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Lymphoma is one of the most frequent hematopoietic tumors in dogs and shares similar features with human counterparts. MicroRNAs (miRNA, small non-coding RNAs) are pivotal in gene regulation fine tuning and cancer hallmarks are influenced by their aberrant expression. Consequently, miRNA biomarkers may assist predicting therapeutic response and clinical outcome by providing less-invasive novel diagnostics tools. The aim of this study was to detect dysregulated miRNAs in lymphomatous lymph node tissues in comparison to lymph node material or PBMCs from healthy control dogs. Potential significant differences in miRNA expression profiles between four lymphoma entities were evaluated. A customized PCR array was utilized to profile 89 canine target miRNAs. Quantification was performed using qPCR, relative expression was determined by the delta-delta Ct method, and p-values were calculated with student's t-test. In the 14 diffuse large B-cell lymphoma (DLBCL) patients, 28 and 24 different miRNAs were significantly dysregulated compared to lymph node material or PBMCs. Sixteen miRNAs occurred in both control groups, with 12 miRNAs being down- and four miRNAs being upregulated. The six peripheral T-cell lymphoma (PTCL) samples showed 24 and 25 dysregulated miRNAs when compared to the healthy controls. A combined analysis of DLBCL and PTCL samples revealed seven shared and 19 differently expressed miRNAs. Potential biomarkers in T- and B-cell lymphoma could be the miRNA-17/92 cluster and miRNA-181-family together with miRNA-34a and miRNA-150. Diagnostic utility of potential biomarkers must be validated in larger, prospective cohorts of canine lymphoma cases and in higher numbers of physiological patient material.
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Gastrointestinal lymphoma is the most common form of lymphoma in domestic cats. Aggressive phenotypes are much less common but do bear and unfavorable prognosis. Immunophenotyping by flow cytometry (FCM) is not systematically performed in these patients, because of difficulties in the acquisition of suitable sample material from the gastrointestinal tract. A multimodal diagnostic approach is recommended to improve identification of subtypes targeting patient tailored therapeutic strategies. The aim of this prospective study was to present results of multicolor FCM immunophenotyping in surgically removed gastrointestinal mass and relate them with histopathology using the World Health Organization (WHO) classification and clonality PCR testing. Thirty-two patients were included. Eight cats (25%) had gastric, 23 (72%) had intestinal lymphoma and 1 (3%) had gastric/jejunal lymphoma. Intestinal lymphoma sites were represented by 18 small intestinal, 4 ileocaecal, 1 large intestinal. All gastric lymphomas were diffuse large B-cell lymphoma (DLBCL). Small intestinal lymphomas were 10 enteropathy associated T-cell lymphoma type I (EATL I), 2 enteropathy associated T-cell lymphoma type II (EATL II), 2 peripheral T-cell lymphoma (PTCL), 3 DLBCL and one DLBCL+EATL II. The most common small intestinal FCM T-cell phenotype was CD3+CD21- CD4-CD8-CD18+ CD5-CD79- in 7/10 EATL I and one EATL II. The most frequent FCM B-cell phenotype was CD3-CD21+ CD4-CD8-CD18+ CD5-CD79+ in 13/17 DLBCL and the DLBCL+EATL II. Clonality PCR results were positive in 87.5% (28/32) of all cases. No cross-lineage rearrangement was observed. IHC and FCM results agreed in 87.5% (28/32) of all cases. When all 3 methods were combined, consistent results were seen in 75% (24/32). This is the first demonstration of a multicolor FCM approach set in context to the gold standard histopathology and clonality testing results.
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Lymphoma is the most common tumour of domestic cats, developing most frequently in the small intestine. Feline small intestinal lymphoma predominantly demonstrates a T-cell immunophenotype identified by standard immunopositivity for T cells with CD3 or immunopositivity for B cells with CD20. In contrast, a wide spectrum of immunohistochemical antibodies are applied in humans to diagnose the various specific lymphoma subtypes according to the WHO classification. Our aim was to augment our knowledge of immunophenotypes in feline non-B-cell lymphomas forming macroscopic masses in the intestinal tract. We evaluated the combined immunohistochemistry and flow cytometry findings from 15 cases. Neoplastic lymphoid cells were immunopositive for CD3 in 93% (14/15), granzyme B in 87% (13/15), CD5 in 20% (3/15), CD8 in 13% (2/15), CD4 in 7% (1/15) and CD56 in 7% (1/15) of cases. Cytotoxic granules indicating a cytotoxic origin of the neoplastic cells were identified by histopathology only in 13% (2/15) and by cytology in 47% (7/15) of the cases. Without immunohistochemical labelling of the cytotoxic protein granzyme B, the cytotoxic status would have been missed in 46% (6/13) of the cytological and in 85% (11/13) of the histopathological slides. These findings suggest that more complex immunophenotyping may advance our understanding and help prognosticate small intestinal T-cell lymphoma in cats.
