FLOW CYTOMETRY FOR THE DIAGNOSIS AND THE CHARACTERIZATION OF CANINE LYMPHOPROLIPHERATIVE TUMORS

ABSTRACT Flow cytometry (FC) is a diagnostic technique in continuous development and, to date, it plays a fundamental role in human medicine for the diagnosis and the classification of hematopoietic tumors. Several studies state that the information obtained from the FC analysis in addition to defining the immunophenotype of the tumor cells, hence the origin of the neoplasia, can help to predict its biological behavior (Swerdlow et al., 2016). FC has thus become a simple and objective method to characterize hematopoietic tumors in order to obtain also valuable prognostic information. In veterinary medicine, FC is increasingly adopted for the diagnosis, staging, and monitoring of hematopoietic tumors, but despite advances in the generation and validation of antibodies for the use in dogs, the characterization of such neoplasms remains challenging (Wilkerson et al., 2005; Comazzi and Gelain, 2011). The aim of this doctoral project is to describe some less frequent subtypes of lymphomas and leukemia of the dog via FC, in order to define its biological behavior and to investigate whether there is any variable of prognostic value among all the factors analyzed. For this purpose, four studies will be illustrated; the first is a retrospective work, aiming to evaluate pre-analytical factors that may affect the diagnostic utility of FC in samples of lymph node aspirates. The work included 987 cases selected in the period 2009-2015, in which a lymph node aspirate was sent to our laboratory with suspect of lymphoproliferative disease. In order to define any possible bias affecting the outcome of the FC diagnosis, the variables analyzed were related to the animal (breed, sex, age), related to the operator (year, season, method of delivery to the laboratory, referring veterinarian) and related to the sample (type of material, cell concentration, presence of cytological slides, presence of artifacts). Of the factors considered, the sample cellularity and the presence of dead cells were the ones that most influenced the possibility of obtaining an adequate diagnosis. FC was, however, conclusive in almost all the samples, that were characterized by good quality and adequate sampling conditions. The study focused on TZL, a peculiar canine lymphoma with an indolent behavior, aimed to characterize this entity from a clinical and pathological point of view. The first phase of the work was retrospective, with the aim of describing clinical presentation and outcome of 51 cases selected between 2009 and 2014. The second phase of the study was aimed at clarifying the origin of the peculiar CD45 negative T-immunophenotype (Martini et al., 2013; Seelig et al., 2014); specifically, we confirmed the absence of the surface protein by means of two different techniques (flow cytometry and immunohistochemistry) and verified whether the transcript and the gene encoding the protein were present. The results confirmed that this type of lymphoma has indolent behavior with long survival times, despite being often diagnosed at the V stage of the disease. Furthermore, we can note that the origin of the phenotypic aberration is probably attributable to transcription factors, given the absence of transcription associated with the presence of the corresponding genomic tract. The objective of the third study was focused on the description of the biological behavior of nodal-type marginal lymphoma (nMZL). Although in literature it is classified as indolent lymphoma, some publications reported cases with a rather aggressive behavior (Flood-Knapik et al., 2012; Valli et al., 2013; Aresu et al., 2015; Marconato et al., 2015). Clinical information was collected from 35 retrospectively selected nMZL cases, with complete staging and standardized therapies. In our cohort, this lymphoma did not show and indolent behavior, with a generalized involvement and with short survival times, almost overlapping with the high-grade diffuse B-cell lymphoma (DLBCL). Thanks to the results of this work, discussions could be opened on the correct therapeutic approach. The latest study aimed to evaluate new antigens for the diagnosis and stratification of patients with CLL-B. The expression of the ZAP70 and CD38 markers in human medicine is closely related to the progression of the disease, with much lower survivals in patients with expression over specific thresholds (Rossi et al., 2010; Sulda et al., 2012). In the present project, these markers were evaluated for the first time in 37 blood samples of dogs with chronic B-cell leukemia, together with CD25 and ki67. Clinical data of the cases were obtained and survival analysis finally revealed that ZAP70 is a potential prognostic marker, providing bases for further studies with larger case studies and standardized therapy. The results of my doctoral project confirm that FC is a good technique for the study of the clinical-pathological aspects of lymphoproliferative tumors of dogs, and provides useful information to complete the biological profile of these tumors, also laying the foundations for future investigations on the usefulness of the proposed new markers.