M a k i n g S e n s e o f Lym p h o m a D i a g n o s t i c s in Sm a l l A n i m a l P a t ients Mary Jo Burkhard,
a, DVM, PhD *,
Dorothee Bienzle,
b DVM, PhD
KEYWORDS Lymphoma Cytology Immunophenotyping Flow cytometry PCR for clonal antigen receptor gene rearrangement (PARR) Immunohistochemistry/immunocytochemistry KEY POINTS Cytologic assessment is diagnostic in most cases of diffuse large B-cell and diffuse lymphoblastic lymphoma in dogs. The cytologic diagnosis of lymphoma is more challenging in cats than in dogs. Although cytology is useful for staging lymphoma, histopathology is necessary for classification and grading. Immunophenotyping by flow cytometry allows evaluation of lymphocyte populations using a panel of antibodies, and serves as an adjunctive tool for both diagnosis and prognosis. Polymerase chain reaction (PCR) to detect clonal antigen receptor gene rearrangement (PARR) is a relatively new test in veterinary medicine that has strong potential for supporting the diagnosis of lymphoma. However, false-positive and false-negative results may confound the diagnosis, and PARR is less sensitive in cats than in dogs. Immunohistochemistry and immunocytochemistry should not be used as stand-alone diagnostic techniques, nor should an interpretation be based on a single antibody label.
INTRODUCTION
Lymphoma is the most common hemolymphatic malignancy in dogs and cats and, similar to lymphoma in people, is a heterogeneous disease with variable clinical signs and response to therapy.1 Patient genetics, immunocompetence, location, and morphologic subtype all contribute to the heterogeneity of the disease and prognosis (Box 1).
Funding Sources: None. Conflict of Interest: None. a Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, 1925 Coffey Road, Columbus, OH 43210, USA; b Department of Pathobiology, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G 2W1, Canada * Corresponding author. E-mail address: [email protected] Vet Clin Small Anim 43 (2013) 1331–1347 http://dx.doi.org/10.1016/j.cvsm.2013.07.004 vetsmall.theclinics.com 0195-5616/13/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved.
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Box 1 Clinical evaluation of lymphoma Several options are available for the diagnosis and characterization of lymphoma. This review helps in choosing the best test or tests for the patient.
Anatomically, lymphoma can be characterized as multicentric, alimentary, mediastinal, or extranodal. In dogs, multicentric lymphoma accounts for 80% to 85% of reported cases, and diffuse large B-cell lymphoma is the most common histomorphologic variant.2 However, other types of B-cell lymphomas as well as T-cell lymphomas also are frequently diagnosed. The prognosis of lymphoma variants in dogs depends not only on the type of neoplastic lymphocyte but also on the location, characteristics of the cells, and the stage of disease. In cats, the diagnosis is more challenging because lymphoma more commonly affects extranodal sites, particularly the alimentary and upper respiratory tract.3,4 Lymphoma affecting the gastrointestinal tract is common but particularly challenging to diagnose, owing to the relative inaccessibility for sampling and potential progression from lymphocytic inflammation to neoplasia. Other types of lymphoma in cats often contain a heterogeneous population of neoplastic lymphocytes plus reactive lymphocytes, plasma cells, and other inflammatory cells. The diagnosis of lymphoma classically depended on morphologic characteristics identified by cytology and/or histopathology. In recent years, additional assays such as immunocytochemistry (ICC), immunohistochemistry (IHC), immunophenotyping by flow cytometry, and polymerase chain reaction (PCR) to detect clonal antigen receptor gene rearrangement (PARR) have been used to assist in the diagnosis of lymphoma and to classify lymphoma for prognostic purposes. Diagnostic tests are not perfectly sensitive or specific; therefore multiple assays are often used in conjunction or in sequence to enhance the accuracy of diagnosis and assist with prognosis. This article considers the utility and pitfalls of each diagnostic tool (Box 2). CYTOLOGY