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Enfermedades de los Gatos , Inmunofenotipificación , Neoplasias Intestinales , Gatos , Animales , Inmunofenotipificación/veterinaria , Enfermedades de los Gatos/patología , Enfermedades de los Gatos/inmunología , Neoplasias Intestinales/veterinaria , Neoplasias Intestinales/patología , Inmunohistoquímica , Masculino , FemeninoRESUMEN
OBJECTIVES: Cerebrospinal fluid (CSF) analysis is used in the diagnostic investigation of cats with epileptic seizures. The aim of this retrospective study was to evaluate the diagnostic value of CSF analysis in cats with epileptic seizures that have unremarkable brain MRI or only hippocampal signal changes. METHODS: Unremarkable brain MRI or MRI studies with signal alterations in the hippocampus only in cats with suspected epilepsy and CFS analysis performed at the Small Animal Internal Department or Diagnostic Imaging Department at Vetmeduni Vienna, Austria, between 2011 and 2017 were reviewed. Total nucleated cell count, total protein, blood contamination and cytology data from CSF analysis were evaluated. RESULTS: In total, 87 cats were included. Seventy cats (80.5%) had unremarkable MRI, five (5.7%) had hippocampal signal changes with contrast enhancement and 12 (13.8%) had hippocampal signal changes without contrast enhancement. Overall, four cats (4.6%) had abnormalities on CSF analysis; all (100%) had an increased total nucleated cell count (22 cells/µl, 7 cells/µl, 6 cells/µl and 6 cells/µl, respectively), and no cat had increased total protein (100%), although in one cat total protein was not evaluated. Three of these cats had unremarkable MRI and one had hippocampal signal changes without contrast enhancement. The median duration of epileptic signs prior to the MRI study was 2 days. CONCLUSIONS AND RELEVANCE: Our results show that, in our cohort of epileptic cats with unremarkable brain MRI or with hippocampal signal changes, CSF analysis was usually normal. This should be considered before performing a CSF tap.
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Enfermedades de los Gatos , Epilepsia , Gatos , Animales , Estudios Retrospectivos , Epilepsia/diagnóstico por imagen , Epilepsia/veterinaria , Convulsiones/veterinaria , Hipocampo/diagnóstico por imagen , Imagen por Resonancia Magnética/veterinaria , Enfermedades de los Gatos/diagnóstico por imagenRESUMEN
OBJECTIVES: Flow cytometric (FCM) immunophenotyping of lymphoid tissue aspirates is an available adjunct for feline lymphoma diagnostics. Reference data have only been established for feline peripheral blood. Studies investigating the composition of normal and mildly reactive feline lymph nodes (LNs) are lacking. The aim of this prospective study was to establish reference data for lymphocyte subpopulations in normal and mildly reactive feline peripheral LNs using a standardised multicolour panel of antibodies. METHODS: Macroscopically inconspicuous mandibular and/or popliteal LNs from 31 adult cats, which were euthanased for reasons other than haematological diseases, were excised and processed within 5 h after death. Multicolour flow cytometry using eight different feline-specific, anti-canine and human cross-reactive monoclonal antibodies used in current diagnostic marker panels was performed after cytological exclusion of pathological states and complemented by lymphocyte clonality testing, histopathology and immunohistochemistry (IHC) to ensure the absence of lymphoid disease. RESULTS: Of 31 cats, the immunophenotyping data of 24 individuals could be included as histopathology and clonality testing excluded a pathological condition. Lymphocyte populations showed the following positive antibody reactions: CD18+ 86.3% ± 13.86%, CD3+ 54.81% ± 11.10%, CD5+ 57.39% ± 12.66%, CD21+ 40.42% ± 12.40%, CD79alphacy+ (CD79αcy) 30.41% ± 13.49% and CD14+ 0.75% ± 1.35%. There were 30.88% ± 13.48% CD4+ and 12.91% ± 6.68% CD8+ cells. Cytology revealed a mixed population of mostly lymphoid cells in all samples. The absence of a monoclonal/oligoclonal neoplastic population was confirmed by lymphocyte clonality testing. Histopathology and IHC showed a normal or mildly reactive pattern in all cases. CONCLUSIONS AND RELEVANCE: This study establishes FCM immunophenotyping data of lymphocyte populations of normal and mildly reactive feline peripheral LNs. For the first time, anti-CD5, CD4, CD8 and CD21 reference data in normal and mildly reactive feline peripheral LNs are presented. CD18, CD3, CD14 and CD79αcy have been used to establish reference data for the first time in any feline material.
