Human lymphoproliferative diseases are a heterogeneous group of disorders comprised of over 50 subtypes. It is essential to distinguish among the different forms because each subtype has discrete risk factors, epidemiologic characteristics, and outcomes. For example, there are several types of mature T-cell lymphomas. One form, angioimmunoblastic T-cell lymphoma (AITL), is uniquely associated with hyperglobulinemia and immunologic dysregulation. This basis for this association recently was explained by the discovery that this tumor arises from follicular helper T cells—T cells whose function is to provide help for B-cell proliferation, isotype switching, and somatic hypermutation. Survival with this disease is less than 5 years. In contrast to AITL, lymphoblastic T-cell lymphoma/leukemia (T-LBL) is derived from immature T cells and, although it is clinically aggressive, unlike AITL it is curable. AITL is seen almost exclusively in adults, whereas T-LBL is seen in both children and adults. A large number of other subtypes of T-cell lymphoma, including peripheral T-cell lymphoma, adult T-cell leukemia/lymphoma, and anaplastic large cell lymphoma, all have unique diagnostic features, outcomes, and treatments. Contemporary classification of human lymphoma utilizes the World Health Organization scheme, which includes morphologic, genetic, and immunophenotypic characteristics.
A recent study validated the reproducibility of this system for the successful classification of lymphoma in dogs, demonstrating that a large group of veterinary pathologists without specific expertise in hematopathology could reach consensus on a series of over 200 lymphomas. This study described the histologic features of 3 aggressive lymphomas in dogs as well as 3 subtypes of indolent lymphomas. The indolent lymphomas were marginal zone lymphoma and follicular lymphoma (both B-cell diseases) and a single T-cell disease called T-zone lymphoma (TZL). Human TZL is a morphologic variant of peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) characterized by clonal expansion of T-zone lymphocytes that manifest a unique architectural and cytomorphologic pattern. Although the true incidence of canine TZL is unknown, 2 publications suggest that it is relatively common, comprising between 15.5 and 62% of all canine indolent lymphomas.[7, 8]
Although there are very few studies addressing the clinical outcomes of histologically defined subsets of canine lymphoproliferative diseases, those that are available illustrate the clinical utility of classification.[9, 10] Ponce et al demonstrated that a small clear-cell variant of TCL (n = 5), which was most analogous to TZL, has a prolonged survival (median overall survival, 21 months), whereas other histologic forms of TCL had a significantly shorter survival, with the lowest being plasmacytoid TCL (median survival, 3 months).[10, 11] Similarly in a recent, larger study, Flood-Knapik et al demonstrated a 33-month overall survival for 37 cases of TZL. These descriptions of a biologically indolent variant of canine TCL stand in stark contrast to the more commonly reported 6-month survival time for grouped canine TCL and serve as a powerful reminder of the importance of lymphoma classification.[12, 13]
Many, and perhaps most, cases of lymphoma in dogs are diagnosed by fine-needle aspiration cytology (FNAC), in large part because of the greater expense and invasiveness of biopsy and histopathology. Unfortunately, there are no data evaluating canine FNAC samples for their utility in subclassification by the current WHO algorithm. However, FNAC samples are amenable to immunophenotyping, either by flow cytometry (FC) or immunocytochemistry. Thus, if histologic subtypes of lymphoma could be accurately identified by FC immunophenotyping using a constellation of surface markers, vital diagnostic and prognostic information could be obtained without the need for a surgical procedure. Moreover, in light of recent reports indicating that dogs with indolent lymphoma, including TZL, are likely to undergo multiple lymph node aspirates yielding either inconclusive or erroneous results, demonstration of the use of FC in the diagnosis of TZL could result in a more rapid, less invasive primary diagnostic tool.[5, 8]
Multiparameter FC immunophenotyping is used routinely in the diagnosis of human lymphoproliferative disease and is increasingly being used in veterinary medicine to provide important diagnostic and prognostic information. Most of the entities in the WHO classification scheme require the detection of multiple cell surface proteins. For example, chronic lymphocytic leukemia/small cell lymphoma can be distinguished from mantle cell lymphoma (both B-cell diseases) by the levels of expression of a series of proteins, including CD5, CD10, CD20, CD22, and CD23.
