Diagnosis<\/span><\/h2>\nStep 1: Evaluate the Patient<\/h3>\n
The first step in approaching a suspected leukemia is to assess the patient\u2019s history and clinical presentation.<\/p>\n
Chronic Lymphocytic Leukemia<\/b><\/p>\n
CLL is frequently diagnosed incidentally, <\/i>and the patient is often clinically healthy with no reported abnormal clinical signs and a normal physical examination. Some dogs have mild peripheral lymphadenopathy or palpable splenomegaly. If a chronic leukemia progresses, clinical signs develop, ranging from mild lethargy to more severe signs similar to those seen in dogs with acute leukemias.<\/p>\n
CLL mostly occurs in older, small-breed dogs (median age, 10 to 11 years) with no sex predilection.1<\/sup> A classic scenario is when an otherwise healthy geriatric dog undergoes routine laboratory tests for an unrelated issue or anesthesia and is found to have an elevated lymphocyte count with no clinical abnormalities.<\/p>\nEnglish bulldogs, a notable exception among larger-breed dogs, tend to develop CLL at a younger age (median age, 6 years).3<\/sup> Additionally, the syndrome of persistent lymphocytosis, often accompanied by splenomegaly, has been described in this breed.3<\/sup> This phenomenon indicates a non-neoplastic, polyclonal expansion of B cells rather than true CLL, and these conditions can be distinguished by PCR for antigen receptor rearrangement (PARR) testing.3,4<\/sup> Hyperglobulinemia is present in approximately 25% of CLL cases, with a notably higher prevalence in English bulldogs (about 50% of cases).3,5<\/sup><\/p>\nAcute Leukemia<\/b><\/p>\n
Dogs with acute leukemia usually show vague systemic signs such as lethargy, pyrexia, anorexia, or gastrointestinal signs; more specific physical examination findings include organomegaly (spleen larger than liver) and lymphadenopathy. Affected dogs can show signs of myelophthisis, manifesting as petechiae, pale mucous membranes, epistaxis, and other signs of spontaneous bleeding. It is rare to incidentally diagnose acute leukemia in an otherwise healthy dog.<\/p>\n
Patients with acute leukemias are usually middle-aged (7 to 8 years), although disease can occur at any age. There is no sex predilection.6<\/sup> <\/b>The progression of the disease is usually rapid. If lymphadenopathy presents, it is usually less pronounced than in cases of high-grade multicentric lymphoma.<\/p>\nStep 2: Evaluate the Slides<\/h3>\n
The most valuable diagnostic test in cases of suspected leukemia is a CBC with blood smear examination by a clinical pathologist. The leukocyte count and differential reveal the magnitude of leukocytosis and the types of cells involved. In suspected leukemia, examination of a well-made blood smear is invaluable, as cell morphology can help establish a tentative diagnosis; however, for definitive diagnosis, more advanced diagnostics, including flow cytometry and cytochemistry with routine imaging tests, are required. In-house automated hematology analyzers might fail to detect neoplastic cells and can misclassify them (e.g., identifying acute leukemia as CLL).7<\/sup><\/p>\nIn addition to identifying neoplastic cells, the CBC reveals any concurrent cytopenias. In CLL, the remainder of the CBC is often normal at diagnosis aside from the lymphocytosis. Mild anemia is present in some dogs with CLL, but severe cytopenias are uncommon. Acute leukemias commonly cause anemia (usually nonregenerative) and thrombocytopenia due to overcrowding of the bone marrow.<\/p>\n
In rare instances, leukemia cannot be diagnosed solely by evaluating peripheral blood. Neoplastic cells might be confined to the bone marrow with no circulating neoplastic cells (aleukemic leukemia) or be present in low numbers despite a normal or decreased white blood cell count (subleukemic leukemia). This lack of neoplastic cells occurs more commonly, though not exclusively, in AML, making bone marrow evaluation essential for diagnosis in some cases.1<\/sup><\/p>\nChronic Lymphocytic Leukemia<\/b><\/p>\n