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Ganglios Linfáticos , Subgrupos Linfocitarios , Animales , Anticuerpos Monoclonales , Gatos , Citometría de Flujo/veterinaria , Inmunofenotipificación/veterinaria , Ganglios Linfáticos/citología , Estudios ProspectivosRESUMEN
Gastrointestinal lymphomas are uncommon in dogs and little is known about their distinct subtypes or proliferation rate. The aim of this study was to stratify 33 canine gastrointestinal lymphoma samples according to the latest World Health Organization classification and to determine the Ki67 proliferation index by manual counting, digital image analysis and visual estimation. The Ki67 index was then correlated with subtype, immunophenotype, mitotic index, grade and tumour location. The mitotic index correlated positively with the Ki67 index. A significantly higher number of Ki67-positive cells was found in enteropathy-associated T-cell lymphoma type I and in diffuse large B-cell lymphoma compared with enteropathy-associated T-cell lymphoma type II. There was also a significant difference in Ki67 immunolabelled cells between grade 1 and grade 2 lymphomas. Moderate agreement was found between the Ki67 index as obtained by manual counting and visual estimation, but there was strong agreement between manual counting and digital image analysis. The user-friendly digital imaging system used in this study could have potential for future determination of the Ki67 index in lymphoid neoplasms.
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Enfermedades de los Perros , Neoplasias Gastrointestinales , Linfoma de Células B Grandes Difuso , Animales , Proliferación Celular , Perros , Neoplasias Gastrointestinales/veterinaria , Antígeno Ki-67 , Linfoma de Células B Grandes Difuso/veterinaria , Índice Mitótico/veterinariaRESUMEN
Differentiation between resident mature lymphocyte populations and small-cell lymphoma cannot be made by cytological review alone and remains challenging in histopathological review. These cases warrant application of complementary tools like PCR-based immunoglobulin (IG) and T-cell receptor (TCR) clonality testing for confirmation. Although primer coverage of potential IG gene rearrangements in feline B-cell neoplasms constantly improves, the possibility of false negative and false positive test results still poses a problem. In this retrospective study, we assessed diagnostic sensitivity and specificity of a novel developed multiplex PCR assay for routine diagnosis of B-cell clonality. Therefore, 24 feline patients were subjected to comparative clonality testing by using different primer sets. Feline lymphoma cell lines and confirmed patient material served as positive control. Compared to previous studies, this novel developed multiplex PCR assay showed positive effects on diagnostic sensitivity, specificity, accuracy, and positive predictive value accompanied by a slight impairment of negative predictive value. Notably, none of the primer sets was superior; hence, we recommend the combined application of the herein tested primer sets in routine diagnostics. However, a more in-depth-evaluation of the dynamic of assay specific parameters in dependency on primer set usage requires prospective studies on larger cohorts of feline patients.
