Factors correlating with circulating T-zone-like cells of undetermined significance (TZUS) in dogs resident in Italy.

T-zone-like cells of undetermined significance (TZUS) share the same phenotypic pattern (CD45-CD5+) with T-zone lymphoma cells and were first described a few years ago in the peripheral blood (PB) of healthy aged American Golden retrievers (GR). History of bladder and eye disease increased the odd of circulating TZUS in the American GR population. Since differences among dogs may exist according to the geographical region of origin, herein we screened 489 PB samples to assess potential factors predisposing to the presence of circulating TZUS in dogs living in Italy. Overall, TZUS were found in 174 (35.6%) samples. Among 83 clinical variables, significant associations emerged with sex, age, diagnosis of neoplasia, history of neoplasia, history of infectious or parasitic disease, history of osteoarticular disease, presence of traumatic lesions or foreign bodies, and lymphocytes count. Only age and history of neoplasia retained significance at multivariate analysis (p=0.019 and p=0.036, respectively). Thus, older age and history of neoplasia are the main factors associated with circulating TZUS in Italian dogs. Future studies should focus on elucidating the biological role of TZUS and determining reproducible criteria for their identification, distinguishing them from infiltrating TZL.

Prognostic value of peripheral blood and bone marrow infiltration assessed by flow cytometry in dogs with de novo nodal peripheral T-cell lymphoma receiving alkylating-rich chemotherapy.

Peripheral T-cell lymphoma (PTCL) is highly aggressive in dogs and demonstrates a poor response to traditional chemotherapy. The aim of this retrospective study was to assess the prognostic significance of peripheral blood (PB) and bone marrow (BM) infiltration evaluated by flow cytometry (FC) in dogs with treatment-naïve and histologically confirmed PTCL. To be included, dogs had to undergo complete staging, including FC on lymph nodes, PB and BM samples. Additionally, dogs had to receive an alkylating-rich protocol and have a complete follow-up. Treatment response was evaluated based on RECIST criteria at each chemotherapy session, and the end-staging was conducted at the completion of treatment. Endpoints were time to progression (TTP) and lymphoma-specific survival (LSS). The relationship between TTP/LSS and the percentage of PB and BM infiltration, categorized as > 1%, > 3%, > 5%, > 10%, > 15% and > 20% was investigated. Fifty dogs were included: based on imaging and FC, 78.0% had stage 5 disease, 14.0% had stage 4, 6.0% had stage 3 and 2.0% had stage 1. By multivariable analysis, the CD4-negative phenotype was the only factor associated with a shorter TTP (P = 0.049), while BM infiltration was significantly associated with LSS (P = 0.037). Dogs with BM infiltration > 5% had shorter median LSS (114 days; 95%CI: 0-240) compared to dogs with BM infiltration ≤ 5% (178 days; 95%CI: 145-211). Lack of complete response (P = 0.039) and administration of corticosteroids before chemotherapy (P = 0.026) also significantly worsened LSS. BM flow cytometric evaluation could be considered an essential part of staging work-up for dogs with PTCL and has prognostic relevance.

Acute myelomonocytic leukemia with multifocal manifestation and spinal cord infiltration in a dog.

INTRODUCTION: Few cases of myelomonocytic leukemia associated with neurological signs have been described in dogs; none have been related to intraparenchymal spinal cord infiltration by neoplastic cells. This short communication describes a case of acute myelomonocytic leukemia subtype M4 in a dog with spinal cord infiltration. A 3-year-old male Golden Retriever was presented with a history of hyperthermia, lymphadenomegaly, leukocytosis with circulating blast cells, anemia and thrombocytopenia, and acute onset paraplegia. Immunophenotyping of peripheral blood by flow cytometry was consistent with acute myelomonocytic leukemia subtype M4. The dog was euthanized because of clinical deterioration and unfavourable prognosis. Postmortem examination revealed multi-organ neoplastic infiltration, including the spinal cord. To our knowledge, this is the first case of acute myelomonocytic leukemia subtype M4 in a dog with spinal cord infiltration. Our findings hold importance for including myelomonocytic leukemia in the differential diagnosis of patients with neurological signs due to spinal cord localisation.