In CLL, the neoplastic cells are often morphologically indistinguishable from normal mature lymphocytes. They are typically small, with nuclei smaller than those of neutrophils, and contain dense, clumped chromatin and minimal cytoplasm. As a result, a blood smear in CLL may appear relatively unremarkable, aside from a marked increase in the number of small lymphocytes.<\/p>\n
Acute Leukemia<\/b><\/p>\n
\u201cIf the cells are big, it\u2019s bad.\u201d<\/i> In acute leukemia, the circulating neoplastic cells are predominantly immature and are larger than normal neutrophils (the nucleus is larger than that of a neutrophil). Some clinical pathologists describe them as \u201cbig blue cells.\u201d8<\/sup> These cells typically have fine to reticulated chromatin, 1 or more visible nucleoli, and a high nuclear-to-cytoplasm ratio with basophilic cytoplasm. Lymphoblasts and myeloblasts have a similar large, immature appearance, making it challenging to distinguish between them based on cytologic features alone. Nevertheless, the presence of a significant population of large, immature, hematopoietic cells in the blood should raise immediate concern for acute leukemia or stage V lymphoma. Mitotic figures might occasionally be observed on the smear in acute leukemia due to the high rate of cell proliferation.<\/p>\nIs This Leukemia?<\/b><\/p>\n
An important initial step in diagnosing leukemia is to differentiate true leukemias from non-neoplastic causes of extreme leukocytosis. The 2 common white blood cell types that are responsible for leukocytosis are neutrophils and lymphocytes.<\/p>\n
Neutrophilic leukocytosis<\/b> in dogs is far more often reactive (e.g., severe infection or inflammation causing a leukemoid reaction) than neoplastic; true chronic neutrophilic leukemia is exceedingly rare. Thus, an unexplained extreme mature neutrophilic leukocytosis (segmented neutrophils \u2265\u2009<\/span>50\u2009<\/span>\u00d7\u2009<\/span>103<\/sup> cells\/\u00b5L) should prompt a thorough search for underlying infection, inflammation, or marrow stimulation before leukemia is considered as a diagnosis.9,10<\/sup> Neutrophilic leukocytosis can also be a paraneoplastic syndrome secondary to a cancer other than leukemia, such as carcinoma, intestinal lymphoma, metastatic fibrosarcoma, and even rectal polyps.1<\/sup><\/p>\nLymphocytic leukocytosis<\/b> <\/i>can occur secondary to antigenic stimulation (e.g., Ehrlichia canis<\/i> infection) or Addison\u2019s disease, but such reactive lymphocytoses are usually mild to moderate in magnitude. Persistent lymphocyte counts above 20\u2009<\/span>000 to 30\u2009<\/span>000\/\u00b5L are uncommon in chronic infections and raise concern for lymphoproliferative neoplasia.<\/p>\nAs mentioned, English bulldogs can present with a unique syndrome of non-neoplastic small B-cell lymphocytosis that mimics CLL. Although the degree of lymphocytosis (median, ~22\u2009<\/span>000\/\u00b5L) is typically less marked than in true CLL, the clinical presentation is similar.3<\/sup><\/p>\nMost non-neoplastic causes of persistent lymphocytosis in dogs are rare, and most dogs with sustained, marked small-cell lymphocytosis have CLL or stage V indolent lymphoma.2<\/sup> In practice, a high lymphocyte count that is repeatable and unexplained by other diseases should prompt diagnostic evaluation for leukemia or lymphoproliferative disease.<\/p>\nStep 3: Establish a Definitive Diagnosis<\/h3>\n
Flow Cytometry<\/b><\/p>\n
Cellular morphology aids in the diagnosis of leukemias; however, for a definitive diagnosis, flow cytometry is the preferred diagnostic tool (Box 1<\/b><\/span>). Flow cytometry evaluates cells by their size (forward scatter) and granularity (side scatter) and can detect various cell surface (or intracellular) markers to identify the cell type. It is worth noting that flow cytometry does not directly determine clonality; instead, clonality can only be inferred from flow cytometry results. Definitive determination of clonality is achieved with PARR. Cellular markers are used to differentiate various types of leukemia (Box 2<\/b><\/span>).11-13<\/sup><\/p>\nBOX 1 When Morphology and Flow Cytometry Results Don\u2019t Match <\/div>
