Granulocytic neoplasm suggestive of primary myeloid sarcoma in 3 dogs.

Myeloid sarcoma (MS) is a solid tumor of granulocytic origin with extramedullary localization. This tumor is rare in humans and animals. The diagnostic approach is heterogeneous, and the definitive diagnosis may be difficult to achieve. Primary MS has never been described as a spontaneous neoplasm in companion dogs. Two purebred and 1 mixed-breed dogs, 6- to 11-year-old, developed round cell tumors in the mediastinum, lymph nodes (LNs) and tonsils, and LNs, respectively. Granulocytic origin and exclusion of lymphoid lineage were confirmed by flow cytometry, supported by immunohistochemistry or immunocytochemistry. Pivotal to the diagnosis were positive labeling for myeloid (CD11b, CD14) and hematopoietic precursors (CD34) markers, along with negative labeling for lymphoid markers. Blood and bone marrow infiltration were not detected at initial diagnosis, excluding acute myeloid leukemia. The behavior of these tumors was aggressive, resulting in poor clinical outcomes, even when chemotherapy was attempted.

A method to test antibody cross-reactivity toward animal antigens for flow cytometry.

The availability of cross-reacting antibodies and/or of antibodies working in flow cytometry is a major issue in the veterinary field. One of the main problems is the availability of certain positive controls. With this brief communication, we report an method to quickly screen a wide number of products without the need to look for positive biological samples. We propose this approach as a first step to select the best antibodies to test on biological specimens.

Thromboelastometric assessment of hemostasis following hydroxyethyl starch (130/0.4) administration as a constant rate infusion in hypoalbuminemic dogs.

BACKGROUND: The primary aim was to evaluate by means of thromboelastometry (ROTEM) the effects of hydroxyethyl starch (HES) 130/0.4 administered as a constant rate infusion (CRI) on hemostasis in hypoalbuminemic dogs. The second aim was to use ROTEM analysis to detect whether all hypoalbuminemic dogs of our population were hypercoagulable. RESULTS: The study sample was 20 hypoalbuminemic dogs (albumin < 2 g/dl) with normal perfusion parameters and requiring intravenous fluid therapy. In order to support plasma colloid osmotic pressure, in addition to crystalloid, HES 130/0.4 was administered as a constant rate infusion at 1 ml/kg/h (group 1, n = 11) or 2 ml/kg/h for 24 h (group 2, n = 9). Blood samples were collected at baseline (T0) and 24 h postinfusion (T1); coagulation was assessed by standard coagulation profile (prothrombin time, activated partial thromboplastin time, and fibrinogen), and ROTEM analysis (in-TEM®, ex-TEM® and fib- TEM® profile). No statistically significant differences in ROTEM values in group 1 were observed (P > 0.05), whereas in group 2 statistically significant differences (P < 0.05) were found at T1 in the in-TEM® profile [decrease in clot formation time (P = 0.04) and increase in α angle (P = 0.02)] and in the ex-TEM® profile [increase in maximum clot firmness (P = 0.008) and α angle (P = 0.01)]; no changes were identified in the fib-TEM® profile. In both groups, a statistically significant decrease (P = 0.007) in hematocrit was noted, whereas no statistically significant differences in platelet count and standard coagulation profile were found. In group 2, a statistically significant increase in TS values (P = 0.03) was noted at T1. ROTEM tracings indicating a hypercoagulable state were observed in 7/20 dogs at T0 (5/11 in group 1 and 2/9 in the group 2). CONCLUSION: Our findings suggest that HES 130/0.4 administered as CRI does not cause hypocoagulability in hypoalbuminemic dogs. A trend toward hypercoagulability, probably related to the underlying diseases, was observed in group 2 at T1. Although all dogs were hyoalbuminemic, only 7/20 were hypercoagulable at T0, confirming the lack of correlation between albumin level and prothrombotic state.

A two-step immunomagnetic separation of somatic cell subpopulations for a gene expression profile study in bovine milk.

