Marzia Cozzi

Canine small clear cell/T-zone lymphoma: clinical presentation and outcome in a retrospective case series

Published studies, taken together, suggest the existence of a single canine lymphoma entity, with a small clear cell appearance by cytological evaluation, a histopathological T-zone pattern and an aberrant CD45-negative T-cell phenotype, mostly characterized by long-term survival. We describe clinical presentation and outcome in a retrospective case series of canine small clear cell/T-zone lymphoma. Despite the reported predisposition of Golden retriever, this breed was not represented in our case series. Most dogs presented with stage V disease, whereas only few had clinical signs or peripheral cytopenias. Blood was almost always more infiltrated than bone marrow. Median survival confirmed the favourable prognosis described in literature, but a few dogs died within a short time. Also, a subgroup of dogs developed second malignancies, eventually leading to death. We did not investigate possible prognostic factors because of the wide variety in treatments, and further studies are needed to identify high-risk animals.

Prognostic factors in canine acute leukaemias: a retrospective study

Canine acute leukaemias (ALs) have a poor prognosis, with reported survival times (ST) of only a few weeks or months. Also, clinical studies assessing prognostic factors are lacking. This study aims to retrospectively assess variables that predict ST in dogs with AL, and to identify correlations between outcome and therapeutic protocols. Diagnosis and sub-classification into AL subtypes was made based on haematological findings, morphological assessment and flow cytometric immunophenotyping. Clinical-pathological features of AL subtypes at presentation concurred with those described in the literature. A normal neutrophil count at presentation significantly prolonged ST (P = 0.027). Additionally, there was a trend for anaemic dogs to have shorter survival compared with those without anaemia, and the incorporation of cytosine in the chemotherapy protocol produced a moderate but not significant increase in median ST for dogs with AL. Further prospective studies with standardized treatments are needed to confirm and improve our results.

Flow cytometry for feline lymphoma: a retrospective study regarding pre-analytical factors possibly affecting the quality of samples

Objectives Flow cytometry (FC) is becoming increasingly popular among veterinary oncologists for the diagnosis of lymphoma or leukaemia. It is accurate, fast and minimally invasive. Several studies of FC have been carried out in canine oncology and applied with great results, whereas there is limited knowledge and use of this technique in feline patients. This is mainly owing to the high prevalence of intra-abdominal lymphomas in this species and the difficulty associated with the diagnostic procedures needed to collect the sample. The purpose of the present study is to investigate whether any pre-analytical factor might affect the quality of suspected feline lymphoma samples for FC analysis. Methods Ninety-seven consecutive samples of suspected feline lymphoma were retrospectively selected from the authors’ institution’s FC database. The referring veterinarians were contacted and interviewed about several different variables, including signalment, appearance of the lesion, features of the sampling procedure and the experience of veterinarians performing the sampling. Statistical analyses were performed to assess the possible influence of these variables on the cellularity of the samples and the likelihood of it being finally processed for FC. Results Sample cellularity is a major factor in the likelihood of the sample being processed. Moreover, sample cellularity was significantly influenced by the needle size, with 21 G needles providing the highest cellularity. Notably, the sample cellularity and the likelihood of being processed did not vary between peripheral and intra-abdominal lesions. Approximately half of the cats required pharmacological restraint. Side effects were reported in one case only (transient swelling after peripheral lymph node sampling). Conclusions and relevance FC can be safely applied to cases of suspected feline lymphomas, including intra-abdominal lesions. A 21 G needle should be preferred for sampling. This study provides the basis for the increased use of this minimally invasive, fast and cost-effective technique in feline medicine.

Looking at genetic structure and selection signatures of the Mexican chicken population using single nucleotide polymorphism markers

Abstract Genetic variation enables both adaptive evolutionary changes and artificial selection. Genetic makeup of populations is the result of a long‐term process of selection and adaptation to specific environments and ecosystems. The aim of this study was to characterize the genetic variability of Méxicos chicken population to reveal any underlying population structure. A total of 213 chickens were sampled in different rural production units located in 25 states of México. Genotypes were obtained using the Affymetrix Axiom® 600 K Chicken Genotyping Array. The Identity by Descent (IBD) and the principal components analysis (PCA) were performed by SVS software on pruned single nucleotide polymorphisms (SNPs). ADMIXTURE analyses identified 3 ancestors and the proportion of the genetic contribution of each of them has been determined in each individual. The results of the Neighbor‐Joining (NJ) analysis resulted consistent with those obtained by the PCA. All methods utilized in this study did not allow a classification of Mexican chicken in distinct clusters or groups. A total of 3,059 run of homozygosity (ROH) were identified and, being mainly short in length (<4 Mb), these regions are indicative of a low inbreeding level in the population. Finally, findings from the ROH analysis indicated the presence of natural selective pressure in the population of Mexican chicken. The study indicates that the Mexican chicken clearly appear to be a unique creole chicken population that was not subjected to a specific artificial selection. Results provide a genetic knowledge that can be used as a basis for the genetic management of a unique and very large creole population, especially in the view of using it in production of hybrids to increase the productivity and economic revenue of family farming agriculture, which is widely present in México.