Morphology and flow cytometry results can yield discordant results. For example, some cases show large cells on blood smear yet are diagnosed as chronic lymphocytic leukemia (CLL) by flow cytometry. In contrast, other leukemias, particularly acute leukemias, might exhibit coexpression of both T-cell and B-cell markers. In such instances, clinical signs and disease progression should be carefully considered. While flow cytometry is regarded as the gold standard for diagnosis, it is essential to inform owners that some cases diagnosed as CLL might behave more aggressively and progress rapidly, resembling acute leukemia in their clinical course.<\/p>\n
<\/div><\/div>\n
BOX 2 Common Cellular Markers in Leukemia<\/div>
<\/p>\n
\n- CD3 (T lymphocytes)<\/li>\n
- CD4 (helper T cells)<\/li>\n
- CD8 (cytotoxic T cells)<\/li>\n
- CD34 (progenitor cells)<\/li>\n
- CD79a (B lymphocytes)<\/li>\n
- MHC class II (leukocytes)<\/li>\n<\/ul>\n
CD\u2009=\u2009cluster of differentiation; MHC\u2009=\u2009major histocompatibility complex<\/em><\/p>\n<\/div><\/div>\n
Sample Handling<\/b><\/span><\/em><\/p>\nFlow cytometry requires live cells; therefore, proper sample collection and handling are critical. Viability is usually assessed early in the analysis using stains such as propidium iodide, as data from dead cells can be misleading. Most laboratories reject samples with more than 50% nonviable cells. Blood or bone marrow should be collected in EDTA and ideally arrive at the laboratory for processing within 24 to 48 hours. Samples should be refrigerated (not frozen) and shipped early in the week to avoid delays. Results are accompanied by an interpretive summary from the clinical pathologist, which can be very helpful in understanding the diagnosis.11,12<\/sup><\/p>\nChronic Lymphocytic Leukemia<\/b><\/span><\/em><\/p>\nCanine CLL is typically classified into 2 major immunophenotypic subtypes: T-cell CLL (T-CLL), which accounts for approximately 75% of cases, and B-cell CLL (B-CLL), which makes up the remaining 25%.2<\/sup> This distribution contrasts with human CLL, which is almost exclusively of B-cell origin. Among canine T-CLLs, most are CD8+<\/sup>, and many exhibit large granular lymphocyte morphology. CD4+<\/sup> T-CLL is rare. Canine B-CLL is characterized by the expansion of CD21+ B cells. Unlike human B-CLL, which frequently shows aberrant expression of the T-cell marker CD5, canine B-CLL typically lacks CD5 expression.14<\/sup><\/p>\nAcute Leukemia<\/b><\/span><\/em><\/p>\nMost acute leukemias express CD34, a cell surface antigen that is expressed on early hematopoietic progenitor cells and lost as cells mature. Its expression on neoplastic cells has been associated with a poor prognosis.15<\/sup> Some ALLs are CD34-negative; therefore, the absence of CD34 does not exclude a diagnosis of acute leukemia if other diagnostic features are supportive. Additionally, some dogs with lymphoma have neoplastic lymphocytes that aberrantly express CD34.1,11,16,17<\/sup><\/p>\nFlow cytometry is essential for subclassifying acute leukemias. Neoplastic cells expressing lymphoid markers are categorized as ALL, while those expressing myeloid markers are considered AML. In some cases, neoplastic cells lack definitive lineage markers, consistent with acute undifferentiated leukemia, or coexpress markers from multiple lineages, consistent with mixed-phenotype acute leukemia.18<\/sup> Despite these distinctions, all subtypes carry a poor prognosis, and in many primary care settings, the differentiation might not substantially affect immediate management. However, distinguishing between AML and ALL might be clinically relevant, as treatment strategies differ. ALL in dogs is almost exclusively of T-cell origin, and some believe that true B-cell ALL is rarely, if ever, encountered in this species.15,19<\/sup><\/p>\nImmunocytochemistry\/Cytochemistry<\/b><\/p>\n