The objective of this study was to demonstrate the usefulness of an immunomagnetic method to purify subpopulations of milk somatic cells. The experiment was conducted on milk samples collected from healthy cows (n = 17) and from cows with clinical mastitis (n = 24) due to a Staphylococcus aureus natural infection. A two-step immunomagnetic purification was applied to simultaneously separate three somatic cell subpopulations from the same milk sample. Total RNA was extracted and qPCR was performed to determinate mRNA levels of innate immunity target genes in purified somatic cell subpopulations. Good quality and quantity of RNA allowed the reference gene analysis in each cell subpopulation. An up-regulation of the main genes involved in innate immune defence was detected in separated polymorphonuclear neutrophilic leucocytes-monocytes and lymphocytes of mastitic milk. These results and flow cytometric analysis suggest that the immunomagnetic purification is an efficient method for the isolation of the three populations from milk, allowing the cells to be studied separately.

The role of vascular endothelial growth factor and matrix metalloproteinases in canine lymphoma: in vivo and in vitro study.

BACKGROUND: Canine lymphoma represents the most frequent haematopoietic cancer and it shares some similarities with human non-Hodgkin lymphoma. Matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) play a coordinated role during invasion and proliferation of malignant cells; however, little is known about their role in canine haematologic malignancies. The aim of this study was to investigate the mRNA and protein expression of VEGF and the most relevant MMPs in canine lymphoma. Lymph node aspirates from 26 B-cell and 21 T-cell lymphomas were collected. The protein expression levels of MMP-9, MMP-2 and VEGF-A were evaluated by immunocytochemistry, and the mRNA levels of MMP-2, MMP-9, MT1-MMP, TIMP-1, TIMP-2, RECK, VEGF-A and VEGF-164 were measured using quantitative RT-PCR. RESULTS: MT1-MMP, TIMP-1 and RECK mRNA levels were significantly higher in T-cell lymphomas than in B-cell lymphomas. Higher mRNA and protein levels of MMP-9 and VEGF-A were observed in T-cell lymphomas than in B-cell lymphomas and healthy control lymph nodes. A positive correlation was found between MMP-9 and VEGF-A in T-cell lymphomas. Moreover, MMP-9, MT1-MMP, TIMP-1 and VEGF-A were expressed at the highest levels in high-grade T-cell lymphomas. CONCLUSIONS: This study provides new information on the expression of different MMPs and VEGF in canine lymphoma, suggesting a possible correlation between different MMPs and VEGF, immunophenotype and prognosis.

Flow Cytometric Features of B- and T-Lmphocytes in Reactive Lymph Nodes Compared to Their Neoplastic Counterparts in Dogs.

An in-depth knowledge of non-neoplastic patterns is fundamental to diagnose neoplasia. In the present study, we described the flow cytometric (FC) cell size (FSC) and fluorescence intensity (MFI) of B- and T-lymphocytes in 42 canine reactive lymph nodes and 36 lymphomas. Proliferative activity (Ki67%) in reactive lymph nodes was also reported. Reactive lymph nodes were composed of a mixed population of small and large T (CD5+) and B (CD21+) cells. Small T-cells were larger in size than small B-cells, and large T-cells were larger than large B-cells. Small T-cells were composed of CD5+CD21- and CD5+CD21+dim subpopulations. Large B-cells were <20% in reactive lymph nodes and >20% in lymphomas and showed a higher FSC in lymphomas than in reactive lymph nodes. Large T-cells were <4% in reactive lymph nodes and >4% in lymphomas and showed a higher CD5 MFI in lymphomas (if expressed) compared to reactive lymph nodes. A subset of CD5+CD21+dim lymphocytes was recognized in addition to CD5+CD21- and CD5-CD21+ cells. In T-zone lymphomas, neoplastic cells had higher FSC and CD21 MFI values than small CD5+CD21+dim cells in reactive lymph nodes. Ki67% values were higher than those reported in normal lymph nodes, and largely overlapped with those reported in low-grade lymphomas and partially in high-grade lymphomas. Our results may contribute to making a less operator-dependent FC differential between lymphoma and reactive lymph nodes.

Development of a flow cytometric panel to assess prognostic biomarkers in fine needle aspirates of canine cutaneous or subcutaneous mast cell tumors.

