Abstract: Chronic lymphocytic leukemia (CLL) is a hematologic disorder in dogs, but studies on prognostic factors and clinical outcome are lacking. In people, several prognostic factors have been identified and currently are used to manage patients and determine therapy.
Abstract: MicroRNAs (miRNAs) are posttranscriptional regulatory noncoding RNAs used to profile human hematopoietic tumors. In this study, some mature miRNAs was quantitated in peripheral blood from dogs with chronic lymphocytic leukemia (CLL). Relative expression data were normalised against four endogenous controls (let-7a, miR-17-5p, miR-26b, and miR-223) selected by geNorm analysis. The results revealed distinct miRNA patterns in CLL depending on the immunophenotype. Also in dogs, the different miRNAs expression could reflect developmental lineage and tumor differentiation. The similar genetics, physiology and exposure to environment in dogs and humans make the miRNA expression study in canine CLL attractive for comparative oncology.
Abstract: The elucidation of microRNA (miRNA) expression pattern in canine lymphoma is attractive for veterinary and comparative oncology due to similar genetics, physiology and exposure to environment in dogs and humans. In this work, the expression of a panel of mature miRNAs was quantitated in fresh-frozen and formalin-fixed paraffin-embedded (FFPE) lymph nodes from canine lymphoma. The major findings were: the detection of a panel of miRNAs expressed in canine lymph node; the identification of three suitable endogenous controls (let-7a, miR-16, and miR-26b) by NormFinder and geNorm analysis; the concordance between results obtained from fresh-frozen and FFPE samples; the detection of upregulation of miR-17-5p and miR-181a in B- and T-cell lymphomas respectively. This is the first study aimed to the application of miRNAs analysis in canine lymphoma.
Abstract: The diagnosis of canine lymphoma is a multi-step process involving clinical examination, diagnostic imaging, cytology, haematology, biochemical profiling, histopathology and ancillary techniques such as flow cytometry (FC). In human medicine, FC (in addition to cytology) is reported to increase the accuracy of diagnosis of most lymphoma sub-types. In dogs, FC can add a number of useful diagnostic features to the morphological evaluation of lymphoma including the evaluation of B or T cell lineage, antigen quantification and evaluation of aberrant patterns, the assessment of clonality, staging and the evaluation of minimal residual disease. In comparison to other immunophenotyping techniques, FC is rapid and easy to perform and 'gating' techniques can resolve mixed cell populations although the use of fresh samples is required and the appropriate equipment and its maintenance are quite expensive. The use of FC to refine cytological diagnosis could be further enhanced by the use of a multi-parametric approach and by the development of a wider panel of standardised canine-specific antibodies.
Abstract: Three dogs of different breeds, ages, and genders were presented with pale mucous membranes, depression, anorexia, and splenomegaly. Observed were severe normocytic, nor-mochromic, nonregenerative anemia, thrombocytopenia, and leukopenia. Blood smears contained large, atypical cells with blue vacuolated cytoplasm, cytoplasmic blebs, round to oval central nuclei, and elevated numbers of cytoplasmic fragment resembling macroplatelets. Bi- and multinucleated atypical cells were found mainly in spleen, lymph nodes, and bone marrow. A final diagnosis of acute megakaryoblastic leukemia (AMegL) was made based on morphology and positivity to the megakaryocyte-derived cell-specific markers von Willebrand factor and CD61. In case nos. 1 and 2, no treatment was initiated, and the dogs died on days 4 and 3, respectively. Case no. 3 received supportive therapy with prednisone, and after a brief improvement the dog died spontaneously 35 days after initial presentation. Only 11 cases of AMegL have been reported in dogs, and the specific diagnostic criteria have not been well established. The presence of vacuolization, cytoplasmic blebs, central round nuclei, cytoplasmic fragments, and multinucleated cells in these three cases were considered useful to differentiate AMegL from other hematopoietic neoplasms.
Abstract: Classification of leukemias requires specialized diagnostic techniques. Automated preliminary indicators of neoplastic cells in blood would expedite selection of appropriate tests.