INTRODUCTION: Peu de cas de leucémie myélomonocytaire associés à des signes neurologiques ont été décrits chez le chien ; aucun n'était lié à une infiltration intraparenchymateuse de la moelle épinière par des cellules néoplasiques. Cette courte communication décrit un cas de leucémie aiguë myélomonocytaire de sous-type M4 chez un chien avec infiltration de la moelle épinière. Un Golden Retriever mâle de 3 ans a été présenté avec une anamnèse d'hyperthermie, de lymphadénomégalie, de leucocytose avec des cellules blastiques circulantes, d'anémie et de thrombocytopénie et de paraplégie d'apparition aiguë. L'immunophénotypage du sang périphérique par cytométrie de flux était compatible avec une leucémie myélomonocytaire aiguë de sous-type M4. Le chien a été euthanasié en raison de la détérioration de son état clinique et du pronostic défavorable. L'examen post-mortem a révélé une infiltration néoplasique multi-organique, y compris la moelle épinière. À notre connaissance, il s'agit du premier cas de leucémie aiguë myélomonocytaire de sous-type M4 chez un chien avec infiltration de la moelle épinière. Nos résultats sont importants pour inclure la leucémie myélomonocytaire dans le diagnostic différentiel chez les patients présentant des signes neurologiques dus à une localisation médullaire.

Utility of flow cytometry in canine primary cutaneous and matched nodal mast cell tumor.

Mast cell tumors (MCT) are among the most frequent tumors in dogs, but studies regarding canine mast cell immunophenotype are lacking. The aim of this study was to assess the feasibility of flow cytometric analysis of MCTs, to describe canine MCTs immunophenotype(s), and to evaluate the ability of flow cytometry to detect mast cells in lymph node aspirates. Thirty-four primary canine MCTs and 12 draining lymph nodes were evaluated regarding the expression of CD117, IgE, CD11b, CD18, CD44, CD34, CD25 and CD45. Distinct populations attributable to mast cells and eosinophils were recognized based on light scatters and CD117 positivity. Common antigens (CD18, CD45, CD44) and CD117 were detected in all cases; positivity for IgE and CD11b was found in 28 (82%) and 23 (68%) cases respectively, while CD34 and CD25 were occasionally expressed. A single multicolor tube (IgE/CD117/CD11b/CD21/CD5) allowed the identification of mast cells in lymph nodes, showing a high correlation with cytology in quantifying mast cells infiltration. In conclusion, flow cytometric analysis can be applied to characterize canine MCTs and can be used to detect the presence of mast cells in lymph nodes. The immunophenotype abnormalities observed may be useful to confirm the neoplastic nature of such mast cells but the diagnostic usefulness of atypical antigen expression remains to be clarified.

Flow Cytometric Characterization of S-phase Fraction and Ploidy in Lymph Node Aspirates from Dogs with Lymphoma.

Canine lymphoma is a multifaceted disease encompassing numerous entities with different prognosis. Objective assessment of the proliferation rate is of importance from the pathological and clinical perspectives. Different methods have been described in the literature to assess proliferation rate, including evaluation of Ki67 expression in fresh lymph node (LN) aspirates measured by flow cytometry (FC). This test has a high accuracy in discriminating between low- and high-grade lymphomas, and provides prognostic information among high-grade B-cell lymphomas. DNA content analysis is less expensive and suitable for well-preserved samples. We describe DNA-content analysis using LN aspirates from 112 dogs with lymphoma. S-phase fraction (SPF) accurately discriminated between low- and high-grade lymphomas, with 3.15% being the best discriminating cut-off value. SPF values strongly correlated with Ki67 expression as assessed by FC. Survival analyses were restricted to 33 dogs with high-grade B-cell lymphoma receiving standardized multi-agent chemotherapy, but no significant result was obtained for SPF. We also describe a subset of aneuploid cases and their respective follow-up. We conclude that DNA content analysis may be combined with morphological examination of LN aspirates to improve the objectivity in lymphoma subtype classification in dogs. Further studies are needed to assess the possible prognostic role of SPF and ploidy status within specific lymphoma subtypes in dogs.

Canine nodal marginal zone lymphoma: Descriptive insight into the biological behaviour.