The current study was initiated because of observations made during an investigation of CD4 TCL. This investigation is described in the accompanying paper, in which we found that although most CD4+ TCL have poor outcome, a subset of these dogs has an indolent clinical course. All 6 indolent lymphomas were CD45−, whereas the aggressive cases were all CD45+. Because of the indolent clinical course, we hypothesized that these cases were TZL. The objective of the study described here was to determine if this novel immunophenotypic characteristic could be used as a tool for the diagnosis of canine TZL.
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Our study demonstrated that 100% of 20 cases of T-cell neoplasia characterized by loss of CD45 expression were histologically defined as TZL, and none of the CD45+ cases were given this histologic diagnosis. The group of dogs in our study resembles dogs in a previous report of TZL in that Golden Retrievers were the dominant breed, 50% of the dogs had lymphocytosis, and the disease followed an indolent course. Furthermore, an earlier report by Valli et al demonstrated similar prolonged survival in 10 dogs with TZL. These parallels demonstrate good consistency in the histologic definition of TZL by different pathologists.
This study advances our ability to recognize canine TZL by describing consistent immunophenotypic features that can be used to identify this disease by FC. The characteristic CD45− T cells are readily identified with 2 color flow cytometry, and knowledge of this phenotype can help resolve cases where the distinction between lymphoid hyperplasia and lymphoma is difficult to make histologically. The high levels of expression of CD21 and class II MHC, which are components of many flow cytometry panels, can further establish the TZL phenotype. Our findings closely mirror a recent study in which cases described cytologically as “small clear cell” have a consistent CD45−, CD21-high phenotype. The small clear-cell cytologic appearance is thought to indicate T-zone lymphoma, although histology was not available in this study. An earlier study, however, suggested that a subset of cases defined as “small clear cell” by histology lacked expression of CD45. Taken together, these studies, carried out by 2 different institutions using different flow cytometry panels, indicate consistent identification of this constellation of antigen expression. To avoid potentially misclassifying such tumors as B cell in origin by flow cytometry, it is important to be aware that a subset of T-cell lymphomas expresses high levels of CD21. The immunophenotype of this T-cell disorder contrasts sharply with the immunophenotype of more aggressive CD4 TCL described in the companion paper making the distinction between these 2 types of T-cell lymphoma straightforward.
The disease described in this population of dogs is called “T zone” because of its histologic similarity to the human disease of the same name. Histologically, human TZL is characterized by infiltration of affected tissues with a uniform population of small- to medium-sized cells with an abundant volume of clear cytoplasm, which expands existing T zones. Morphologically, the canine cases presented here mimicked their human counterpart and, moreover, they mimicked previous reports of canine TZL. However, despite these morphologic similarities, we recognize the hazards of applying a well-defined, human classification scheme developed over many years of experience to a much less well-characterized disease in dogs, and that the 2 diseases may not be comparable. In people, TZL is not a distinct classification, but a rare variant of a broader category of TCL called PTCL-NOS (peripheral T-cell lymphoma—not otherwise specified), which includes a number of other entities without a distinct category.
In human medicine, TZL comprises only 1.5% of all cases of PTCL-NOS. Although we do not have histologically confirmed incidence or prevalence data in dogs, 10% of all suspected lymphoproliferative disorders submitted to the Clinical Immunology service for immunophenotyping had the characteristic features of TZL (T-cell, loss of CD45). Two other publications indicate that TZL is relatively common, comprising between 15.5 and 62% of all canine indolent lymphomas, which themselves comprise up to 29% of all canine lymphomas.[7, 8] The very limited data available suggest that the overall survival for TZL in people (14 months, 20–30 months) is similar to what is seen in dogs (21 months, this study, and 33 months). Owing to their shorter natural life span, although this outcome in people is considered poor, in dogs it is considered good.
The neoplastic T cells exhibit aberrant antigen expression in that they do not express CD45. There does not appear to be a normal, CD45− T-cell counterpart described in mice or people, and in the course of immunophenotyping canine lymphomas and leukemias, we have not seen evidence for CD45− T cells in the blood or lymph nodes in normal dogs or in reactive lymph nodes. Thus, it seems likely that loss of CD45 is an event related to neoplastic transformation of these cells, but we do not know if it plays a role in this process, or if it is an epiphenomenon related to other changes. Preliminary data provided no evidence of loss of the telomeric end of chromosome 7, where the CD45 gene is located (M. Breen and S. Culver, personal communication).
