1: Cytology of (WHO) Lymphoma

The cytology of (WHO) lymphoma Dr Bill Vernau BSc, BVMS, DVSc, PhD, Diplomate ACVP Professor of Clinical Pathology, VM PMI, School of Veterinary Medicine, University of California, Davis, USA 95616. [email protected]. Lymphoma has been classified in animals, primarily dogs, in many ways, often using adaptations of schemes developed for classification of human lymphoma. All major classification schemes distinguish follicular from diffuse lymphomas. Diffuse nodal lymphomas are the most common form of lymphoma in animals, and they may be of B or T cell origin. In most species, including dogs, B cell tumors account for 60-80% of all lymphomas. The majority of B cell lymphomas originate from cells present in follicular structures, and most commonly from cells present in the germinal centers of secondary (stimulated) follicles. Immunoglobulin (Ig) V segment somatic hypermutation occurs in the germinal center, in the process of affinity maturation (of immunoglobulin production). However, the processes involved in Ig V segment somatic hypermutation are known to target genes other than Ig genes. Constitutively greater rates of hypermutation probably explains the (much) greater incidence of B cell lymphomas compared to T cell lymphomas. Lymphomas may arise in lymphoid tissues or in non-lymphoid tissues such as intestinal mucosa and skin. Lymphomas often infiltrate multiple sites as a consequence of cell trafficking. In people, major problems existed with many of the (previously used) lymphoma classification schemes, especially poor intra and inter-observer reproducibility. Additionally, none of these systems was shown to be superior in predicting survival, the major goal of all classification schemes. Furthermore, recognition of new entities rendered many of these systems obsolete. In the early 1990’s, an informal group of 19 hematopathologists from Europe, the United States and Asia had a series of meetings and reached consensus on a new approach to lymphoma classification. It was decided that all available information would be assessed and possibly used to define lymphomas. The data included clinical features, tumor topography, cell morphology, immunophenotype, genetic and molecular genetic characteristics and clinical behavior. Different criteria were given variable importance in defining each of the different lymphoma subtypes. The cellular morphology was always important, and in some instances was the defining feature. Architecture or pattern was singularly important in defining follicular lymphomas. The emphasis on defining diseases based on utilization of all available information represented a new paradigm in recognizing lymphoma subtypes. And so, the Revised European American Lymphoma (REAL) classification was born. The inter and intra-observer reproducibility of this classification system (>85%) was shown to be better than for all other previous lymphoma classification schemes. Subsequently, the World Health Organization (WHO) convened more than 50 pathologists from around the world, as well as a large clinical advisory committee. This group largely adopted the REAL classification system, that, in 2001, became the new, revised World Health Organization (WHO) classification of tumors of hematopoietic and lymphoid tissues. This classification scheme has been widely accepted and utilized in human medicine. A number of publications have demonstrated the use of the WHO system in animals and this was the new approach utilized by the lymphoma study group of the American College of Veterinary Pathologists (ACVP). It is clear from this work that there are distinctive subtypes of canine and feline lymphoma that correspond to equivalent human entities as defined by the WHO lymphoma classification scheme. An important issue is that lymphoma is an encompassing term for a number of discrete and different clinical and morphological entities with quite different outcomes. Yet treatment strategies in animals often do not take this heterogeneity into account, typically treating most lymphomas with the same or similar protocols. It is likely that the WHO scheme will also become the pervasive scheme in veterinary medicine. Consequently, a generic diagnosis of lymphoma will no longer be sufficient as clinicians increasingly demand more specific information in order to better manage patients with lymphoma. Ideally, a specific WHO lymphoma type is diagnosed by a veterinary pathologist and that specific lymphoma type is considered when making decisions regarding therapy. In applying the WHO classification scheme to canine and feline lymphomas, it is apparent that some critical assessments are ARCHITECTURAL eg. follicular lymphoma, marginal zone lymphoma, and hence confirmation CANNOT be made via cytology alone, although cytologic findings