Canine nodal marginal zone lymphoma (nMZL) is classified as an indolent lymphoma. Such lymphomas are typified by low mitotic rate and slow clinical progression. While the clinical behaviour of canine splenic MZL has been described, characterized by an indolent course and a good prognosis following splenectomy, there are no studies specifically describing nMZL. The aim of this study was to describe the clinical features of and outcome for canine nMZL. Dogs with histologically confirmed nMZL undergoing a complete staging work-up (including blood analysis, flow cytometry [FC] on lymph node [LN], peripheral blood and bone marrow, imaging, histology and immunohistochemistry on a surgically removed peripheral LN) were retrospectively enrolled. Treatment consisted of chemotherapy or chemo-immunotherapy. Endpoints were response rate (RR), time to progression (TTP) and lymphoma-specific survival (LSS). A total of 35 cases were enrolled. At diagnosis, all dogs showed generalized lymphadenopathy. One-third was systemically unwell. All dogs had stage V disease; one-third also had extranodal involvement. The LN population was mainly composed of medium-sized CD21+ cells with scant resident normal lymphocytes. Histology revealed diffuse LN involvement, referring to "late-stage" MZL. Median TTP and LSS were 149 and 259 days, respectively. Increased LDH activity and substage b were significantly associated with a shorter LSS. Dogs with nMZL may show generalized lymphadenopathy and an advanced disease stage. Overall, the outcome is poor, despite the "indolent" designation. The best treatment option still needs to be defined.

Altered Erythropoiesis in Mouse Models of Type 3 Hemochromatosis.

Type 3 haemochromatosis (HFE3) is a rare genetic iron overload disease which ultimately lead to compromised organs functioning. HFE3 is caused by mutations in transferrin receptor 2 (TFR2) gene that codes for two main isoforms (Tfr2α and Tfr2ß). Tfr2α is one of the hepatic regulators of iron inhibitor hepcidin. Tfr2ß is an intracellular isoform of the protein involved in the regulation of iron levels in reticuloendothelial cells. It has been recently demonstrated that Tfr2 is also involved in erythropoiesis. This study aims to further investigate Tfr2 erythropoietic role by evaluating the erythropoiesis of two Tfr2 murine models wherein either one or both of Tfr2 isoforms have been selectively silenced (Tfr2 KI and Tfr2 KO). The evaluations were performed in bone marrow and spleen, in 14 days' and 10 weeks' old mice, to assess erythropoiesis in young versus adult animals. The lack of Tfr2α leads to macrocytosis with low reticulocyte number and increased hemoglobin values, together with an anticipation of adult BM erythropoiesis and an increased splenic erythropoiesis. On the other hand, lack of Tfr2ß (Tfr2 KI mice) causes an increased and immature splenic erythropoiesis. Taken together, these data confirm the role of Tfr2α in modulation of erythropoiesis and of Tfr2ß in favoring iron availability for erythropoiesis.

Prognostic significance of Ki67 evaluated by flow cytometry in dogs with high-grade B-cell lymphoma.

Ki67 can discriminate between high- and low-grade canine lymphomas, but its prognostic role in specific subtypes of the neoplasm is unknown. We assessed the prognostic significance of Ki67% (percentage of Ki67-positive cells), evaluated by flow cytometry, in 40 dogs with high-grade B-cell lymphoma, treated with a modified Wisconsin-Madison protocol (UW-25). The following variables were investigated for association with lymphoma specific survival (LSS) and relapse free interval (RFI): Ki67%, breed, sex, age, stage, substage, complete remission (CR). By multivariate analysis, Ki67% (P = 0.009) and achievement of CR (P = 0.001) were independent prognostic factors for LSS. Dogs with intermediate Ki67% (20.1-40%) presented longer LSS and RFI (median = 866 and 428 days, respectively) than dogs with low (median = 42 days, P < 0.001; median = 159 days, P = 0.014) or high (median = 173 days, P = 0.038; median = 100 days, P = 0.126) values. Determination of Ki67 is a prognostic tool that improves the clinical usefulness of flow cytometric analysis in canine high-grade B-cell lymphoma.

Chronic lymphocytic leukemia transformation into high-grade lymphoma: a description of Richter's syndrome in eight dogs.

Richter's syndrome (RS) is the development of an aggressive lymphoma in patients with chronic lymphocytic leukaemia (CLL). In humans, RS occurs in 2-20% of CLL, which transform into diffuse large B-cell lymphoma but reports in dogs are scarce. This study retrospectively describes eight dogs with CLL progressing into RS. A database including 153 dogs with CLL (93T CD8+ and 55 B-CLL) was interrogated and RS was demonstrated in eight cases (representing 5.2% of total CLL): two with T-cell (2.2% of T CLL) and six with a B-cell immunophenotype (10.9% of B-CLL). When RS occurred, lymphocytes were decreased compared to CLL. Five dogs had anaemia and two dogs thrombocytopenia. Frequent clinical signs included lymph node swelling, coughing, vomiting, neurological signs and weight loss. Independently from the therapy, RS was associated with a short survival (median 41 days). RS should be considered as an unfavourable evolution in canine CLL.