Cytologic examination of blood films and samples obtained by fine-needle aspiration (FNA) of tissues or fluids is commonly used in the diagnosis of lymphoma in dogs and cats. Advantages of cytology are that sample collection and slide examination are rapid and can be performed in-house with the minimal resources of glass slides, a Romanowsky stain such as Diff Quick or Wright Giemsa, and a high-quality microscope. Limitations of FNA in comparison with an incisional or excisional biopsy are that FNA does not allow assessment of tissue architecture, and material for additional studies (eg, IHC) is unavailable. Blood sampling and aspiration of superficial masses Box 2 Tools available for the diagnosis and characterization of lymphoma Cytology Histopathology Immunocytochemistry and immunohistochemistry Phenotyping by flow cytometry Polymerase chain reaction to detect clonal antigen receptor gene rearrangement (PARR)
Lymphoma Diagnostics for Small Animals
and peripheral lymph nodes are safe and simple techniques for most patients. FNA of internal organs such as the liver or spleen or abdominal or thoracic masses is more challenging, but is still commonly performed and often is diagnostically rewarding. Lymphocyte Morphology
Lymphocyte cytomorphology is characterized by cell size, nuclear features, and cytoplasmic features (Fig. 1). It is important when using cytomorphology to examine only those cells that are both intact and adequately spread out on the slide. Diagnosis of Lymphoma by Cytology
The cytologic diagnosis of lymphoma is the most straightforward if the tumor is diffuse and the entire node is replaced by a uniform population of large neoplastic lymphocytes; this is the most common type of lymphoma in dogs. Most diffuse lymphomas yield FNA that consist entirely or predominantly of large neoplastic cells. However, diffuse lymphomas composed of small or intermediate cells can be difficult to diagnose by cytology, because the cells more closely resemble benign lymphocytes. In contrast to diffuse lymphoma, follicular lymphoma, which is uncommon in dogs, may yield a heterogeneous population of benign and malignant cells of variable size and morphology, which is challenging to interpret by cytology.
Fig. 1. Lymphocyte cytomorphology. Fine-needle aspiration (FNA) of lymph node from a dog (Wright Giemsa stain, original magnification 1000). (A) One neutrophil (lower left) and 4 small lymphocytes: these lymphocytes are smaller than a neutrophil (arrow) and have a dense round nucleus that comprises the majority of the cell. Nucleoli are not seen. Cytoplasm is scant (sometimes only a very thin rim is visible). (B) One intermediate lymphocyte (arrow) and 2 small lymphocytes (arrowheads): intermediate lymphocytes are similar in size to neutrophils, have more abundant cytoplasm that is typically lightly basophilic, and may contain small azurophilic granules. Nuclei are often slightly eccentric within the cell and have less condensed chromatin. Indistinct nucleoli may be seen. (C) Four large lymphocytes and one neutrophil (arrow): large lymphocytes, also called lymphoblasts, are equal to or greater in size than neutrophils and contain round to oval nuclei with fine or stippled chromatin. Nucleoli are commonly seen. A rim of deeply basophilic cytoplasm surrounds the nucleus. Occasionally (especially in cats) the cytoplasm may contain punctate vacuoles, as shown in the 2 large lymphocytes at the bottom of this panel. (D) One reactive lymphocyte (arrow), 2 small lymphocytes (arrowheads), and 1 neutrophil: reactive lymphocytes are similar in morphology to small lymphocytes but slightly larger, and have more abundant, more deeply basophilic cytoplasm. (E) Several plasma cells: plasma cells are medium to large cells that contain small, round, eccentrically placed nuclei with coarsely clumped chromatin. Cytoplasm is abundant, deeply basophilic, and often contains a prominent perinuclear, clear zone that corresponds to the Golgi apparatus. The Golgi apparatus is clearly shown in the 2 plasma cells in the center.