CD45 is a tyrosine phosphatase with a complex role in the regulation of signaling through the T-cell receptor, and in the regulation of cytokine receptor activation. It is a heavily glycosylated protein that is recognized by the carbohydrate-binding protein gal-1, a member of the galectin family.  One possible mechanism linking the absence of CD45 with TCL is that binding of surface CD45 by galectin in both immature (thymic) and activated T cells induces apoptosis. The loss of CD45 expression may allow T cells to escape deletion in the thymus or to evade apoptotic signals in the periphery leading to eventual neoplastic transformation. The role of CD45 in T-cell signaling and apoptosis is extraordinarily complex, however, and any number of additional mechanisms may be proposed.
The CD45− T cells have many features of activated T cells. First, they express higher levels of class II MHC, a characteristic of activated human T cells. In the dog, T cells express class II MHC constitutively, but an increase in the level of expression may be seen with antigen activation. Second, the T cells express high levels of CD25 when compared with nonneoplastic T cells in the same sample. CD25 is the alpha chain of the interleukin 2 receptor, and is expressed on activated effector CD4 and CD8 T cells as well as regulatory T cells. The heterogeneity in phenotype (CD4 and CD8 expression) suggests that the tumor cells are more likely to have arisen from activated effectors than regulatory T cells, but gene expression profiling and functional studies would be necessary to draw conclusions regarding the true lineage of these cells. Finally, the T cells express high levels of CD21. CD21 is a complement receptor, a receptor for Epstein-Barr virus, and a receptor for interferon alpha. In mice, CD21 is expressed at low levels on naïve T cells, and is upregulated significantly on memory T cells. Taken together, this constellation of antigen expression suggests that these neoplastic T cells may arise from an activated precursor T cell. Additional studies focused on the origin of these cells will be very useful in identifying potential triggers for neoplastic transformation.
In this study, 53% of dogs with TZL presented with a lymphocytosis at the time of diagnosis, which is comparable to the data reported by Flood-Knapik, who reported lymphocytosis in 47.5% of their cases. In addition, the median absolute lymphocyte count between the 2 reports (7,753 lymphocytes/μL versus 9,212 lymphocytes/μL) is similar. Surprisingly, 100% of the dogs with histologically confirmed TZL in which peripheral blood was available for FC analysis (n = 12) had neoplastic cells in the peripheral blood (detected by the presence of CD45− T cells), although nearly half had a normal absolute lymphocyte count. While there may be some academic debate regarding the nomenclature ascribed to the question of whether to call a disease stage V lymphoma with peripheral blood involvement or leukemia, these findings emphasize the importance of not using peripheral blood count as a screening tool for the absence of neoplastic cells.
Dogs that present without evidence of lymph node involvement have the same epidemiologic characteristics as those that do. Thus, we hypothesize that both types of presentation (primary disease in the lymph node and primary disease in the blood) are manifestations of a single disease entity. Definitive demonstration of this would require (1) full clinical staging, including bone marrow examination and follow-up on both types of cases, and (2) more detailed molecular description of the T cells involved using gene expression profiling to establish that these cells have a common origin. Nonetheless, the fact that both groups of patients have T cells with an identical, aberrant phenotype and share epidemiologic characteristics suggests that further pursuing this idea would be worthwhile. A similar situation is seen in human B-cell chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma, which is now considered a single entity regardless of the major site of involvement. At least 1 group of veterinary pathologists has proposed merging B-cell CLL and small cell lymphoma in dogs.
One remarkable finding of our population study was that Golden Retrievers comprise almost half of the cases of TZL. This observation points to a strong genetic risk factor for this disease. The unique immunophenotype of the neoplastic cells makes it feasible to recognize even small numbers of these cells in the peripheral blood, and it is likely that the disease could be identified long before it is clinically apparent. Prospective analysis of healthy Golden Retrievers as they age could help determine if early diagnosis is possible, and would provide a powerful model system in which to follow progressive changes in neoplastic cells from their earliest detection.
When viewed with the accompanying report, it is clear that T-cell lymphoproliferative disease is heterogeneous, and thus determining only if a dog has T-cell disease (using clonality studies or immunocytochemistry) without further characterization by histology or flow cytometry can create a misleading clinical picture. We demonstrate here that flow cytometry can be used to accurately identify a common form of indolent T-cell lymphoma, providing clinicians with a minimally invasive way of obtaining a specific, clinically relevant diagnosis.