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EQUINE VETERINARY EDUCATION / AE / AUGUST 2019
IGH2
* IGH3 † Clonal control * KDEi
Fig 4: A multilobular, irregular mass (asterisk; lymphoma), which measured approximately 23 3 18 3 18 cm, enveloped significant vasculature, and caused compression of the pharynx, was located (asterisk) lateral and adjacent to the larynx (†: epiglottis) within the left retropharyngeal region.
sites have also been infrequently reported in regions such as the tongue (Rhind and Dixon 1999), palatine tissues (Lane 1985), urethra (Montgomery et al. 2009), bladder and spleen (Tanimoto et al. 1994). This report adds an additional case of solitary extranodal lymphoma. Equine lymphoma has also been subtyped into 14 categories based on the World Health Organization (WHO) classification criteria (Durham et al. 2013). The current case was diagnosed as TCRLBCL. TCRLBCL is the most common form of lymphoma in the horse, representing up to 87% of equine lymphoma diagnoses (Durham et al. 2013), and is frequently multicentric. Lymphoma can present diagnostic challenges in animals
(Burba et al. 1991; Saulez et al. 2004; Meyer et al. 2006; Marques et al. 2012; Gress et al. 2016), and PCR-based clonality testing has been helpful in other species (Gress et al. 2016). The process of clonal assessment by PARR testing has been previously described (Burnett et al. 2003; Thalheim et al. 2013; Keller et al. 2016), and has recently been described for lymphoma in horses (Meichner et al. 2017). Briefly, PARR testing assesses DNA from cells to ascertain if they come from the same cell line, termed monoclonal (typically neoplastic), or from different cell lines, termed polyclonal (typically reactive) (Avery 2009). Because PCR testing is an assay in which DNA is amplified, assessment of clonality can be accomplished on small sample size with a high level of sensitivity (Avery 2009). PARR testing is advantageously sensitive, identifying even one neoplastic cell in 100 cells (Avery 2009). PCR primers analyse for the conserved regions of receptors or genes (use the most specific lymphoid receptors) that flank hypervariable regions. PCR products are then separated by capillary gel electrophoresis. A single sized or sequence PCR product indicates clonality, while multiple PCR products indicate polyclonality, or a reactive process. The addition of immunophenotypic, genomic and molecular advancements have allowed for an ever-growing selection of diagnostics to characterise and more definitively diagnose lymphoma, particularly in animals (Strauchen 2004; Meyer et al. 2006). PARR testing is the only diagnostic available to
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Poyclonal control TCRG
Fig 5: Molecular clonality assessment of genomic DNA from a large retropharyngeal tumour in a horse was performed using capillary electrophoresis and BioCalculator analysis. Clonal and polyclonal controls (left) are shown for comparison. Electrophore- tograms from the horse’s tumour cells (right) for IgH2, IgH 3, KDEi and TCRG primers demonstrate reproducible (done in triplicate) clonal spikes using immunoglobulin heavy chain (IgH) and KDEi (B cell) primers (top three images on right). There is a broad polyclonal result when the neoplastic cells were assessed with T cell receptor gamma (TCRG) primers (bottom image on right). These findings supported the presence of a B cell neoplasm with T cell proliferation consistent with T cell rich, large B cell lymphoma. The presence of the second clonal peak (KDEi and TCGR) is a common artefact seen with capillary electrophoresis of this type (Keller et al. 2016).
veterinarians to verify cellular clonality. Final diagnosis in the current case was eventually made through a combination of cytology, histology, immunohistochemistry and PARR testing. Recently, PARR has been assessed using molecular techniques in horses with a rare condition of monoclonal gammopathy, B cell leukaemia and concurrent lymphadenopathy (lymphoma/leukaemia) (Badial et al. 2015). Additionally, PARR has been used to successfully identify diffuse intermediate to large B cell lymphoma with a robust haematogenous phase (TCRLBCL) in both blood and infiltrative tissue from a horse with multicentric disease (Meichner et al. 2017). To the authors’ knowledge, this is the first documented use of PARR in equine solitary extranodal lymphoma. In the dog, lymphoma is staged (I–V) for prognostication,
and recently, PARR has been used to provide a more sensitive testing for the presence of neoplastic cells in the
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