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Linfocitos B , Enfermedades de los Gatos , Linfoma de Células B , Animales , Enfermedades de los Gatos/diagnóstico , Gatos , Linfoma de Células B/genética , Linfoma de Células B/veterinaria , Reacción en Cadena de la Polimerasa/veterinaria , Estudios RetrospectivosRESUMEN
Recent literature suggests conventional flow cytometric (FCM) immunophenotyping complemented by Ki-67 FCM assessment as a reliable tool to classify canine lymphomas. Ki-67 expression assessed by FCM is rarely reported in canine lymphoma cases and reference data for normal canine lymph nodes are missing. Moreover, nothing is known about the Ki-67 expression within the occasionally observed remnant cell population within the gates of normal lymphocytes in lymphoma cases. Aim of this study was to compare flow cytometric Ki-67 expression of lymphocyte populations from normal canine lymph nodes, lymphoma cells from World-Health-Organisation (WHO) classified lymphoma patient samples and their neighboring normal remnant cell population. Cryopreserved lymphocyte cell suspensions from normal lymph nodes from eight dogs free of lymphoma served as reference material. Fourteen cases diagnosed by cytology, FCM, clonality testing, histopathology including immunohistochemistry consisting of 10 DLBCL, 1 MZL, 1 PTCL and 2 TZL showed a residual small lymphocyte population and were investigated. The Ki-67 expression in normal canine lymphoid tissue was 3.19 ± 2.17%. Mean Ki-67 expression in the malignant cell populations was 41 ± 24.36%. Ki-67 positivity was 12.34 ± 10.68% in the residual physiologic lymphocyte population, which otherwise exhibited a physiologic immunophenotype pattern. This ratio was equivalent (n = 3) or lower (n = 11) than the Ki-67 expression of the malignant cell population within the sample. This is the first report of FCM derived Ki-67 expression combined with immunophenotype patterns in normal canine lymph nodes, compared with lymphoma cell populations and residual normal cell populations of lymphoma cases diagnosed by state of the art technology.
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Recent literature suggests a combination of flow cytometric determination of Ki-67 and immunophenotype as a reliable tool to classify canine lymphomas. Immunohistochemistry (IHC) on histological samples is the gold standard technique assessing Ki-67 index. Agreement between IHC and FCM derived Ki-67 indices has never been investigated. The aim of this study was to investigate the agreement between IHC and FCM in the assessment of Ki-67 expression/index, in order to evaluate whether FCM may serve as a non-invasive alternative method for the estimation of proliferative activity in canine lymphoma. Dogs with previously untreated canine lymphoma undergoing diagnostic lymphadenectomy were prospectively enrolled. Ki-67 expression/index was assessed by FCM and IHC and expressed as percentage of positive cells. 39 dogs classified by histopathology matched the inclusion criteria. With both methods, Ki-67 expression/index was higher in intermediate/high-grade lymphomas. Spearman's coefficient of correlation was ρ = 0.57; (95% CI0.33-0.75) suggesting a moderate correlation. A Bland-Altman plot revealed a negative constant bias of -3.55 (95% CI: -10.52 to 3.42) with limits of agreement from -45.71 to 38.61. The study confirmed agreement albeit with wide confidence intervals between the values of Ki-67 expression/index assessed with FCM and IHC. Discrepancies were observed in a subset of cases. Possible explanation could be that Ki-67 index in IHC is determined in the most proliferative areas of the slide, which could introduce kind of sampling bias, whereas FCM evaluates many more cells in cell suspension. Further studies are warranted to investigate this phenomenon.
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Enfermedades de los Perros , Linfoma no Hodgkin , Animales , Enfermedades de los Perros/diagnóstico , Perros , Citometría de Flujo/veterinaria , Inmunohistoquímica , Antígeno Ki-67 , Linfoma no Hodgkin/diagnóstico , Linfoma no Hodgkin/veterinariaRESUMEN
Feline lymphoma, one of the most important malignant tumors in domestic cats, is also increasingly diagnosed in non-domestic felines, most notably, African lions (Panthera leo). The gold standard for the diagnosis of lymphoma is histopathological evaluation. As an additional tool, the PCR for antigen receptor gene rearrangement (PARR) has been established. To support the diagnosis on a molecular level, the PCR-based clonality assay is designed to distinguish between reactive and neoplastic lymphocyte populations. In general, PARR primers are used to target complete immunoglobulin heavy chain V-D-J (IGH-VDJ) and T-cell receptor gamma V-J (TRG-VJ) chain gene rearrangements. In this study, we validated the primer sets used in routine diagnostics of domestic cats for the application in non-domestic felines. Clonality testing was used in 41 non-domestic feline species and the results were interpreted in the light of their clinical history and their pathology. In total, clonality could be detected in 8 non-domestic felines (19.4%), including 3 lymphoma cases confirmed by histopathology. These results confirmed the successful application of domestic feline-specific PARR primers in non-domestic feline species. Diagnostic sensitivity and specificity of the clonality assay were 100% and 88%, respectively. Finally, the overall diagnostic accuracy was 89%.