Mast cell tumor (MCT) is a common skin cancer in dogs that has a wide range of clinical behaviors. The purpose of this study was to develop a novel multicolor flow cytometry (FC) panel that will enable the quantification of candidate prognostic markers (Ki-67 and pKIT) in fine needle aspirate (FNA) samples prior to surgical removal of the tumors. FNA of canine MCTs and the NI-1 cell line were utilized to develop a FC panel that includes a viability dye (FVS620, BD Biosciences; 7-AAD, Invitrogen) and the following primary conjugated antibodies: CD117-PE (ACK45, BD Biosciences), pKIT-A647 (polyclonal bs-3242R, BIOSS) and Ki-67-FITC (20Raj1, eBioscience; MIB-1, DAKO). A total of nine FNA samples of canine MCTs were collected, seven out which produced sufficient cells for FC analysis. The Ki-67 antibody clone 20Raj1 produced a positive signal when applied to blood leukocytes but failed to provide robust labeling of neoplastic mast cells. The Ki-67 antibody clone MIB-1 delivered a superior staining quality in both the NI-1 cells and primary MCT cells. CD117-PE signal was adequate post fixation and permeabilization and in the combination of 7-AAD. pKIT produced non-specific staining and was not suitable for this multicolor FC panel. In conclusion, FNA samples of canine MCTs can often yield adequate cell numbers for FC analysis, and a multicolor FC panel was developed that can detect Ki-67 in canine mast cells. This would permit further studies into the potential use of this panel for canine cutaneous and subcutaneous MCT prognostication purposes.

Comparison of 3 Tfr2-deficient murine models suggests distinct functions for Tfr2-alpha and Tfr2-beta isoforms in different tissues.

Transferrin receptor 2 (TFR2) is a transmembrane protein that is mutated in hemochromatosis type 3. The TFR2 gene is transcribed in 2 main isoforms: the full-length (alpha) and a shorter form (beta). alpha-Tfr2 is the sensor of diferric transferrin, implicated in the modulation of hepcidin, the main regulator of iron homeostasis. The function of the putative beta-Tfr2 protein is unknown. We have developed a new mouse model (KI) lacking beta-Tfr2 compared with Tfr2 knockout mice (KO). Adult Tfr2 KO mice show liver iron overload and inadequate hepcidin levels relative to body iron stores, even though they increase Bmp6 production. KI mice have normal transferrin saturation, liver iron concentration, hepcidin and Bmp6 levels but show a transient anemia at young age and severe spleen iron accumulation in adult animals. Fpn1 is strikingly decreased in the spleen of these animals. These findings and the expression of beta-Tfr2 in wild-type mice spleen suggest a role for beta-Tfr2 in Fpn1 transcriptional control. Selective inactivation of liver alpha-Tfr2 in KI mice (LCKO-KI) returned the phenotype to liver iron overload. Our results strengthen the function of hepatic alpha-Tfr2 in hepcidin activation, suggest a role for extrahepatic Tfr2 and indicate that beta-Tfr2 may specifically control spleen iron efflux.

Performance of lymph node cytopathology in diagnosis and characterization of lymphoma in dogs.

BACKGROUND: Cytopathology is a minimally invasive and convenient diagnostic procedure, often used as a substitute for histopathology to diagnose and characterize lymphoma in dogs. OBJECTIVES: Assess the diagnostic performance of cytopathology in diagnosing lymphoma and its histopathological subtypes in dogs. ANIMALS: One-hundred and sixty-one lymph node samples from 139 dogs with enlarged peripheral lymph nodes. METHODS: Based only on cytopathology, 6 examiners independently provided the following interpretations on each sample: (a) lymphoma vs nonlymphoma; (b) grade and phenotype; and (c) World Health Organization (WHO) histopathological subtype. Histopathology and immunohistochemistry (IHC) findings were used as reference standards to evaluate diagnostic performance of cytopathology. Clinical, clinicopathologic, and imaging data also were considered in the definitive diagnosis. RESULTS: Classification accuracy for lymphoma consistently was >80% for all examiners, whereas it was >60% for low grade T-cell lymphomas, >30% for high grade B-cell lymphomas, >20% for high grade T-cell lymphomas, and <40% for low grade B-cell lymphomas. Interobserver agreement evaluated by kappa scores was 0.55 and 0.32 for identification of lymphoma cases, and of grade plus immunophenotype, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: Cytopathology may result in accurate diagnosis of lymphoma, but accuracy decreases when further characterization is needed. Cytopathology represents a fundamental aid in identifying lymphoma and can be used as a screening test to predict grade and phenotype. However, these results must be confirmed using other ancillary techniques, including flow cytometry, histopathology, and immunohistochemistry (IHC).

