Diagnosis of Canine Lymphoid Neoplasia Using Clonal Rearrangements of Antigen Receptor Genes

Abstract

Abstract. Although the diagnosis of canine leukemia and lymphoma in advanced stages is usually uncomplicated, some presentations of the disease can be a diagnostic challenge. In certain situations, lymphoma and leukemia can be difficult to distinguish from a benign reactive proliferation of lymphocytes. Because clonality is the hallmark of malignancy, we have developed an assay that uses the polymerase chain reaction to amplify the variable regions of immunoglobulin genes and T-cell receptor genes to detect the presence of a clonal lymphocyte population. The assay detected clonally rearranged antigen receptor genes in 91% of the 77 dogs with lymphoid malignancy. Of the 24 dogs tested, that were either healthy or had clearly defined conditions not related to lymphoid malignancy, a clonally rearranged antigen receptor gene was found in one (a dog with Ehrlichia canis infection). Gene rearrangement was appropriate for the immunophenotype (immunoglobulin gene rearrangement in B-cell leukemias and T-cell receptor gene rearrangement in T-cell leukemias). Dilution analysis showed that the clonal rearrangement could be detected when 0.1-10% of the DNA was derived from neoplastic cells, depending on the source tissue. Potential applications of this assay include the diagnosis of lymphoma or leukemia in biopsy samples, cavity fluids, fine needle aspirates, bone marrow and peripheral blood; the determination of lineage (B or T cell); staging of lymphoma; and detection of residual disease after chemotherapy.Key words: Dogs; gene rearrangement; genes-immunoglobulin; leukemia; lymphoma; myeloma; receptorsantigen-T-cell.Detection of canine lymphocytic malignancies relies on the cytologic assessment of circulating lymphocytes or lymphoid tissue or on the histologic examination of the lymphoid tissue. The diagnosis is often straightforward but there are situations that present a diagnostic challenge. These include the early stages of lymphoma that may be difficult to distinguish from lymphoid hyperplasia; cavity fluids that contain large numbers of small, mature-appearing lymphocytes; cases with chronic or mild lymphocytosis; cases diagnosed by biopsy where the biopsy does not fully represent the lesion; and the finding of a small number of atypical lymphocytes in a fine needle aspirate or cavity fluid. In addition, mass aspirates can be limited by inadequate cell numbers or by aspirating a site that does not represent the lesion. Therefore, more sensitive and objective assays are desirable for detecting lymphoid malignancies in dogs.Because all lymphomas, lymphocytic leukemias, and myelomas are clonal expansions of lymphocytes, 1 Present address: Center for Cancer Causation and Prevention, AMC Cancer Research Center, Denver, CO. each particular neoplasm contains DNA regions that are unique in both length and sequence. The CDR3 region of both immunoglobulin and T-cell receptor (TCR) genes encodes the antigen-binding region of the respective receptor and contains the majority of this unique sequence. In B cells, CDR3 is produced ...

Establishment of Five Canine Lymphoma Cell Lines and Tumor Formation in a Xenotransplantation Model

“…PARR was performed as described previously [1,3]. Briefly, each sample was amplified by two sets of primers (major and minor) for IgH and one set of primers for TCRγ.…”

Applied Protein and Molecular Techniques for Characterization of B Cell Neoplasms in Horses

“…Here, we provide the first report of two molecular techniques that confirm the diagnosis of mature B cell lymphoma/leukemia in horses. Assessments of B cell IGHV clonality and sequencing have widely been used in human patients and infrequently in dogs with lymphoid neoplasia (leukemia and lymphoma) (5,39,40). Rearrangement of the IGHV region and expression of a functional B cell receptor occur during B cell development in the bone marrow, and mutations may follow during antigen-dependent development (41).…”

Development of High-Grade B-Cell Lymphoma Concurrent with T-Cell Chronic Lymphocytic Leukemia in a Dog

“…However, this contradicted the clinical finding in the peripheral blood (PB), which showed CLL. Therefore, a polymerase chain reaction for antigen receptor rearrangement (PARR) analysis (Kahotechno, Fukuoka, Japan) of the LN and PB samples was performed to detect the clonal expansion of lymphocytes [2]. Both samples exhibited rearrangements of the immunoglobulin heavy chain (IgH) gene (B cells) and the T cell receptor (TCR) γ gene (T cells) (Fig.…”

“…Both samples exhibited rearrangements of the immunoglobulin heavy chain (IgH) gene (B cells) and the T cell receptor (TCR) γ gene (T cells) (Fig. 3A, B) [2]. We also examined the immunophenotype of the LN sample using flow cytometry.…”

“…PARR analysis on days 1 and 8. On day 1, LN (A) and PB (B) samples showed IgH (major) [2] and TCRγ gene rearrangements. On day 8, PB (C) showed only TCRγ gene rearrangements.…”