may suggest the (high) likelihood of a particular entity whose confirmation subsequently requires histopathologic assessment. Additionally, in applying the WHO classification scheme to animal lymphomas, it is apparent that morphological assessments often require immunochemical stains to enable an accurate diagnosis. In some instances, molecular clonality determination by lymphocyte antigen receptor gene rearrangement analysis (see below) is also needed to confidently diagnose lymphoma versus lymphoid hyperplasia or inflammation. WHO classification of tumors of hematopoietic and lymphoid tissues The following table lists the entities that were reproducibly recognized by non-specialist veterinary pathologists in the ACVP lymphoma study group. B cell neoplasms T cell neoplasms Diffuse large B cell lymphoma - centroblastic, Lymphoblastic T cell lymphoma immunoblastic, anaplastic variants T cell / histiocyte rich large B cell lymphoma Peripheral T cell lymphoma (unspecified) Marginal zone lymphoma T-zone lymphoma Follicular lymphoma Anaplastic large T cell lymphoma Mantle cell lymphoma Enteropathy associated T cell lymphoma Lymphoblastic B cell lymphoma Mycosis fungoides / Sezary syndrome Extramedullary plasmacytoma Hepatosplenic T cell lymphoma Subcutaneous panniculitis-like T cell Multiple myeloma lymphoma Burkitt-like lymphoma Lymphomatoid granulomatosis (controversial) As stated above, while few WHO lymphoma types can be diagnosed definitively with the use of cytology alone, cytologic findings can be highly suggestive for several of the entities. This is indicated below where appropriate. Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma in dogs. DLBCL typically arises in lymph nodes from centroblasts in the dark zone of the germinal center. The lesion rapidly effaces the cortical architecture of the lymph node(s). Splenic involvement is also common. Neoplastic lymphocytes are large with nuclei > 2-4 red blood cells in diameter, large prominent nucleoli and a variable, often large, volume of deep blue cytoplasm. Nuclei tend to be very round, with less nuclear membrane irregularity than is often present with other lymphoma types (especially T cell types). Three major cytologic variants are recognized – centroblastic with neoplastic lymphocytes having multiple large prominent peripheralized nucleoli (90% of cases), immunoblastic, with >90% of neoplastic lymphocytes having a single, large, central, prominent nucleolus and anaplastic. The cytologic appearance of these types is quite distinctive and cytologic assessment can be very suggestive of this lymphoma type. However, at a minimum, adjunctive immunophenotyping is required and architectural assessment excludes other, more uncommon, possibilities. DLBCL is a high grade lymphoma with a high proliferative fraction; it is treated with aggressive chemotherapy. T cell and histiocyte rich B cell lymphoma (TCRBCL) is a another variant of DLBCL in which the bulk of the tumor is composed of reactive T cells and histiocytes; the neoplastic B cells are usually <10% of the cellular infiltrate. T cell / histiocyte rich BCL is most recognized in the horse where it usually occurs as single or multiple masses in the skin and subcutis. The lesions can wax and wane for years and may terminally progress to more aggressive DLBCL with dissemination beyond the skin. TCRBCL is uncommon in dogs and therefore poorly characterized at this time. As the lymphoma consists mostly of small, mature appearing lymphocytes with lower numbers of admixed large immature lymphocytes and other leukocytes, the diagnosis cannot be confirmed with cytology or histopathology alone. However, TCRBCL can be one of the differential diagnoses based on routine morphologic assessment, and confirmed with immunophenotyping and molecular clonality assessments. The immunophenotyping and molecular clonality assessments can be done on remaining cytologic smears / material or done on formalin fixed, paraffin embedded material if histopathologic biopsy is done subsequent to cytology. Marginal zone lymphoma (MZL) is a B cell lymphoma, which may involve lymph nodes, spleen or extranodal sites (gastrointestinal tract and respiratory tract). In the spleen, MZL may infiltrate the white pulp diffusely and / or it can lead to mass formation as a solitary lesion, presenting as a large splenic nodule (most common). Different morphologic cell types may be seen in MZL but the most common has nuclei approximately 1.5-2 red cells in diameter with a high nucleus to cytoplasmic ratio, single, large, central nucleoli and a continuous rim of cytoplasm. Despite this morphology, MZL is an indolent lymphoma with a low proliferative fraction (few mitoses); treatment of this lymphoma type with high grade lymphoma

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