Immunocytochemistry (ICC) or cytochemistry (CC) is the application of specific dyes or antibodies to cytology preparations, analogous to immunohistochemistry on histopathology samples. The key difference is that CC uses chemical dyes that react with general cellular components (e.g., alkaline phosphatase staining in suspected canine AML) while ICC employs antibodies to detect specific antigens (e.g., CD3 or CD79a to distinguish canine ALL subtypes).17<\/sup> These techniques highlight targets on or within cells, aiding in classification. ICC\/CC can be performed on unstained or previously stained cytology slides. It is not widely available; however, some specialty diagnostic laboratories offer it as an add-on to cytology.<\/p>\nHistorically, ICC\/CC was the only tool for distinguishing AML from ALL in dogs. Today, it has largely been supplanted by flow cytometry, which provides more comprehensive immunophenotyping. Still, ICC\/CC remains valuable, either as a complement to flow cytometry or when fresh samples suitable for flow cytometry are unavailable.<\/p>\n
PARR<\/b><\/p>\n
PARR detects clonality and is primarily used to distinguish neoplastic from non-neoplastic conditions. For example, PARR can help differentiate the polyclonal lymphocytosis seen in English bulldogs from true CLL.3<\/sup> Unlike flow cytometry, PARR does not require viable cells. It can be performed on a variety of tissues, including stained cytology slides sent for clinical pathologist evaluation, making it an attractive option. However, it is not recommended for immunophenotyping, as results might be misleading. Neoplastic cells may exhibit clonality across multiple lineages, complicating interpretation. In addition, more than half of AML cases might have aberrant PARR-positive results, which could lead to misclassification as ALL.16<\/sup><\/p>\nDifferentiating Acute Leukemia Versus Stage V Lymphoma<\/h3>\n
Distinguishing between acute lymphoblastic leukemia and stage V high-grade lymphoma can be diagnostically challenging. Both conditions can present with elevated counts of large immature lymphocytes in circulation. No single test definitively differentiates acute leukemia from stage V lymphoma. Flow cytometry can be helpful, and the expression of CD34 supports a diagnosis of acute leukemia. However, not all cases of acute leukemia express CD34. Lymphadenopathy is generally less pronounced than in patients with lymphoma, while the total white blood cell count tends to be markedly higher in leukemia. However, exceptions can occur.<\/p>\n
A mediastinal mass can be detected in both lymphomas and acute leukemias. Its presence does not favor a diagnosis of lymphoma over leukemia\u2014nearly half of dogs with AML present with a mediastinal mass.18<\/sup> This distinction does not significantly impact initial management, but it can influence the prognosis. Both ALL and stage V high-grade lymphoma are treated with multiagent injectable chemotherapy protocols (e.g., CHOP). Dogs with stage V lymphoma (especially B cell) sometimes respond to chemotherapy and achieve remissions measured in months, whereas acute leukemia often responds transiently, and the disease progresses rapidly.<\/p>\nManaging Chronic Lymphocytic Leukemia<\/span><\/h2>\nWhen to Treat Versus When to Monitor<\/h3>\n
The first decision to be made in managing dogs with CLL is whether to initiate treatment. Generally accepted indications to begin treatment include:<\/p>\n
\n- The patient develops clinical signs attributable to CLL (e.g., lethargy, poor appetite, unexplained weight loss).<\/li>\n
- There is evidence of bone marrow compromise (e.g., moderate to severe anemia not due to other causes, thrombocytopenia developing as the lymphocyte count rises).<\/li>\n
- Significant organomegaly is present (e.g., enlarged spleen or liver causing discomfort, markedly enlarged lymph nodes).<\/li>\n
- The lymphocyte count is excessively high (commonly a threshold of around 30\u2009<\/span>000 to
\n60\u2009<\/span>000\/\u00b5L is used) or rising rapidly over successive rechecks.<\/li>\n<\/ul>\nIf any of these criteria are met, treatment should be initiated. If none are present, a conservative approach is appropriate, including monthly physical examinations and CBCs, or every 2 to 3 months in stable cases. It is essential to educate clients that CLL is typically a slow-progressing disease and that immediate treatment is not required unless clinical signs are present. This helps prevent overtreatment and avoids exposing dogs to unnecessary adverse effects.<\/p>\n