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Lymphoma in cats less commonly affects lymph nodes, and more commonly affects other tissues such as the intestine, nasal cavity, kidneys, liver, stomach, thymus, and spinal cord. In addition, those feline lymphomas that involve 1 or several peripheral lymph nodes often comprise a heterogeneous population of small and large lymphocytes as well as plasma cells and macrophages, making the diagnosis of lymphoma in cats by cytology more challenging than in dogs. Hence, when lymph node FNA from either dogs or cats yield heterogeneous cell populations, reactive hyperplasia must be considered as a differential diagnosis (Table 1). Histopathology, in conjunction with ancillary diagnostic tests, should be used for additional characterization of the cell population. Supporting Cytologic Features
There are several cytologic features (Fig. 2) that are more often associated with lymphoma than with benign lymphocyte proliferations, and these may be helpful in establishing a diagnosis. However, these features may be seen in both neoplastic and reactive populations and are not pathognomonic. Staging of Lymphoma by Cytology
The World Health Organization staging scheme is based on the degree of metastasis, invasiveness, and presence of clinical signs. Cytology is useful in the staging of lymphoma by helping to identify the degree of metastasis (Box 3). Limitations of Cytology
Diagnosis of small-cell and intermediate-cell lymphoma by cytology is challenging. Diagnosis in these cases may require additional supportive evidence such as generalized lymphadenopathy, identification of cells with similar cytologic features in multiple tissues, and lack of detection of infectious agents. Additional diagnostic tests such as histopathologic evaluation, flow-cytometric analysis, or PARR may be required for diagnosis. Another cytologic diagnostic challenge is differentiating early lymphoma and reactive hyperplasia. In both processes, the percentage of lymphoblasts (large lymphocytes) will be increased within an overall heterogeneous population of lymphocytes. In these cases, examination by flow cytometry may also reveal a heterogeneous lymphocyte population, and therefore may be less specific than histopathology or PARR. Many lymphomas in cats are composed of such heterogeneous cell populations, and are therefore challenging to diagnose by cytology. Sample Requirements
Sample collection and processing for FNA cytology of lymphoid tissue is relatively straightforward, and has been described in numerous texts and continuing education
Table 1 Differentiating lymphoma and reactive hyperplasia Lymphoma
Hyperplasia
50%, often >80/90% of cells are composed of homogeneous lymphoblasts or atypical lymphocytes Plasma cells and other inflammatory cells are rare
a, DVM, PhD *,
Dorothee Bienzle,
b DVM, PhD
KEYWORDS Lymphoma Cytology Immunophenotyping Flow cytometry PCR for clonal antigen receptor gene rearrangement (PARR) Immunohistochemistry/immunocytochemistry KEY POINTS Cytologic assessment is diagnostic in most cases of diffuse large B-cell and diffuse lymphoblastic lymphoma in dogs. The cytologic diagnosis of lymphoma is more challenging in cats than in dogs. Although cytology is useful for staging lymphoma, histopathology is necessary for classification and grading. Immunophenotyping by flow cytometry allows evaluation of lymphocyte populations using a panel of antibodies, and serves as an adjunctive tool for both diagnosis and prognosis. Polymerase chain reaction (PCR) to detect clonal antigen receptor gene rearrangement (PARR) is a relatively new test in veterinary medicine that has strong potential for supporting the diagnosis of lymphoma. However, false-positive and false-negative results may confound the diagnosis, and PARR is less sensitive in cats than in dogs. Immunohistochemistry and immunocytochemistry should not be used as stand-alone diagnostic techniques, nor should an interpretation be based on a single antibody label.
INTRODUCTION
Lymphoma is the most common hemolymphatic malignancy in dogs and cats and, similar to lymphoma in people, is a heterogeneous disease with variable clinical signs and response to therapy.1 Patient genetics, immunocompetence, location, and morphologic subtype all contribute to the heterogeneity of the disease and prognosis (Box 1).
Funding Sources: None. Conflict of Interest: None. a Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, 1925 Coffey Road, Columbus, OH 43210, USA; b Department of Pathobiology, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G 2W1, Canada * Corresponding author. E-mail address: [email protected] Vet Clin Small Anim 43 (2013) 1331–1347 http://dx.doi.org/10.1016/j.cvsm.2013.07.004 vetsmall.theclinics.com 0195-5616/13/$ – see front matter Ó 2013 Elsevier Inc. All rights reserved.