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Felidae , Linfoma/veterinaria , Reacción en Cadena de la Polimerasa/veterinaria , Animales , Estudios de Cohortes , Femenino , Reordenamiento Génico , Linfocitos , Linfoma/diagnóstico , Linfoma/genética , Masculino , Receptores de Antígenos de Linfocitos T gamma-delta , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Canine 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. RESULTS: A 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. CONCLUSION: Further 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.
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Enfermedades de los Perros/etiología , Linfoma/veterinaria , Animales , Enfermedades de los Perros/epidemiología , Perros , Europa (Continente)/epidemiología , Linfoma/epidemiología , Linfoma/etiología , Linfoma de Células T/epidemiología , Linfoma de Células T/etiología , Linfoma de Células T/veterinaria , Prevalencia , Estudios Retrospectivos , Factores de Riesgo , Especificidad de la EspecieRESUMEN
In dogs as well as humans, lymphoma is one of the most common hematopoietic malignancies. Furthermore, due to its characteristics, canine lymphoma is recognized as a clinically relevant in vivo model to study the corresponding human disease. Immortalized cell lines are widely used as in vitro models to evaluate novel therapeutic agents and characterize their molecular mechanisms. However, it is known that long-term cultivation leads to clonal selection, genetic instability, and loss of the initial heterogenic character, limiting the usefulness of cell lines as preclinical models. Herein, we present a systematic characterization and comparison of the transcriptomic landscape of canine primary B- and T-cell lymphomas, five lymphoid cell lines (CLBL-1, CLBL-1M, GL-1, CL-1, and OSW) and four non-neoplastic control samples. We found that lymphomas and cell lines exhibit a common "differentiation and proliferation signature". However, our analysis also showed that, independently of the cell of origin, the transcriptional signatures of lymphomas are more similar to each other than they are to those of cell lines. In particular, we observed that not all common therapeutic targets are similarly expressed between lymphomas and lymphoid cell lines, and provide evidence that different lymphoid cell-lines should be used to model distinct aspects of lymphoma dysregulation.
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Enfermedades de los Perros/patología , Secuenciación del Exoma/métodos , Linfoma/patología , Transcriptoma/genética , Animales , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Modelos Animales de Enfermedad , Enfermedades de los Perros/clasificación , Enfermedades de los Perros/genética , Perros , Regulación Neoplásica de la Expresión Génica , Humanos , Linfoma/clasificación , Linfoma/genética , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de SeñalRESUMEN
Objectives The purpose of this study was to specify lymphoma subtypes according to the World Health Organization (WHO) classification in a group of cats and to investigate their potential prognostic value. Methods Records of cats from the University of Veterinary Medicine Vienna suffering from lymphoma were reviewed in this retrospective study. To diagnose various subtypes specified in the WHO classification, histopathological and immunohistochemical examinations, as well as clonality assays in some cases, were performed. Results Of the 30 cats included in this study and classified according to the WHO guidelines, peripheral T-cell lymphoma was the most prevalent lymphoma subtype (37% of cases; n = 11), followed by diffuse large B-cell (23%; n = 7), intestinal T-cell (10%; n = 3), T-cell-rich B-cell (10%; n = 3), large granular lymphocytic (7%; n = 2), anaplastic large T-cell (7%; n = 2), B-cell small lymphocytic (3%; n = 1) and T-cell angiotropic lymphoma (3%; n = 1). The median survival time (MST) was 5.4 months (range 6 days to 2.2 years), with two cats still alive after 1.7 and 2.0 years, respectively. Treating cats prior to chemotherapy with glucocorticoids did not worsen their prognosis. Adding to chemotherapy, radiotherapy or surgery did not improve the clinical outcome. We observed that patients with intestinal T-cell lymphoma lived significantly longer (MST 1.7 years) than those with a diffuse large B-cell (MST 4.5 months) or peripheral T-cell lymphoma (MST 6.1 months). Cats with T-cell-rich B-cell lymphoma survived significantly longer (MST 1.2 years) than those with a diffuse large B-cell lymphoma. Conclusions and relevance A detailed diagnosis of feline lymphoma can be obtained by allocating different subtypes according to the WHO classification. From the eight detected lymphoma subtypes, two, intestinal T-cell lymphoma and T-cell-rich B-cell lymphoma, showed promising survival times in cats.