DNA methylation and targeted sequencing of methyltransferases family genes in canine acute myeloid leukaemia, modelling human myeloid leukaemia.

Tumours shows aberrant DNA methylation patterns, being hypermethylated or hypomethylated compared with normal tissues. In human acute myeloid leukaemia (hAML) mutations in DNA methyltransferase (DNMT3A) are associated to a more aggressive tumour behaviour. As AML is lethal in dogs, we defined global DNA methylation content, and screened the C-terminal domain of DNMT3 family of genes for sequence variants in 39 canine acute myeloid leukaemia (cAML) cases. A heterogeneous pattern of DNA methylation was found among cAML samples, with subsets of cases being hypermethylated or hypomethylated compared with healthy controls; four recurrent single nucleotide variations (SNVs) were found in DNMT3L gene. Although SNVs were not directly correlated to whole genome DNA methylation levels, all hypomethylated cAML cases were homozygous for the deleterious mutation at p.Arg222Trp. This study contributes to understand genetic modifications of cAML, leading up to studies that will elucidate the role of methylome alterations in the pathogenesis of AML in dogs.

European canine lymphoma network consensus recommendations for reporting flow cytometry in canine hematopoietic neoplasms.

BACKGROUND: Flow cytometry (FC) is assuming increasing importance in diagnosis in veterinary oncology. The European Canine Lymphoma Network (ECLN) is an international cooperation of different institutions working on canine lymphoma diagnosis and therapy. The ECLN panel of experts on FC has defined the issue of reporting FC on canine lymphoma and leukemia as their first hot topic, since a standardized report that includes all the important information is still lacking in veterinary medicine. METHODS: The flow cytometry panel of the ECLN started a consensus initiative using the Delphi approach. Clinicians were considered the main target of FC reports. A panel of experts in FC was interrogated about the important information needed from a report. RESULTS: Using the feedback from clinicians and subsequent discussion, a list of information to be included in the report was made, with four different levels of recommendation. The final report should include both a quantitative part and a qualitative or descriptive part with interpretation of the salient results. Other items discussed included the necessity of reporting data regarding the quality of samples, use of absolute numbers of positive cells, cutoff values, the intensity of fluorescence, and possible aberrant patterns of antigen expression useful from a clinical point of view. CONCLUSION: The consensus initiative is a first step toward standardization of diagnostic approach to canine hematopoietic neoplasms among different institutions and countries. This harmonization will improve communication and patient care and also facilitate the multicenter studies necessary to further our knowledge of canine hematopoietic neoplasms. © 2016 International Clinical Cytometry Society.

Identification of a suitable internal control for fluorescence analysis on canine peripheral blood samples.

Reliable detection of fluorescence intensity (FI) by flow cytometry (FC) is fundamental. FI depends on instrument settings and sample processing procedures: thus, measurements should be done using internal controls with known FI. Commercially available beads-based standards are expensive, thus reducing their usability in the veterinary practice. Cell subsets with stable mean FI (MFI) within the population have been proposed as acceptable surrogates in human medicine. In veterinary medicine, no data exist about stability of antigen expression among different subjects or upon sample storage. The aim of the present study was to evaluate MFI variability of main lymphocytes antigens among the lymphoid cells within each subject, among different subjects, and upon 24-h storage, in order to identify the antigen most suitable as stable internal control in MFI analyses. Peripheral blood samples from 18 healthy dogs were analysed by FC within 3h from sampling to assess the expression of CD3, CD5, CD4, CD8, CD21 and cyCD79b using conjugated monoclonal antibodies. Analyses were restricted to the lymphoid population. Fluorescent microbeads were added to each tube, and antigen MFI was calculated as Relative Fluorescence Intensity RFI (CD/beads). Fluorescence histogram CV (fhCV) for each CD was regarded as an index of the variability of expression among lymphocytes within each subject (cell-to-cell variability); whereas the CV of RFI was regarded as an index of inter-subjects variability (dog-to-dog variability). In 11 cases, FC analyses were repeated after 24h storage at 4°C and RFI and CVs of fresh and stored samples were compared to assess variability linked to storage. CD4 was identified as the best antigen to be used as an internal control for MFI analyses in canine peripheral blood samples because of low cell-to-cell and dog-to-dog variability, and optimal stability upon 24-h storage. Blood samples from a second group of 21 healthy dogs were labelled only with CD4, in order to assess the influence of breed, sex and age on the expression of CD4 in a larger case series. Based on univariate GLMs, none of these variables influenced CD4 RFI. Normalizing fluorescence data using lymphoid CD4 MFI as a reference would improve the comparison of results obtained by different laboratories, patients or times in diagnostic and research analyses of FI. Further studies are needed to confirm our results with different FC approaches.

