Bio-Rad Laboratories Inc., Hercules, CA, USA), mouse anti-cat CD4 FITC (vpg34 clone; Bio-Rad Laboratories Inc.), mouse anti-cat CD5 FITC (FE1.1B11 clone; Bio-Rad Laboratories Inc.), mouse anti-cat CD8 alpha/beta (vpg9 clone; Bio-Rad Laboratories Inc.), mouse anti-human CD79a FITC (HM57 clone; Bio-Rad Laboratories Inc.) and mouse anti-canine CD21 (CA2.1D6 clone; Bio-Rad Laboratories Inc.) (3).

FC analysis indicated that peripheral lymphocytes did not express either T-cell markers (CD3–/CD4–/CD5–/CD8–) or B-cell markers (CD21–/CD79a–) (Fig. 2). PARR assay (IDEXX laboratory; referral test performed at the Clinical Immunopathology Laboratory at Colorado State University) using the peripheral blood revealed the presence of clonal rearrangements for both the immunoglobulin and T-cell receptor genes.

Figure 2. Immunophenotyping results of the peripheral blood by flow cytometry. Dot plots show the population of lymphocytes (72.6%). Majority of the lymphocytes are negative for B-cell markers (CD21–/CD79a–) and T-cell markers (CD3–/CD4–/CD5–/CD8–).

The cat was diagnosed with non-B, non-T ALL, and the owner did not opt for further examination or treatment because of the poor prognosis. Follow-up was lost afterward.

In the present case, the presence of intermediate-large lymphocytes in the peripheral blood, along with cytopenias (such as non-regenerative anemia, neutropenia, and thrombocytopenia), nonspecific clinical signs, splenomegaly, and hepatomegaly without lymphadenopathy suggested the possibility of lymphoblastic leukemia. However, further diagnostic tests were necessary to determine the specific type and origin of the disease. FC analysis revealed a non-B, non-T immunophenotype, as indicated by the absence of CD3, CD4, CD5, CD8, CD21, and CD79a markers. These findings suggested that the ALL in the cat did not arise from B-cells or T-cells. However, the presence of clonalities for both the immunoglobulin and T-cell receptor genes in the PARR assay confirmed lymphoid malignancy.

Immunophenotyping analysis results must be carefully reviewed because PARR and FC results can be contradictory. While immunohistochemistry (IHC) and histopathology are considered the gold standard methods for diagnosing lymphoproliferative diseases, FC and PARR have also been commonly employed for lineage assignment (4,8,10). One possibility of discrepancy is that the tumors may arise from precursor cells, as in ALL. In such cases, clonal rearrangements detected through PARR may not necessarily represent the cell of origin (13). Another possibility is that the lymphocytes may be derived from natural killer (NK) cells. NK cells are a subset of granular lymphocytes commonly observed in normal cats. These cells typically lack expression of T-cell and B-cell markers based on FC. A case of feline NK cell leukemia described the negative expression of both B- and T-cell markers on FC, the absence of clonal rearrangement on PARR, and the subsequent diagnosis of NK cell leukemia through additional immunohistochemistry testing (5). Given the presence of granular lymphocytes in the peripheral blood smear and the negative expression of T-cell and B-cell markers, the possibility of tumors derived from NK cells can be considered. Further research and improved diagnostic methods are necessary to accurately diagnose and characterize non-B, non-T lymphoid leukemias in cats. Additionally, comprehensive studies exploring the prognosis and outcomes associated with specific immunophenotypes are warranted to enhance our understanding and management of these conditions.

Several studies compared the agreement between FC and PARR (1,7,11). In the study of canine lymphoid neoplasia, 4% of B-cell lymphomas and 7% of T-cell lymphomas that were flow cytometrically confirmed exhibited rearrangements of both immunoglobulin heavy chain and T-cell receptor gamma (TRG) genes (1). Similarly, the study focused on feline lymphoma evaluated 12 cases of flow cytometrically confirmed B-cell lymphoma, but the PARR assay revealed 1 of the 12 cases had a clonal TRG rearrangement (7). Furthermore, the study evaluating the agreement between FC, PARR, and IHC in dogs with lymphoma found a higher percent agreement between FC and IHC (94%) compared to PARR and IHC (69%). This study indicated that FC results were more concordant with the gold standard IHC results, while there was a lower level of agreement between PARR and IHC. The percent agreement between FC and PARR was even lower (63%), suggesting some discordance between the two techniques (11). Thus, PARR can be reliable to confirm the presence of lymphoid leukemia, while FC supported the diagnosis of non-B/non-T lymphoid leukemia phenotyping.

The presented case had several limitations that should be taken into consideration: 1) The lack of continuous follow-up limited the assessment of prognosis and response to treatment. 2) Bone marrow evaluation was absent. As mentioned above, bone marrow evaluation is crucial for diagnosing leukemia. In this case, the lack of bone marrow assessment limited the diagnostic process of the disease. 3) NK cell leukemia confirmation was inconclusive.

In conclusion, the presented case sheds light on the existence of non-B, non-T ALL in cats and highlights the importance of considering this rare subtype in the differential diagnosis of feline lymphoproliferative diseases. This case provides valuable insights into the clinical symptoms, laboratory test results, and immunophenotyping analysis associated with non-B, non-T ALL in cats. However, due to the limited number of reports on this specific subtype, further studies are necessary to validate and expand upon these findings. By raising awareness of this rare subtype and sharing clinical experiences, this case can contribute to the knowledge base and encourage future research in this area.

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2020R1C1C1008675).

The authors have no conflicting interests.