Flow Cytometry in the Diagnosis of Canine B-Cell Lymphoma.

B cell lymphoma (BCL) is a heterogeneous group of lymphoid malignancies which comprise the majority of canine lymphomas. Diffuse large B cell lymphoma is the most common lymphoma subtype in dogs but other subtypes (e.g., marginal zone lymphoma, follicular lymphoma, mantle cell lymphoma, and others) have been described. This review aims to explore the use of flow cytometry to refine the diagnosis of canine BCL. Particular emphasis will be given to the possible identification of peculiar immunotypes, putative prognostic markers, staging and minimal residual disease.

Flow Cytometry in the Diagnosis of Canine T-Cell Lymphoma.

T cell lymphoma (TCL) is a heterogenous group of lymphoid malignancies representing about 30-40% of all canine lymphomas and often harboring a very aggressive behavior. WHO classification identifies the majority of TCLs as peripheral TCL, but other subtypes with peculiar presentation and outcome have been recognized. This review aims to explore the use of flow cytometry for refining the diagnosis of canine TCL, putting a particular emphasis on the identification of some peculiar immunotypes, such as T zone lymphoma; on the investigation of putative prognostic markers; and on the evaluation of lymphoma stage and of the minimal residual disease.

Variation of apoptotic and proliferative activity among lymphoma subtypes in dogs: A flow cytometric study.

Tumor growth depends on both proliferative and apoptotic rate of neoplastic cells. High proliferation index is a well-known negative prognostic factor in canine lymphomas, whereas little is known about apoptotic activity. We describe proliferative and apoptotic rates in different canine lymphoma subtypes at diagnosis. Flow cytometry (FC) was used to assess the percentage of proliferating cells (Ki67%) and of apoptotic cells (AnnV%) in 128 lymph node (LN) aspirates from dogs with lymphoma. Proliferation/apoptosis ratio (PAR) and turnover index (TI; Ki67% + AnnV%) were then calculated for each case. High-grade B-cell lymphomas showed high values for both Ki67% and AnnV%, low-grade B-cell lymphomas showed low Ki67% and high AnnV%, high-grade T-cell lymphomas showed high Ki67% and low AnnV%, and low-grade T-cell lymphomas showed low levels of both parameters. Lymphoblastic lymphomas had the highest PAR values. High-grade B-cell lymphomas had the highest TI values while small clear cells lymphomas the lowest. The panorama of proliferative and apoptotic activity widely varies among lymphoma subtypes. Our results lay the ground for future clinical and pharmacological studies.

Cell blocks in veterinary medicine: A comparison of two methods (cell tube and agar) in 52 effusions from dogs and cats.

BACKGROUND: Cell blocks are alternative preparations of fluid cytological specimens. They can be used for immunochemical studies as complementary tools or when other techniques (eg, immunocytochemistry, flow cytometry) are not available. OBJECTIVES: We aimed to provide comparative morphologic, immunohistochemical, and technical features of agar-based cell blocks (ACBs) and cell tube blocks (CTBs) from cavitary effusions. METHODS: Agar-based cell blocks and CTBs were obtained from canine and feline effusions with neoplastic/atypical cells or with packed cell volumes ≥3%. Cellularity, RBC separation, and cellular features were evaluated on digitalized H&E slides with evaluators blinded to the method. The immunohistochemical intensity and nonspecific background were assessed on pan-cytokeratin and vimentin-stained slides. Overall yield was calculated, and morphologic and immunohistochemical features were compared among paired samples. Technical and cellular features were also described. RESULTS: Agar-based cell blocks and CTBs yielded evaluable sections in 100% (52/52) and 98% (51/52) of the cases, respectively. Cellularity and RBC separation scores were significantly higher in CTBs. Similar staining intensities were observed, and background staining was more frequently seen in pan-cytokeratin-stained ACBs. Only basic materials and equipment were required for both methods. Agar-based cell block preparations were more operator dependent and difficult to standardize, whereas CTBs were easier to prepare, but laboratory processing was more demanding. CONCLUSIONS: Both methods can be used to produce good sections for immunohistochemistry staining with no significant differences. Cell tube blocks are beneficial for RBC-rich samples, and little additional training is required to prepare the blocks. Both types of cell blocks are reliable, cost-effective methods that could be introduced in diagnostic laboratories to further characterize canine and feline effusions.