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.

Enhanced therapeutic effect of APAVAC immunotherapy in combination with dose-intense chemotherapy in dogs with advanced indolent B-cell lymphoma.

The aim of this non-randomized controlled trial was to compare time to progression (TTP), lymphoma-specific survival (LSS), and safety of an autologous vaccine (consisting of hydroxyapatite ceramic powder and Heat Shock Proteins purified from the dogs' tumors, HSPPCs-HA) plus chemotherapy versus chemotherapy alone in dogs with newly diagnosed, clinically advanced, histologically confirmed, multicentric indolent B-cell lymphoma. The vaccine was prepared from dogs' resected lymph nodes and administered as an intradermal injection. Forty-five client-owned dogs were enrolled: 20 dogs were treated with dose-intense chemotherapy, and 25 received concurrent immunotherapy. Both treatment arms were well tolerated, with no exacerbated toxicity in dogs also receiving the vaccine. TTP was significantly longer for dogs treated with chemo-immunotherapy versus those receiving chemotherapy only (median, 209 versus 85 days, respectively, P=0.015). LSS was not significantly different between groups: dogs treated with chemo-immunotherapy had a median survival of 349 days, and those treated with chemotherapy only had a median survival of 200 days (P=0.173). Among vaccinated dogs, those mounting an immune response had a significantly longer TTP and LSS than those with no detectable response (P=0.012 and P=0.003, respectively). Collectively these results demonstrate that vaccination with HSPPCs-HA may produce clinical benefits with no increased toxicity, thereby providing a strategy for enhancing chemotherapy in dogs with advanced indolent lymphoma.

Flow cytometric evaluation of ki67 for the determination of malignancy grade in canine lymphoma.

Ki67 is a nuclear antigen significantly correlated with degree of malignancy in human non-Hodgkin lymphomas. We wanted to assess the ability of flow cytometric evaluation of Ki67 index (Ki67I) in differentiating the grade of malignancy in canine lymphomas. Ki67I was determined on lymph node aspirates of 90 immunophenotyped lymphomas classified according to the updated Kiel classification: 80 high grade (HG, 62 B cell and 18 T cell) and 10 low grade (LG, 3 B cell and 7 T cell) lymphomas. HG lymphomas showed significantly higher Ki67I compared with LG lymphomas (P < 0.0001). A significant difference in HG lymphomas was detected between B- and T-immunophenotypes. Receiver operating characteristic (ROC) curve highlighted a high accuracy of Ki67I in recognizing HG lymphomas [area under the curve (AUC) = 99.4] and a cut-off value of 12.2% was established (sensitivity = 96.3% and specificity = 100%). Thus, we suggest the combination of Ki67I flow cytometric determination and immunophenotype as a reliable tool to classify canine lymphomas.

Flow-cytometric detection of phenotypic aberrancies in canine small clear cell lymphoma.

Histopathology and immunohistochemistry are mandatory to solve the differential between canine low-grade lymphoma and reactive hyperplasia. However, clinicians and owners often show reluctance toward these invasive tests. However, molecular biology techniques are still not sensitive and specific enough to be regarded as a reliable tool for final diagnosis. In humans, flow cytometry (FC) allows a definitive diagnosis of T-cell lymphoma based on high prevalence of antigen aberrancies. We describe here the immunophenotype of 26 cases of suspect canine small-clear cell lymphoma, determined by multi-colour FC. All cases showed antigen aberrancies and therefore neoplasia was always confirmed. As a consequence, we argue that the combined use of cytology and FC allows solving the differential diagnosis between small clear cell lymphoma and non-neoplastic reactive conditions when histopathology is not available. Further studies are needed to establish if any aberrancy can be considered indicative of specific histotypes.

Haematological and physiological responses of Piemontese beef cattle to different housing conditions.