Treatment Protocol<\/h3>\n
If treatment is warranted, the most commonly used and well-tolerated protocol for canine CLL is a combination of chlorambucil and prednisone. The authors use the following treatment protocol:<\/p>\n
\n- Chlorambucil:<\/b> 6 to 8 mg\/m\u00b2 PO q48h or 20\u202f<\/span>mg\/m\u00b2 every 2 weeks. For long-term maintenance, a lower dose (e.g., 2 mg\/m\u00b2 q48h) can be used, with adjustments made based on clinical response and bone marrow tolerance.<\/li>\n
- Prednisone:<\/b> Administered concurrently at an initial immunosuppressive dose of 1 mg\/kg q24h for 1 to 2\u00a0weeks, then tapered to 0.5 mg\/kg q48h.<\/li>\n<\/ul>\n
Most dogs show a favorable response within a few weeks to months, with declining lymphocyte counts and a reduction in lymphadenopathy or splenomegaly. Approximately 70% to 80% of dogs achieve a partial or complete remission with this combination.14<\/sup> There is no consensus on the optimal duration of treatment. The authors typically continue therapy for up to 2\u00a0years; however, this remains an area of debate, and some oncologists elect to stop treatment about 1\u00a0year after remission while some continue treatment indefinitely. In nonresponders, vincristine can be added or cyclophosphamide substituted for chlorambucil.<\/p>\nMonitoring<\/h3>\n
Unlike more intensive chemotherapy protocols, chlorambucil at these doses rarely causes significant myelosuppression. However, mild neutropenia or thrombocytopenia occasionally occurs; therefore, periodic monitoring of CBCs is recommended. Prednisone-related adverse effects, such as polyuria, polydipsia, and polyphagia, are common, particularly during the initial stages of treatment, but are generally manageable for most owners, especially as the dose is tapered over time.<\/p>\n
At the authors\u2019 institutions, follow-up for dogs with CLL begins with baseline diagnostics before initiating treatment, including a CBC, serum biochemistry profile, and urinalysis. After starting therapy, CBC and body weight are monitored every 2 weeks for the first month. If the patient remains stable, monitoring continues monthly until 6 months after treatment initiation. At this point, both a CBC and biochemistry panel are repeated. If the patient remains in remission, CBC and body weight checks are extended to every 2\u00a0months for 1 year after starting treatment. A complete reevaluation, including CBC, biochemistry, and urinalysis, is then performed.<\/p>\n
If remission is maintained, follow-up can occur every 3\u00a0months for the first year and then every 6 months for the second year. In select cases where remission persists 2 years after starting therapy, the authors consider discontinuation of chlorambucil, tapering of prednisone, and continued monitoring every 3 months. See CASE EXAMPLE<\/strong><\/span>\u00a01<\/b><\/span> for a real-world case report of CLL.<\/span><\/p>\nCASE EXAMPLE 1 Chronic Lymphocytic Leukemia in a Maltese<\/div>
History and Presentation <\/strong><\/p>\nMartha, a 12-year-old spayed female Maltese, was presented to her primary care veterinarian for a dental procedure. On physical examination, she had severe gingivitis, dental tartar, and halitosis. Mildly enlarged mandibular lymph nodes were detected, which were initially attributed to her dental disease. No organomegaly was detected on abdominal palpation, and rectal examination was unremarkable.<\/p>\n
Routine preanesthetic blood analysis revealed lymphocytosis (9546 cells\/\u00b5L). No other CBC abnormalities were noted. Serum biochemistry and urinalysis were unremarkable. An in-house blood smear revealed small, mature lymphocytes. The dental procedure was postponed pending clarification of the lymphocytosis.<\/p>\n
Diagnosis and Treatment <\/strong><\/p>\nA blood sample was sent to a clinical pathologist, who confirmed mature lymphocytosis and recommended further evaluation with flow cytometry (Figure A<\/strong>). A fresh EDTA blood sample was collected and shipped on ice. Flow cytometry documented a CD8+ T-cell lymphocytosis, consistent with chronic lymphocytic leukemia (CLL).<\/p>\n
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