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Enfermedades de los Gatos/mortalidad , Linfoma/veterinaria , Estadificación de Neoplasias , Animales , Austria/epidemiología , Enfermedades de los Gatos/clasificación , Enfermedades de los Gatos/patología , Gatos , Femenino , Linfoma/clasificación , Linfoma/mortalidad , Masculino , Prevalencia , Pronóstico , Estudios Retrospectivos , Análisis de Supervivencia , Organización Mundial de la SaludRESUMEN
Feline alimentary lymphoma is the most common hematopoietic neoplasia in cats. It affects mainly the small intestines and is most frequently of T-cell origin. Evaluation of a fine needle aspirate is often the first step in the diagnostic work-up. Differentiation between a resident mature lymphocyte population as encountered in inflammatory bowel disease and small cell lymphoma cannot be achieved by cytology alone. Even full thickness biopsies evaluated by histopathology can be inconclusive. These cases warrant the application of complementary tools like PCR-based T-cell receptor (TCR) clonality testing for confirmation. The aim of this study was to optimize the DNA extraction protocol for formalin fixed and paraffin embedded tissues (FFPE) and to establish a heteroduplex analysis to enhance resolution of the PCR fragments of the T-cell receptor gamma (TCRG) V-J gene. The new protocols resulted in improved quantity and quality of the extracted DNA. Heteroduplex analysis of the samples improved the resolution of the electrophoresis results so that rules for interpretation of the different patterns could be established. Application of this improved setup detected clonal rearrangements in at least one TCRG primer reaction in 31 of 36 of our feline intestinal lymphoma samples after DNA quality testing.
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Enfermedades de los Gatos/diagnóstico , Neoplasias Gastrointestinales/veterinaria , Linfoma de Células T/veterinaria , Receptores de Antígenos de Linfocitos T gamma-delta/metabolismo , Animales , Enfermedades de los Gatos/patología , Gatos , Neoplasias Gastrointestinales/diagnóstico , Neoplasias Gastrointestinales/patología , Regulación Neoplásica de la Expresión Génica , Linfoma/genética , Linfoma de Células T/metabolismo , Linfoma de Células T/patología , Mutación , Reacción en Cadena de la Polimerasa/métodos , Estudios RetrospectivosRESUMEN
Protein kinase inhibitors are widely used in chemotherapeutic cancer regimens. Maleimide derivatives such as SB-216763 act as GSK-3 inhibitor targeting cell proliferation, cell death and cell cycle progression.Herein, the two arylindolylmaleimide derivatives PDA-66 and PDA-377 were evaluated as potential chemotherapeutic agents on canine B-cell lymphoma cell lines. Canine lymphoma represents a naturally occurring model closely resembling the human high-grade non-Hodgkin's lymphoma (NHL). PDA-66 showed more pronounced effects on both cell lines. Application of 2.5µM PDA-66 resulted in a significant induction of apoptosis (approx. 11 %), decrease of the metabolic activity (approx. 95 %), anti-proliferative effect (approx. 85 %) and cell death within 48h. Agent induced mode of action was characterized by whole transcriptome sequencing, 12 h and 24 h post-agent exposure. Key PDA-66-modulated pathways identified were cell cycle, DNA replication and p53 signaling. Expression analyses indicated that the drug acting mechanism is mediated through DNA replication and cycle arrest involving the spindle assembly checkpoint.In conclusion, both PDA derivatives displayed strong anti-proliferation activity in canine B-cell lymphoma cells. The cell and molecular PDA-induced effect characterization and the molecular characterization of the agent acting mechanism provides the basis for further evaluation of a potential drug for canine lymphoma serving as model for human NHL.