Breed-associated risks for developing canine lymphoma differ among countries: an European canine lymphoma network study

BackgroundCanine breeds may be considered good animal models for the study of genetic predisposition to cancer, as they represent genetic clusters. From epidemiologic and case collection studies it emerges that some breeds are more likely to develop lymphoma or specific subtypes of lymphoma but available data are variable and geographically inconsistent. This study was born in the context of the European Canine Lymphoma Network with the aim of investigating the breed prevalence of canine lymphoma in different European countries and of investigating possible breed risk of lymphoma overall and/or different lymphoma subtypes.ResultsA total of 1529 canine nodal lymphoma cases and 55,529 control cases from 8 European countries/institutions were retrospectively collected. Odds ratios for lymphoma varied among different countries but Doberman, Rottweiler, boxer and Bernese mountain dogs showed a significant predisposition to lymphoma. In particular, boxers tended to develop T-cell lymphomas (either high- or low-grade) while Rottweilers had a high prevalence of B-cell lymphomas. Labradors were not predisposed to lymphoma overall but tended to develop mainly high-grade T-cell lymphomas. In contrast with previous studies outside of Europe, the European golden retriever population did not show any possible predisposition to lymphoma overall or to specific subtypes such as T-zone lymphoma.ConclusionFurther prospective studies with more precise and consistent subtype identification are needed to confirm our retrospective results and to create the basis for the investigation of possible genes involved in different predispositions.

Prevalence of Golden retriever in European dogs with lymphoma: preliminary data

Introduction. Canine breeds, being genetic clusters, are good models for studies on genetic predisposition. Golden retriever (GR) has been described with a high incidence of both lymphoma overall (19%) and T zone lymphoma (TZL, 40%) with differences in different geographical areas in US. This breed predisposition is confirmed in Japanese but not in European (EU) case series although specific studies are still lacking. Aim of the present study is to investigate the prevalence of GR in a huge case series of canine lymphomas from different EU countries and to compare prevalence of different subtypes with studies in extra-EU countries, in order to support a possible different genetic predisposition. Materials and methods. Signalment data on 1734 consecutive cases of canine lymphoma collected from 9 different European countries are retrospectively analysed. When subtypes are available, cases are furtherly separated in three subtype groups: 1) B-cell lymphoma, 2) T-cell lymphoma-high grade, 3) TZL. Odds ratio (OR) for different lymphoma subtypes are calculated in comparison with mixed breed population, considered as control. Results. Overall prevalence of GR is 5.19% (range 1.59-7.32%) of lymphoma cases and differs from that reported in American and Japanese caseloads. Prevalence slightly varies among EU countries and no subtypes predilection is found if compared with mixed breed. Concerning Italian cohort, GR is not predisposed to develop a lymphoma when normalized for the breed prevalence (OR=1.49, 95% confidence interval=0.87-2.55, p=0.14). Discussion. Prevalence of lymphoma in EU population of GR is much lower than that of US. No predisposition is identified in EU GR for TZL differently from US and Japan. Being genetic of European GR population quite different from American and Japanese ones this suggest a possible different genetic predisposition. Slight differences in GR lymphoma prevalence among European countries likely reflects different breed distribution rather than different genetic predisposition.

Flow cytometry for feline lymphoma: a retrospective study about pre-analytical factors possibly affecting the quality of samples

Introduction Flow cytometry (FC) is an increasingly required technique on which veterinary oncologists rely to have an accurate, fast, minimally invasive lymphoma or leukemia diagnosis. FC has been studied and applied with great results in canine oncology, whereas in feline oncology the use of this technique is still to be experienced. This is mainly due to a supposed discomfort in sampling, because of the high prevalence of intra-abdominal lymphomas. The purpose of the present study is to investigate whether any pre-analytical factor might affect the quality of suspected feline lymphoma samples for FC analysis. Methods 97 consecutive samples of suspected feline lymphoma were retrospectively selected from the authors’ institution FC database. The referring veterinarians were recalled and interrogated about several different variables, including signalling, features of the lesion, features of the sampling procedure and the experience of veterinarians performing the sampling. Statistical analyses were performed to assess the possible influence of these variables on the cellularity of the samples and the likelihood of being finally processed for FC. Results None of the investigated variables significantly influenced the quality of the submitted samples, but the needle size, with 21G needles providing the highest cellularity (Table 1). Notably, the samples quality did not vary between peripheral and intra-abdominal lesions. Sample cellularity alone influenced the likelihood of being processed. About a half of the cats required pharmacological restraint. Side effects were reported in one case only (transient swelling after peripheral lymph node sampling). Conclusions FC can be safely applied to cases of suspected feline lymphomas, even for intra-abdominal lesions. 21G needle should be preferred for sampling. This study provides the bases for the spread of this minimally invasive, fast and cost-effective technique in feline medicine.