Prognostic significance of peripheral blood and bone marrow infiltration in newly-diagnosed canine nodal marginal zone lymphoma.

Canine nodal marginal zone lymphoma (nMZL) is infrequent and is typically diagnosed at an advanced disease stage. However, it is currently unknown whether different levels of peripheral blood (PB) and bone marrow (BM) infiltration may provide prognostic stratification in dogs with nMZL. The aims of the present prospective study were to assess the influence of PB and BM infiltration detected by flow cytometry (FC) on time to progression (TTP) and lymphoma-specific survival (LSS) in dogs with newly-diagnosed multicentric nMZL, and to establish a cut-off value of prognostic significance. Forty-five completely staged and treatment-naïf dogs with histologically-confirmed nMZL were enrolled. After staging, dogs received chemo-immunotherapy or chemotherapy. PB infiltration was significantly associated with TTP (p=0.001): dogs with PB infiltration <30% had a median TTP of 186 days, whereas dogs with PB infiltration ≥30% had a median TTP of 43 days. Additionally, vaccinated dogs had a significantly (p=0.012) longer TTP (399 days) compared with dogs receiving chemotherapy only (211 days). BM infiltration was significantly associated with LSS (p<0.001): dogs with BM infiltration <1% had a median LSS of 1403 days, those with BM infiltration 1-20% of 337 days, and those with BM infiltration ≥20% of 188 days. Normal LDH levels and the administration of chemo-immunotherapy also significantly improved LSS (560 vs 211 days, and 399 vs 211 days, respectively; p<0.001). PB and BM flow cytometric evaluation is an integral part of staging work-up in dogs with nMZL and has prognostic relevance.

Prognostic role of non-neoplastic lymphocytes in lymph node aspirates from dogs with diffuse large B-cell lymphoma treated with chemo-immunotherapy.

Dogs with Diffuse Large B-Cell Lymphoma (DLBCL) benefit from the addition of active immunotherapy to traditional chemotherapy. We hypothesized that immune cells within neoplastic lymph nodes (LNs) may play a role in the tumor pathobiology and treatment response. The present study describes the composition and prognostic role of non-neoplastic lymphocytes in LNs of 59 dogs with treatment-naive DLBCL receiving chemo-immunotherapy. The percentage of small non-neoplastic cells and of CD5+, CD21+, CD4+ and CD8+ small cells was recorded via flow cytometry. CD4+/CD8+ and CD5+/large CD21+ cell ratios were calculated. The likelihood of progression significantly diminished with increasing percentage of small cells, CD5+ and CD8+ small cells, and CD5+/large CD21+ cell ratio, with decreasing CD4+/CD8+ ratio and in non-anemic dogs. Active immunotherapy is more effective in dogs with higher percentage of non-neoplastic lymphocytes at diagnosis. We lay the ground for future studies assessing the role of the immune system in the pathobiology of canine DLBCL.

Minimal residual disease in lymph nodes after achievement of complete remission predicts time to relapse in dogs with large B-cell lymphoma.