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Box 1 Clinical evaluation of lymphoma Several options are available for the diagnosis and characterization of lymphoma. This review helps in choosing the best test or tests for the patient.
Anatomically, lymphoma can be characterized as multicentric, alimentary, mediastinal, or extranodal. In dogs, multicentric lymphoma accounts for 80% to 85% of reported cases, and diffuse large B-cell lymphoma is the most common histomorphologic variant.2 However, other types of B-cell lymphomas as well as T-cell lymphomas also are frequently diagnosed. The prognosis of lymphoma variants in dogs depends not only on the type of neoplastic lymphocyte but also on the location, characteristics of the cells, and the stage of disease. In cats, the diagnosis is more challenging because lymphoma more commonly affects extranodal sites, particularly the alimentary and upper respiratory tract.3,4 Lymphoma affecting the gastrointestinal tract is common but particularly challenging to diagnose, owing to the relative inaccessibility for sampling and potential progression from lymphocytic inflammation to neoplasia. Other types of lymphoma in cats often contain a heterogeneous population of neoplastic lymphocytes plus reactive lymphocytes, plasma cells, and other inflammatory cells. The diagnosis of lymphoma classically depended on morphologic characteristics identified by cytology and/or histopathology. In recent years, additional assays such as immunocytochemistry (ICC), immunohistochemistry (IHC), immunophenotyping by flow cytometry, and polymerase chain reaction (PCR) to detect clonal antigen receptor gene rearrangement (PARR) have been used to assist in the diagnosis of lymphoma and to classify lymphoma for prognostic purposes. Diagnostic tests are not perfectly sensitive or specific; therefore multiple assays are often used in conjunction or in sequence to enhance the accuracy of diagnosis and assist with prognosis. This article considers the utility and pitfalls of each diagnostic tool (Box 2). CYTOLOGY
Cytologic examination of blood films and samples obtained by fine-needle aspiration (FNA) of tissues or fluids is commonly used in the diagnosis of lymphoma in dogs and cats. Advantages of cytology are that sample collection and slide examination are rapid and can be performed in-house with the minimal resources of glass slides, a Romanowsky stain such as Diff Quick or Wright Giemsa, and a high-quality microscope. Limitations of FNA in comparison with an incisional or excisional biopsy are that FNA does not allow assessment of tissue architecture, and material for additional studies (eg, IHC) is unavailable. Blood sampling and aspiration of superficial masses Box 2 Tools available for the diagnosis and characterization of lymphoma Cytology Histopathology Immunocytochemistry and immunohistochemistry Phenotyping by flow cytometry Polymerase chain reaction to detect clonal antigen receptor gene rearrangement (PARR)
Lymphoma Diagnostics for Small Animals
and peripheral lymph nodes are safe and simple techniques for most patients. FNA of internal organs such as the liver or spleen or abdominal or thoracic masses is more challenging, but is still commonly performed and often is diagnostically rewarding. Lymphocyte Morphology
Lymphocyte cytomorphology is characterized by cell size, nuclear features, and cytoplasmic features (Fig. 1). It is important when using cytomorphology to examine only those cells that are both intact and adequately spread out on the slide. Diagnosis of Lymphoma by Cytology
The cytologic diagnosis of lymphoma is the most straightforward if the tumor is diffuse and the entire node is replaced by a uniform population of large neoplastic lymphocytes; this is the most common type of lymphoma in dogs. Most diffuse lymphomas yield FNA that consist entirely or predominantly of large neoplastic cells. However, diffuse lymphomas composed of small or intermediate cells can be difficult to diagnose by cytology, because the cells more closely resemble benign lymphocytes. In contrast to diffuse lymphoma, follicular lymphoma, which is uncommon in dogs, may yield a heterogeneous population of benign and malignant cells of variable size and morphology, which is challenging to interpret by cytology.