Abstract: Zeta-chain-associated protein (ZAP-70) and spleen tyrosine kinase (Syk) are structurally and functionally homologous tyrosine kinases playing a role in the T- and B-cell signal transduction. Their activation can lead to lymphokine production, cytolitic activity, antibody secretion, cell proliferation, differentiation, survival and phagocytosis. Anomalous ZAP-70 and Syk expression is reported to be related to tumor formation and progression, and ZAP-70 immunoreactivity is a good prognostic marker of disease progression in human chronic lymphocytic leukaemia (CLL). Until now, to our knowledge, there are no reports about canine ZAP-70 and Syk expression profiles. In the present study, a RT-PCR procedure for the quali-quantitative evaluation of canine ZAP-70 and Syk transcripts was designed. The expression patterns of canine ZAP-70 and Syk mRNAs were evaluated in canine leukocyte subpopulations and in peripheral whole blood samples from healthy dogs and from dogs with different types of leukaemia. Similarly to humans, normal canine CD4+ and CD8+ T cells showed high expression of ZAP-70, whereas Syk was abundantly expressed in normal CD21+ B cells. The expression profile of ZAP-70 and Syk was markedly different in canine normal and leukaemic blood. Decreased Syk expression was detected in dogs with T-cell CLL, whereas decreased ZAP-70 expression was detected in dogs with B-cell CLL and B-cell acute lymphocytic leukaemia (ALL). The comparison of ZAP-70 and Syk mRNA levels between normal and leukaemic peripheral whole blood showed that the expression ratio ZAP-70/Syk is subjected to modification depending on the leukaemia status of patients. The results of the present work open an interesting topic for leukaemogenesis investigation and are the basis for further studies for a proper evaluation of the potential utility of these parameters for the diagnosis and prognosis of canine T- and B-cell leukaemia.
Abstract: A 6-year-old Bernese Mountain dog was presented with a history of lethargy and weight loss of 2 weeks duration. On physical examination the dog had pale mucous membranes and tachypnea. Ultrasound examination revealed hepatomegaly, splenomegaly, and mesenteric lymphadenomegaly. Results of a CBC included marked normocytic normochromic nonregenerative anemia, marked thrombocytopenia, and moderate leukocytosis with mild neutrophilia and a large population of unclassified round cells (6.2 x 10(3)/microL). The unclassified cells occasionally were bi- or multinucleated and had variably abundant pale basophilic cytoplasm that contained multiple irregular clear vacuoles and occasionally erythrocytes. Fine needle aspirate specimens of the mesenteric lymph nodes and spleen were composed of a population of round pleomorphic cells with the same features as the circulating cells. On flow cytometric analysis of peripheral blood, the unclassified cells expressed CD18, CD45, CD11c, CD1c, and CD14; immunocytochemical analysis of blood smears also indicated the cells were positive for CD1c, CD1a, and CD11c. The dog died a few hours after referral. The histologic interpretation of samples collected from spleen, liver, and lymph nodes was malignant neoplasia of histiocytic origin. Immunohistochemical staining yielded negative results for CD11d, a marker of red-pulp macrophages, ruling out hemophagocytic histiocytic sarcoma. Based on clinical and pathologic findings, the final diagnosis was disseminated histiocytic sarcoma (DHS) with peripheral blood involvement. To our knowledge, DHS in a dog with evidence and immunophenotyping of neoplastic cells in peripheral blood has been reported only rarely.
Abstract: Flow cytometry may be a useful tool to analyze lymphoma samples that are obtained from fine needle aspirations (FNA). This study aimed to determine if flow cytometric analysis add more objective and standardized information on the cellularity and morphology of lymphoma cells to conventional cytology. The typical immunophenotype of different lymphoma subtypes was assessed and leukocyte marker expression was evaluated to determine which antigens were more frequently over- or under-expressed in these lymphoma subtypes. Fifty FNA lymph node samples were evaluated from canine lymphomas. Thirty-one samples were identified to be of B-cell origin, sixteen were identified to be of T/NK-cell origin and three cases were classified as acute lymphoblastic leukaemia with lymph nodes involvement. The most common B-cell lymphoma subtypes were centroblastic lymphomas, whereas three cases were atypical and classified as B-large cell pleomorphic lymphomas. Among the T/NK lymphomas, small clear cells, large and small pleomorphic mixed cells, large granular lymphocytic cells and small pleomorphic cells were identified. Aberrant phenotypes and/or antigen under/over regulation was identified in thirty out of forty-seven lymphoma cases (64%; 18/31 B-cell=58% and 12/16 T-cell=75%). In B-cell lymphomas the most frequent finding was the diminished expression of CD79a (45%). CD34 expression was also observed in four cases (13%). Among T-cell lymphomas the prevalent unusual phenotype was the under-expression or absence of CD45 (25%). These findings reveal flow cytometry may be useful in confirming the diagnosis of lymphoma, as the technique allows one to add useful information about morphology of the neoplastic cells and identify antigenic markers and aberrant phenotypes.