Public concern for animal welfare has progressively grown over the recent years. In this context, stress has a great economical impact on growth of animals and quality of animal products. The development and validation of methods to assess animal stress, particularly at the farm level, are desirable to evaluate animal production systems. Piemontese breed is traditionally tie-stall housed in the fattening period. Hence, the objective of this study was to characterise a profile of physiological and haematological changes of Piemontese beef cattle under different management conditions (tie-stall and loose housing). Our results suggest that the housing system is an important factor in animal welfare. Indeed, the values of the total protein, lysozyme, cortisol, serum and faecal corticosterone concentration and GR-α gene expression indicate that the tie-stall housing is more stressful than the loose system. All the alterations highlighted in this study considered together may be effective biomarkers of stress and disease susceptibility.

CD44 in canine leukemia: analysis of mRNA and protein expression in peripheral blood.

Hyaluronan receptor CD44 mediates interaction between cells and extracellular matrix. The expression of standard form and its variants is dysregulated in human leukemias and is associated with metastasis and prognosis. The aim of this work is the evaluation of CD44 mRNA and protein expression in canine leukemia. Peripheral blood from 20 acute leukemias (AL) (10 acute lymphoblastic, 6 acute myeloid and 4 acute undifferentiated leukemias), 21 chronic lymphocytic leukemias (CLL) and thirteen healthy dogs were collected. The mRNA expression of all CD44 variants presenting exons 1-5 and/or 16-20 (CD44_ex1-5 and CD44_ex16-20) and CD44 protein were determined by real-time RT-PCR and flow cytometry, using the mean fluorescent index (MFI), respectively. CD44 MFI was significantly higher in leukemic samples compared to controls and a higher expression was found in AL in respect with CLL. No significant differences were found when considering different phenotypic subtypes of AL and CLL. CD44_ex1-5 mRNA expression was significantly higher in AL compared to controls, whereas there was no difference in CLL compared to controls and AL. CD44_es16-20 showed the same trend, but without differences among groups. The high CD44 expression found in canine leukemias could be considered a step toward the definition of their molecular features.

VEGF and MMP-9: biomarkers for canine lymphoma.

Vascular endothelial growth factor (VEGF) and metalloproteinase (MMP) 2 and 9 are useful biomarkers in human lymphoma. During cancerogenesis, transforming growth factor beta (TGF-ß) stimulates VEGF and MMPs production. VEGF and TGF-ß plasma levels were tested by ELISA, MMP-2 and MMP-9 by gelatine zymography in 37 dogs with lymphoma, 13 of which were also monitored during chemotherapy. Ten healthy dogs served as control. Lymphoma dogs showed higher act-MMP-9 (P < 0.01) and VEGF (P < 0.05), and lower TGF-ß than controls, and a positive correlation between act-MMP-9 and VEGF (P < 0.001). Act-MMP-9 and VEGF were significantly higher in T-cell lymphomas, and in stage V compared with stages III-IV disease, regardless of immunophenotype. VEGF was higher in high-grade compared with low-grade T-cell lymphomas. No correlation was found between cytokines levels at presentation and outcome. During chemotherapy, act-MMP-9 and VEGF decreased in B-cell lymphomas (P < 0.01), suggesting a possible predictive role in this group of dogs.

Evaluation of tyrosine-kinase receptor c-kit mutations, mRNA and protein expression in canine lymphoma: might c-kit represent a therapeutic target?

c-kit plays an important role in proliferation, survival and differentiation of hematopoietic progenitor cells. In human hematopoietic malignancies, c-kit is mostly expressed by progenitor cell neoplasms and seldom by mature cell neoplasms. Aim of this study was to evaluate c-kit expression in canine lymphoma. Twenty-five B-cell lymphomas and 21 T-cell lymphomas were enrolled in the study. c-kit mRNA and protein expression was measured in lymph node fine needle aspirates by quantitative real-time RT-PCR, flow cytometry and immunocytochemistry, while the occurrence of KIT mutations on exons 8-11 and 17 was investigated by direct cDNA sequencing. KIT mRNA was amplifiable but below the limit of quantification in 76% of B-cell lymphomas and 33% of T-cell lymphomas. Remaining samples showed a very low expression of KIT, except for some high grade (HG) T-cell lymphomas where a comparatively higher mRNA amount was observed. Transcriptional data were confirmed at the protein level. No gain-of-function mutations were observed. Among canine lymphomas, T-cell lymphoma typically shows an aggressive biological behavior, partly being attributable to the lack of efficacious treatment options, and the evidence of c-kit expression in HG T-cell lymphomas might represent the rationale for its routinely diagnostic evaluation and the use of tyrosine kinase inhibitors in future clinical trials.