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Antineoplásicos/farmacología , Indoles/farmacología , Linfoma de Células B , Maleimidas/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Perros , Inhibidores de Proteínas Quinasas/farmacologíaRESUMEN
A 7-year-old male Boxer with a 3.5-year history of atopy and food hypersensitivity was presented with multiple poorly circumscribed nodules and maculae of the skin and tongue, and jaundiced mucosal membranes. Cytologic and histopathologic examination of the skin lesions revealed cutaneous epitheliotropic lymphoma. Cells were CD3(+) and CD8(+) in flow cytometry. The CBC showed a moderate leukocytosis with 16% atypical lymphocytes with irregularly cleaved nuclei. Flow cytometric phenotyping of peripheral blood showed an elevated proportion of the CD8(+) T-lymphocyte subpopulation, indicating a malignant population of T-cell origin, and the electropherogram of the PCR antigen receptor rearrangement produced a monoclonal peak for TCRγ. Liver enzyme activities were markedly increased and abdominal ultrasound examination showed increased echogenicity of the liver and enlarged abdominal lymph nodes. Fine-needle aspirates of the liver confirmed infiltration with lymphocytes exhibiting the same morphology as the cells detected in skin and peripheral blood. Treatment was induced with L-asparaginase, lomustine, and prednisone. Partial clinical remission of the skin and tongue lesions was achieved within 10 days, and hematologic abnormalities resolved. Despite further treatment with L-asparaginase and lomustine, the dog relapsed within one month and was euthanized. Presence of malignant lymphocytes in skin, peripheral blood, and liver indicate a rare variant of leukemic cutaneous T-cell lymphoma, equivalent of Sézary syndrome in a dog. This case report describes the use of flow cytometry as a complementary tool for lymphocyte characterization of skin lesions for the first time.
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Enfermedades de los Perros/patología , Citometría de Flujo/veterinaria , Linfoma Cutáneo de Células T/veterinaria , Síndrome de Sézary/veterinaria , Enfermedades de la Piel/veterinaria , Neoplasias Cutáneas/veterinaria , Animales , Biopsia con Aguja Fina/veterinaria , Perros , Hígado/enzimología , Hígado/patología , Ganglios Linfáticos/patología , Linfocitos/patología , Linfoma Cutáneo de Células T/patología , Masculino , Síndrome de Sézary/patología , Piel/patología , Enfermedades de la Piel/patología , Neoplasias Cutáneas/patologíaRESUMEN
BACKGROUND: Canine lymphoma has lately drawn focus as a model of human non-Hodgkin's lymphoma due to its spontaneous occurrence and similar biological behavior. Cells with stem cell-like characteristics are believed to play a key role in therapeutic failure. Thus, an initial characterization and the possibility of specific detection of such cells could bear significant value. MATERIALS AND METHODS: Expression of 12 stem cell markers were analyzed in two canine B-cell lymphoma cell lines, their generated spheres, and in primary lymphoma samples by quantitative real-time polymerase chain reaction and partially by flow cytometry and immunocytochemistry. RESULTS: Expression of maternal embryonic leucine zipper kinase (Melk) was significantly higher in CLBL-1, CLBL-1M and in primary B-cell lymphoma samples compared to non-neoplastic lymph nodes. Spheres displayed a higher expression of v-myc myelocytomatosis viral oncogene homolog (Myc) and lower expression of Cd44 compared to original cell lines and primary B-cell lymphoma samples. CONCLUSION: The results suggest a potential interesting role of Melk in canine B-cell lymphoma. Furthermore, the up-regulation of Myc in serum-free-generated spheres offers interesting possibilities for functional assays characterizing the specific generated sub-population.