Most dogs with large B-cell lymphoma (LBCL) that undergo chemotherapy and achieve clinical complete remission (CR) eventually relapse. However, time to relapse (TTR) is unpredictable. The aims of this prospective study were to assess the influence of post-chemotherapy lymph node (LN) infiltration by large CD21+ cells using flow cytometry (FC) on TTR, and to establish a cut-off value of prognostic significance. Dogs with newly-diagnosed, completely staged LBCL in CR after treatment were enrolled. Minimal residual disease (MRD) analysis by FC was performed on LN aspirates. TTR was calculated between MRD and relapse. Thirty-one dogs were enrolled: 4% had stage V disease, and diffuse large B-cell lymphoma was the most common histotype (74%). Based on LN infiltration at MRD evaluation, three groups were created: (a) acellular samples, (b) ≤0.5% infiltration and (c) >0.5% infiltration. Overall median TTR was 154 days (range, 31-1974): 22 (71%) dogs relapsed during the study period, whereas 9 (29%) dogs did not. The difference among the three groups was significant (P = 0.042 log-rank test): median TTR was not reached for dogs with LN infiltration ≤0.5% (range, 195-429 days), 164 days (range 63-1974) for dogs with acellular LN samples, and 118 days (range, 31-232) for dogs with LN infiltration >0.5%. These results demonstrate that MRD assessment by FC on LN aspirates in dogs with LBCL in clinical CR predicts TTR. LN infiltration by >0.5% large CD21+ cells after treatment is an unfavourable prognostic factor.

Opportunities and challenges of active immunotherapy in dogs with B-cell lymphoma: a 5-year experience in two veterinary oncology centers.

BACKGROUND: Pet dogs spontaneously develop lymphoma. An anthracycline-based multidrug chemotherapy regimen represents the treatment cornerstone; however, cure is rarely achieved. We have been treating dogs with B-cell lymphoma with an autologous vaccine (APAVAC®) and CHOP-based chemotherapy since 2011. METHODS: To better characterize the safety and efficacy of APAVAC®, and to find the best candidates for immunotherapy, we designed a retrospective study on all dogs treated with chemo-immunotherapy to date and compared them with those dogs treated with chemotherapy only. All dogs were completely staged and re-staged at the end of treatment. The primary endpoint was the effectiveness of chemo-immunotherapy, measured as time to progression (TTP), lymphoma-specific survival (LSS), and 1-, 2-, and 3-year survival rates. The secondary objective was safety. RESULTS: Three hundred dogs were included: 148 (49.3%) received chemotherapy and 152 (50.7%) chemo-immunotherapy. Overall, the latter survived significantly longer (median LSS, 401 vs 220; P <  0.001). Among dogs with diffuse large B-cell lymphoma, the 1-, 2- and 3-year survival rates were 20, 13 and 8% for chemotherapy, and 51, 19 and 10% for chemo-immunotherapy. The benefit of chemo-immunotherapy was particularly relevant in dogs with concurrent high serum LDH, stage V, substage a disease and not previously treated with steroids (median LSS, 480 vs 85 days; P <  0.001). Among dogs with nodal marginal zone lymphoma, those having at least 3 of the aforementioned characteristics significantly benefited from chemo-immunotherapy (median LSS, 680 vs 160 days, P <  0.001). The 1-, 2- and 3-year survival rates were 30, 16 and 10% for chemotherapy, and 55, 28 and 10% for chemo-immunotherapy. Among dogs with follicular lymphoma, lack of immunotherapy administration was the only variable significantly associated with increased risk of tumor-related death. Chemo-immunotherapy was remarkably well tolerated, with no local or systemic adverse events. CONCLUSIONS: Overall, the addition of immunotherapy to a traditional CHOP protocol is associated with improved outcome in dogs with B-cell lymphoma, regardless of histotype and evaluated prognostic factors. Moreover, the identikit of the best candidate for immune-therapy was delineated for the most common histotypes. The study also confirms the excellent tolerability of the vaccine.

Effects of road transportation on lymphocyte subsets in calves.

The effect of transportation on peripheral blood lymphocyte subsets in 24 calves was investigated by flow cytometry. Blood was collected before departure, on arrival, at 24h and 1 week after arrival. Highest leucocyte and neutrophil counts, associated with increased concentrations of cortisol and catecholamines, indicated that stress was maximal upon arrival. At this time, a decrease in the percentages of all T lymphocyte subsets was evident, while they did not decrease as absolute counts. The proportion of CD21(+) cells did not change, indicating that the relative reduction of T lymphocyte subsets was not related to an increase in B lymphocytes. These variations may be due to the increase of a natural killer (NK) cell subset. NK cell expansion, together with increasing lymphocyte count and increasing major histocompatibility complex class II expression, may indicate stress-induced stimulation of the immune system.