Fig. 1. Lymphocyte cytomorphology. Fine-needle aspiration (FNA) of lymph node from a dog (Wright Giemsa stain, original magnification 1000). (A) One neutrophil (lower left) and 4 small lymphocytes: these lymphocytes are smaller than a neutrophil (arrow) and have a dense round nucleus that comprises the majority of the cell. Nucleoli are not seen. Cytoplasm is scant (sometimes only a very thin rim is visible). (B) One intermediate lymphocyte (arrow) and 2 small lymphocytes (arrowheads): intermediate lymphocytes are similar in size to neutrophils, have more abundant cytoplasm that is typically lightly basophilic, and may contain small azurophilic granules. Nuclei are often slightly eccentric within the cell and have less condensed chromatin. Indistinct nucleoli may be seen. (C) Four large lymphocytes and one neutrophil (arrow): large lymphocytes, also called lymphoblasts, are equal to or greater in size than neutrophils and contain round to oval nuclei with fine or stippled chromatin. Nucleoli are commonly seen. A rim of deeply basophilic cytoplasm surrounds the nucleus. Occasionally (especially in cats) the cytoplasm may contain punctate vacuoles, as shown in the 2 large lymphocytes at the bottom of this panel. (D) One reactive lymphocyte (arrow), 2 small lymphocytes (arrowheads), and 1 neutrophil: reactive lymphocytes are similar in morphology to small lymphocytes but slightly larger, and have more abundant, more deeply basophilic cytoplasm. (E) Several plasma cells: plasma cells are medium to large cells that contain small, round, eccentrically placed nuclei with coarsely clumped chromatin. Cytoplasm is abundant, deeply basophilic, and often contains a prominent perinuclear, clear zone that corresponds to the Golgi apparatus. The Golgi apparatus is clearly shown in the 2 plasma cells in the center.
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Lymphoma in cats less commonly affects lymph nodes, and more commonly affects other tissues such as the intestine, nasal cavity, kidneys, liver, stomach, thymus, and spinal cord. In addition, those feline lymphomas that involve 1 or several peripheral lymph nodes often comprise a heterogeneous population of small and large lymphocytes as well as plasma cells and macrophages, making the diagnosis of lymphoma in cats by cytology more challenging than in dogs. Hence, when lymph node FNA from either dogs or cats yield heterogeneous cell populations, reactive hyperplasia must be considered as a differential diagnosis (Table 1). Histopathology, in conjunction with ancillary diagnostic tests, should be used for additional characterization of the cell population. Supporting Cytologic Features
There are several cytologic features (Fig. 2) that are more often associated with lymphoma than with benign lymphocyte proliferations, and these may be helpful in establishing a diagnosis. However, these features may be seen in both neoplastic and reactive populations and are not pathognomonic. Staging of Lymphoma by Cytology
The World Health Organization staging scheme is based on the degree of metastasis, invasiveness, and presence of clinical signs. Cytology is useful in the staging of lymphoma by helping to identify the degree of metastasis (Box 3). Limitations of Cytology
Diagnosis of small-cell and intermediate-cell lymphoma by cytology is challenging. Diagnosis in these cases may require additional supportive evidence such as generalized lymphadenopathy, identification of cells with similar cytologic features in multiple tissues, and lack of detection of infectious agents. Additional diagnostic tests such as histopathologic evaluation, flow-cytometric analysis, or PARR may be required for diagnosis. Another cytologic diagnostic challenge is differentiating early lymphoma and reactive hyperplasia. In both processes, the percentage of lymphoblasts (large lymphocytes) will be increased within an overall heterogeneous population of lymphocytes. In these cases, examination by flow cytometry may also reveal a heterogeneous lymphocyte population, and therefore may be less specific than histopathology or PARR. Many lymphomas in cats are composed of such heterogeneous cell populations, and are therefore challenging to diagnose by cytology. Sample Requirements
Sample collection and processing for FNA cytology of lymphoid tissue is relatively straightforward, and has been described in numerous texts and continuing education
Table 1 Differentiating lymphoma and reactive hyperplasia Lymphoma
Hyperplasia
50%, often >80/90% of cells are composed of homogeneous lymphoblasts or atypical lymphocytes Plasma cells and other inflammatory cells are rare