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Linfoma de Células B/genética , Linfoma no Hodgkin/genética , Células Madre Neoplásicas/patología , Animales , Modelos Animales de Enfermedad , Perros , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Humanos , Receptores de Hialuranos/biosíntesis , Ganglios Linfáticos/patología , Linfoma de Células B/patología , Linfoma de Células B/veterinaria , Linfoma no Hodgkin/patología , Células Madre Neoplásicas/metabolismo , Proteínas Proto-Oncogénicas c-myc/biosíntesisRESUMEN
BACKGROUND: Flow cytometric immunophenotyping of lymph node aspirates has become a standard practice of canine lymphoma diagnostic workup. Ultimately, the combination of flow cytometry data, histopathology, and clinical signs allows consensus classification, and improves prognostic accuracy and therapeutic approaches. OBJECTIVES: Although there is a growing body of information regarding lymphocyte population subsets in various types of lymphoma, only few studies provide information regarding the composition of the normal canine lymph node. The aim of this prospective study was to establish exploratory reference data for lymphocyte subpopulations in normal canine lymph nodes using an extended panel of antibodies. METHODS: Popliteal lymph nodes excised from normal dogs were analyzed by cytology, multi-color flow cytometry using 11 different canine-specific and anti-human cross-reactive monoclonal antibodies, and polymerase chain reaction for antigen receptor rearrangement (PARR). RESULTS: Subpopulations from lymph nodes of 26 dogs were classified according to the following positive antibody reactions: CD11a(+) 92.2 ± 12.3%, CD3(+) 55.0 ± 14.1%, CD3-12(+) 57.3 ± 14%, CD5(+) 52.3 ± 12.7%, CD21(+) 33.9 ± 11.8%, CD79αcγ(+) 46.9 ± 14.8%, CD56(+) 4.9 ± 5.9%, and CD14(+) 5.5 ± 6.8%. There were 58.7 ± 9% CD4(+) and 21.3 ± 7.8% CD8(+) cells inside the gate of CD3(+) cells. Cytology revealed a mixed population of mostly lymphoid cells in all samples. The absence of a monoclonal or oligoclonal neoplastic population was confirmed by PARR. CONCLUSION: This study establishes for the first time flow cytometry data of lymphocyte populations in a larger group of normal canine lymph nodes, including populations positive for some new antibodies against CD3-12, CD5, CD11a, CD56, and CD79αcy.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD/inmunología , Enfermedades de los Perros/diagnóstico , Perros/inmunología , Linfoma/veterinaria , Animales , Biopsia con Aguja/veterinaria , Reacciones Cruzadas , Femenino , Citometría de Flujo/veterinaria , Inmunofenotipificación/veterinaria , Ganglios Linfáticos/inmunología , Subgrupos Linfocitarios , Linfoma/inmunología , Masculino , Estudios Prospectivos , Especificidad de la EspecieRESUMEN
In lagomorphs, lymphocyte subset distributions and the importance of CD4(+) T cell levels has so far only been considered in the frame of rabbit disease models. In this study, the first assessment of CD4(+) T lymphocytes in peripheral blood cells in brown hares (Lepus europaeus L., 1758), a further leporid species using a cross-reactive rabbit anti-CD4 antibody in flow cytometry, is presented. In addition, the entire coding region of the hare CD4 gene (1380 bp) coding for a polypeptide of 459 amino acids has been sequenced. Using generalized least squares fitting by maximum likelihood (GLS) test, significantly (p=0.0095) higher CD4(+) T cell frequencies in males than in females and significantly (p=0.0001) higher frequencies for leverets (younger than 2 months of age) than for subadult and adult (older than 7 months of age) individuals were detected. No significant age influence, however, was found for subadult and adult hares. The study is particularly meant to provide a first step in establishing a toolbox for the assessment of the immune response in this leporid species.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/fisiología , Liebres/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos , Antígenos CD4/genética , Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/citología , Femenino , Masculino , Datos de Secuencia Molecular , Conejos , Especificidad de la Especie , Bazo/citologíaRESUMEN
To evaluate radiosensitivity and the effects of radiation on the expression of vascular endothelial growth factor (VEGF) and VEGF receptors in the canine oral melanoma cell line, TLM 1, cells were irradiated with doses of 0, 2, 4, 6, 8 and 10 Gray (Gy). Survival rates were then determined by a MTT assay, while vascular endothelial growth factor receptor (VEGFR)-1 and -2 expression was measured by flow cytometry and apoptotic cell death rates were investigated using an Annexin assay. Additionally, a commercially available canine VEGF ELISA kit was used to measure VEGF. Radiosensitivity was detected in TLM 1 cells, and mitotic and apoptotic cell death was found to occur in a radiation dose dependent manner. VEGF was secreted constitutively and significant up-regulation was observed in the 8 and 10 Gy irradiated cells. In addition, a minor portion of TLM 1 cells expressed vascular endothelial growth factor receptor (VEGFR)-1 intracellularly. VEGFR-2 was detected in the cytoplasm and was down-regulated following radiation with increasing dosages. In TLM 1 cells, apoptosis plays an important role in radiation induced cell death. It has also been suggested that the significantly higher VEGF production in the 8 and 10 Gy group could lead to tumour resistance.