Abstract: The sialylation pattern of serum alpha1-acid glycoprotein (AGP) in non-symptomatic cats infected by feline coronavirus (FCoV) and its possible relationship with the amount of FCoVs shed in faeces were investigated. Blood from three specific pathogen-free cats (group A) and from 10 non-symptomatic FCoV-positive cats from catteries with low (group B, three cats) or high (group C, seven cats) levels of faecal shedding were collected monthly. AGP was purified from serum and Western blotting followed by lectin-staining of alpha(2,3)-linked and alpha(2,6)-linked sialic acid. Faecal shedding was quantified in group C by quantitative polymerase chain reaction. Variations of AGP sialylation were recorded only in cats from group C, on which viral shedding peaked before the occurrence of feline infectious peritonitis (FIP) in the cattery, and decreased 1 month later, when serum AGP had an increase of alpha(2,3)-linked sialic acid. These results suggest that hypersialylation of AGP may be involved in host-virus interactions.
Abstract: Previous studies have demonstrated that the concentration of alpha1-acid glycoprotein (AGP) transiently increases in asymptomatic cats infected with feline coronavirus (FCoV). In order to establish whether these fluctuations depend on the FCoV status, the serum concentration of AGP and anti-FCoV antibody titres and/or faecal shedding of FCoVs in clinically healthy cats from catteries with different levels of prevalence of FCoV infection were monitored over time. Serum AGP concentrations fluctuated over time in clinically healthy cats from the cattery with the highest prevalence of feline infectious peritonitis (FIP) and significantly increased just before an outbreak of FIP. Further studies are required to clarify whether the observed increase of AGP concentration is a consequence of the increased viral burden or a protective response against mutated viral strains. Nevertheless, the results of the present study suggest that AGP might be useful in monitoring FCoV-host interactions in FCoV-endemic catteries.
Abstract: In dogs, flow cytometry is used in the phenotyping of immunologic cells and in the diagnosis of hemic neoplasia. However, the paucity of specific antibodies for myeloid cells and B lymphocytes and of labeled antibodies for multicolor techniques limits the ability to detect all leukocyte subpopulations. This is especially true for neoplastic and precursor cells. CD18 and CD45 are expressed on all leukocytes and are involved in cell activation, and together could be useful in helping determine cell lineage.
Abstract: In this study, the cytokine profiles of clinically healthy cats naturally infected with feline coronavirus (FCoV), of cats with feline infectious peritonitis (FIP) and of specific pathogen-free (SPF) cats were investigated in whole blood using a traditional reverse-transcriptase polymerase chain reaction (RT-PCR) assay and a semi-quantitative method of analysis based on computerised quantification of positive bands. The low inter-assay coefficient of variation recorded demonstrated that this method is highly repeatable. Compared with SPF cats, cytokine production was upregulated in most of the samples from FCoV-positive non-symptomatic cats. The appearance of a case of FIP in the cattery was associated with an increased expression of cytokines, in particular there was an increased production of IL-1beta and IFN-gamma, suggesting that these cytokines might protect infected cats from the disease. This hypothesis was also supported by the low levels of IFN-gamma recorded in blood from cats with FIP.
