Contents lists available at ScienceDirect Veterinary Immunology and Immunopathology journal homepage: www.elsevier.com/locate/vetimm Research paper Loss of CD45 cell surface expression in canine T-zone lymphoma results from reduced gene expression V. Martini a, ⁎ , M. Cozzi a , A. Aricò b , G. Dalla Rovere b , A. Poggi c , F. Albonico a , M. Mortarino a , E. Ciusani d , L. Aresu b , S. Comazzi a a Department of Veterinary Medicine, University of Milan, via Celoria 10, 20133 Milan, Italy b Department of Comparative Biomedicine and Food Science, University of Padua, via dell’Università 16, 35020 Legnaro, Padua, Italy c Department of Veterinary Sciences, University of Turin, Largo P. Braccini 2, 10095 Grugliasco, Turin, Italy d Laboratory of Clinical Pathology and Medical Genetics, Neurological Institute Foundation “Carlo Besta”, via Celoria 11, 20133 Milan, Italy ARTICLE INFO Keywords: CD45 Protein expression mRNA DNA transcription Lymphoma ABSTRACT Canine T-zone lymphoma (TZL) is a peculiar lymphoma subtype characterized by an indolent clinical course and aberrant CD45-negative phenotype, commonly recognized by flow cytometry (FC). Recent studies have described clinical presentation and behavior, but to date the mechanisms behind the loss of CD45 protein expression have never been investigated. The aims of this study were: 1) to confirm the absence of CD45 in canine TZL via the concomitant use of FC and immunohistochemistry with two different sources of antibody; and 2) to investigate the amount of CD45 transcript and the presence of CD45 gene in the neoplastic cells of dogs affected by TZL. 57 lymph node aspirates were included in the present study: 40 (70.2%) TZLs, 7 (12.3%) high grade T-cell lymphomas and 10 (17.5%) reactive lymph nodes. Neoplastic cells and normal T-cells were isolated from TZL and reactive lymph nodes, respectively, via cell sorting. Immunohistochemistry was performed on 2 TZL, 2 reactive lymph nodes and 2 Peripheral T-cell Lymphomas. Total RNA and genomic DNA were extracted from lymph-nodes aspirates. Two different quantitative real-time PCR experiments were designed, to determine the amount of the CD45 transcript and of the corresponding gene fragment. All TZL cases were negative for CD45 at immunohistochemistry. CD45 transcript amount was significantly lower in TZL compared to controls (p < 0.001). This difference was not significant (p = 0.584) for CD45 DNA load, that was similar between TZL and controls. Moreover, CD45 transcript amount was inversely correlated with the percentage of neoplastic cells in each TZL sample (p = 0.010). These results confirm that CD45 protein is lacking on cell surface irrespective of the technique and antibody source adopted. This phenotypic aberrancy is apparently due to the absence of gene transcription, as CD45 DNA was present, whereas CD45 transcript was virtually absent in the neoplastic cells. The data here reported support further studies investigating possible factors impairing CD45 gene transcription. 1. Introduction Dogs are frequently affected by hematopoietic malignancies, with lymphoma being the most represented. Nevertheless, many different entities are grouped together under the diagnosis of lymphoma, showing different clinical presentation and outcome (Valli et al., 2013). Small clear cell/T-zone lymphoma (TZL) subtype has been recently identified as a unique entity with a peculiar disease distribution at onset and with a typical indolent clinical course (Seelig et al., 2014; Martini et al., 2015a). Histologically, TZL shows neoplastic cells expanding the paracortex and medullary cords without effacing the nodal architec- ture; neoplastic cells are small lymphocytes with sharp, shallow nuclear indentations, unapparent nucleoli and a moderate volume of pale cytoplasm. Mitoses are rare (Valli et al., 2006). Cytological aspirates in dogs diagnosed with TZL are characterized by a prevalent population of small lymphoid cells with a clear cytoplasm and frequent hand mirror shape (“small clear cells” appearance). The mitotic index is low (Fournel-Fleury et al., 2002). Finally, a peculiar phenotype has been identified by flow cytometry (FC), characterized by the expression of a variable number of T-cell markers and loss of expression of CD45 (Martini et al., 2015b; Seelig et al., 2014). CD45 is a transmembrane protein tyrosine phosphatase expressed http://dx.doi.org/10.1016/j.vetimm.2017.03.006 Received 20 May 2016; Received in revised form 28 February 2017; Accepted 23 March 2017 ⁎ Corresponding author. E-mail address: valeria.martini@unimi.it (V. Martini). Veterinary Immunology and Immunopathology 187 (2017) 14–19 0165-2427/ © 2017 Elsevier B.V. All rights reserved. MARK