Genomic variability in Mexican chicken population using copy number variants

Copy number variants (CNVs) are polymorphisms which influence phenotypic variation and are an important source of genetic variability [1]. In Mexico the backyard poultry population is a unique widespread Creole chicken (Gallus gallus domesticus) population, an undefined cross among different breeds brought to Mexico from Europe and under natural selection for almost 500 years [2-3]. The aim of this study was to investigate genomic variation in the Mexican chicken population using CNVs. A total of 256 DNA samples genotyped with Axiom® Genome-Wide Chicken Genotyping Array were used in the analyses. The individual CNV calling, based on log-R ratio and B-allele frequency values, was performed using the Hidden Markov Model (HMM) of PennCNV software on the autosomes [4-5]. CNVs were summarized to CNV regions (CNVRs) at a population level (i.e. overlapping CNVs), using BEDTools. The HMM detected a total of 1924 CNVs in the genome of 256 samples resulting, at population level, in 1216 CNV regions, of which 959 gains, 226 losses and 31 complex CNVRs (i.e. containing both losses and gains), covering a total of 47 Mb of sequence length corresponding to 5,12 % of the chicken galGal4 assembly autosome. A comparison among this study and 7 previous reports about CNVs in chicken was performed, finding that the 1,216 CNVRs detected in this study overlap with 617 regions (51%) mapped by others studies. This study allowed a deep insight into the structural variation in the genome of unselected Mexican chicken population, which up to now has not been never genetically characterized with SNP markers. Based on a cluster analysis (pvclust – R package) on CNV markers the population, even if presenting extreme morphological variation, does not resulted divided in differentiated genetic subpopulations. Finally this study provides a CNV map based on the 600K SNP chip array jointly with a genome-wide gene copy number estimates in Mexican chicken population.

Loss of cd45 protein in canine small clear cell/t-zone lymphoma is due to absence of gene transcription.

Canine small clear cell/T-zone lymphoma (TZL) is a peculiar lymphoma subtype characterized by an indolent clinical course and aberrant CD45-negative phenotype, easily recognized by flow cytometry (FC). Recent studies have described clinical presentation and behavior, but to date the mechanisms for CD45-negativity have never been investigated. Aim of this study is to confirm the lack of surface protein using a different technique from FC and to investigate if CD45-absence in TZL is linked to the lack of the corresponding transcript and gene. 40 TZL cases and 17 controls (7 T-high grade lymphoma, 10 reactive lymphnodes) were included in the present study. Immunohistochemistry was performed with a different antibody respect with FC to confirm CD45 surface protein absence. Total RNA and genomic DNA were extracted from lymph-nodes aspirates. CD45 transcript amount was investigated by quantitative real-time RT-PCR and the corresponding gene fragment was analyzed by quantitative real-time PCR. ΔΔCt method was used for the relative quantification of transcript amount and DNA load compared to housekeeping genes. All TZL cases were negative for CD45 at immunohistochemistry. CD45 transcript amount was significantly lower in TZL compared to controls (p=0.000). This difference was not significant (p=0.584) for CD45 DNA load, that was similar between TZL and controls. These results highlight that CD45 protein is lacking on cell surface and gene transcription is absent in TZL, whereas the corresponding gene is not deleted. The data here reported support further studies for clarifying possible genomic or epigenomic factors involving CD45 gene transcription and for better clarifying the possible role of CD45 in lymphomagenesis.

Evaluation of CD45 protein expression and transcript in canine small clear cell/T zone lymphoma.

Canine small clear cell lymphoma is a peculiar lymphoma entity with T-zone histopathological pattern andindolent clinical course. From an immunophenotypic point of view the main feature is the lack of CD45 staining by flow-cytometry (FC), which accounts for >95% of cases. Underlying mechanisms have never been investigated. Aim of this work was to evaluate CD45 protein and mRNA expression in small clear cell lymphoma. Lymph nodes of 18 cases and 11 controls, with either reactive hyperplasia or CD45-positive high grade T-cell lymphoma, were investigated. FC was performed on lymph node fine needle aspiration and CD45 median fluorescence intensity (MFI) was then evaluated on small clear cells and normal residual T-lymphocytes. CD45 surface expression was also evaluated by immunohistochemical reaction on paraffin wax-embedded lymph node sections. Quantitative real-time RT-PCR was performed on cases and controls. Total RNA was isolated from cell suspension in RNA later. The generated CD45cDNA was amplified and ΔΔCt method was used for the relative mRNA quantification. CD45-MFI in neoplastic cells was <1% compared to normal residual T-lymphocytes in the same sample. Cells were also negative for CD45 stain on histopathological preparations. RT-PCR showed a significantly lower amount of CD45 transcript in neoplastic samples compared to controls, likely due to the residual population. Results showed the lack of CD45 surface antigen and the virtually absence of CD45-mRNA in small clear cell lymphoma. We hypothesize a possible genomic/epigenomic aberration; further studies are in progress to investigate the pathogenesis of this aberrancy and the possible linkage to lymphomagenesis.