Abstract: A 3-year-old, male, domestic shorthaired cat was presented with a 3-day history of anorexia and depression. The cat was moderately dehydrated, had pale, slightly icteric, mucous membranes, oral ulcerations, and mild hepatosplenomegaly. A feline leukemia virus (FeLV) antigen test was positive. CBC results obtained at initial presentation included severe normocytic, normochromic, nonregenerative anemia, severe thrombocytopenia, and marked leukocytosis (>100,000/microL) with 77% eosinophils. After 15 days of treatment with prednisone and doxycycline, the cat had persistent severe nonregenerative anemia (HCT 3.4%), thrombocytopenia (28,000/microL), and extreme eosinophilia (total eosinophils, 123.1 x 10(3)/microL; segmented 103.0 x 10(3)/microL; immature 20.1 X 10(3)/microL). Cytologic examination of aspirates from bone marrow, liver, lymph nodes, and spleen revealed a predominance of mature and immature eosinophils, many with dysplastic changes. The M:E ratio was 96.4. On histopathologic examination, multiple organs were infiltrated by eosinophilic granulocytes. Neoplastic cells in blood and bone marrow stained positive for alkaline phosphatase and were negative for myeloperoxidase, chloroacetate esterase, and alpha-naphthyl acetate esterase. On flow cytometric analysis of peripheral blood, the neoplastic cells were positive for CD11b and CD14. These findings were consistent with chronic eosinophilic leukemia. To our knowledge, this is the first report of chronic eosinophilic leukemia in a cat associated with naturally acquired FeLV infection, in which flow cytometry was used to characterize the neoplastic cells.
Abstract: Flow cytometry is useful to study lymphoid malignancies since it allows both immunophenotyping of neoplastic cells and quantification of antigen expression. CD18 and CD45 are commonly exposed membrane antigens with different levels of expression on blood leukocyte and neoplastic cells. The aim of this retrospective study was to semi-quantitatively evaluate the expression of CD18 and CD45 in dogs with different lymphoid malignancies with blood involvement and to compare results with those from healthy dogs and dogs with reactive diseases. Blood samples from 13 dogs with precursor lymphoid malignancies, 20 with mature neoplasms (either chronic lymphocytic leukaemia or lymphoma), of different immunophenotypes, were compared with 24 healthy dogs and 12 dogs with different reactive diseases. The median fluorescence intensity (MFI) for CD18 and CD45 was recorded on lymphoid and granulocytic populations using dual colour flow cytometry, and the ratio between MFI for lymphoid and granulocytic populations (L/N ratio) was calculated to compare the results obtained in different sessions using an internal control (granulocyte fluorescence intensity). Significant decreases in the L/N ratio were detected in neoplastic samples for both CD18 (either precursors or mature versus controls) and CD45 (either precursors or mature versus control), while using MFI only slight differences were detectable in CD45 between precursors and controls. Neoplastic cells often exhibited lower expression of the L/N ratio for CD18, and mainly for CD45, most likely due to a less mature pattern than normal cells and/or to an aberrant quantitative expression of surface antigen. Moreover, more than 50% of neoplastic lymphoid cells exhibited L/N ratios that were not within the values observed in controls for at least one antigen. Altered L/N ratios, in particular decreases of CD45, were mainly observed in precursor neoplasms and in T-cell neoplasms. Detection of altered expression of common antigens, and in particular a L/N ratio for CD45 lower than a value of 103% may be useful as a confirmation of pseudo-clonality thus helping in differentiating reactive and neoplastic lymphocyte expansions.
Abstract: alpha1-Acid glycoprotein (AGP) is considered one of the major acute phase proteins in cats. In humans, AGP is a heavily glycosylated protein that undergoes several modifications of its glycan moiety during acute and chronic inflammatory pathologies. In this paper we present the feline AGPs (fAGP) glycan moiety modifications in the course of two prevalent feline diseases, the FIV (feline immunodeficiency virus) dependent feline acquired viral immunodeficiency and the feline leukemia virus (FeLV) associated lymphoma. The glycan moiety of fAGP was investigated by means of the binding of its oligosaccharides residues with specific lectins. Four lectins were used: Sambucus nigra agglutinin I and Maackia amurensis agglutinin lectins were used to detect sialic acid residues, Aleuria aurantia lectin was used to detect L-fucose residues and Concanavalin A was used to evaluate the degree of branching. It was found that fAGP undergoes several post-translational modifications of its glycan pattern: in particular the degree of sialylation is increased in FeLV-positive cats diagnosed with lymphoma, while FeLV-positive that did not presented any specific clinical signs cats do not present any increase of expression of sialic acid on the surface. Furthermore, FIV induced a modification of the glycan moiety of fAGP, which however varied widely among individuals. In order to determine the number and the position of oligosaccharide chains, the cDNA sequence of fAGP was also determined. The translation of the mature fAGP coding sequence gave rise to a sequence of 183 residues, with five potential N-glycosylation sites, but also with seven potential phosphorylation sites.
