P. Roccabianca

Classification of Canine Malignant Lymphomas According to the World Health Organization Criteria

A study was carried out to test the accuracy and consistency of veterinary pathologists, not specialists in hematopathology, in applying the World Health Organization (WHO) system of classification of canine lymphomas. This study represents an initiative of the ACVP Oncology Committee, and the classification has been endorsed by the World Small Animal Veterinary Association (WASVA). Tissue biopsies from cases of canine lymphoma were received from veterinary oncologists, and a study by pathologists given only signalment was carried out on 300 cases. Twenty pathologists reviewed these 300 cases with each required to choose a diagnosis from a list of 43 B and T cell lymphomas. Three of the 20 were hematopathologists who determined the consensus diagnosis for each case. The 17 who formed the test group were experienced but not specialists in hematopathology, and most were diplomates of the American or European Colleges of Veterinary Pathology. The overall accuracy of the 17 pathologists on the 300 cases was 83%. When the analysis was limited to the 6 most common diagnoses, containing 80% of all cases, accuracy rose to 87%. In a test of reproducibility enabled by reintroducing 5% of cases entered under a different identity, the overall agreement between the first and second diagnosis ranged from 40 to 87%. The statistical review included 43,000 data points for each of the 20 pathologists.

Canine lymphoma: immunocytochemical analysis of fine-needle aspiration biopsy.

Cytospin preparations of fine-needle aspirates from 21 dogs with peripheral lymphadenopathy (18 with lymphoma and three with lymph node hyperplasia) were studied by combining morphologic and immunocytochemical analysis. Fine-needle aspirates were taken from at least two enlarged lymph nodes, and the diagnosis was based on air-dried smears stained with May-Grünwald Giemsa. Fine-needle aspiration biopsy always provided an adequate quality and quantity of cells to perform morphologic and immunologic studies. Immunophenotyping was performed on cytospin preparations with a panel of eight monoclonal antibodies specific for canine cell surface antigens and one rabbit polyclonal antibody (A452) against human CD3, which cross-reacts with dog antigen. The immunocytochemical study resulted in the diagnosis of 14 B-cell lymphomas (CD21 +, CD3-) and three T-cell lymphomas (all CD3 +, two CD8+). One lymphoma lacked surface antigens specific for the B- or T-cell lineage and was classified as non-B-non-T lymphoma (CD21-, CD3-, CD4-, CD8-). The monoclonal antibodies CA12.10C12, CA4.1D3, and CA1D6 and the polyclonal antibody A452, used as a group, appeared to be the most useful reagents to suggest lymphoid origin and to discriminate between T- and B-cell phenotype. Cytospin preparations in combination with immunocytochemistry provided a practical, economical, and accurate method for the diagnosis and phenotyping of canine lymphoma.

The Spectrum of Canine Cutaneous Perivascular Wall Tumors: Morphologic, Phenotypic and Clinical Characterization

Perivascular wall tumors (PWTs) are defined as neoplasms deriving from mural cells of blood vessels, excluding the endothelial lining. The spectrum of human cutaneous PWT includes glomus tumor, hemangiopericytoma (HEP), myopericytoma, angioleiomyoma/sarcoma, angiomyofibroblastoma, and angiofibroma. The purpose of this study was to revise clinical presentation, cytology, histopathology, and immunohistology of canine cutaneous PWT with cytology typical of canine HEP. Diagnosis was established on the basis of vascular growth patterns (staghorn, placentoid, perivascular whorling, bundles from media) and immunohistology, including 7 smooth muscle markers and the cell membrane ganglioside of unknown origin recognized by the antibody 3G5 (CMG-3G5). Twenty cases were included. Ages ranged from 6 to 13 years; 12 dogs were males and 8 were females, and there was a prevalence of crossbreeds. Tumors arose from a single site with preferential acral location (10/20). Cytology revealed moderate to high cellularity in all cases, cohesive groups of cells (19/20), capillaries (18/20), and bi- to multinucleated cells (18/20). Six myopericytomas, 5 angioleiomyomas, 2 angioleiomyosarcomas, 2 HEP, 1 angiofibroma, and 1 adventitial tumor were identified. A definitive diagnosis was not possible in 3 cases. Smoothelin, heavy caldesmon, desmin, myosin, calponin, and CMG-3G5 were the most valuable markers to differentially diagnose canine PWT. Similar to reports in humans, canine HEP embodied a spectrum of neoplastic entities arising from different vascular mural cells. Before canine PWTs are assimilated into one prognostic category, a consistent classification and characterization of their biology is necessary. As proposed in humans, HEP should also be considered a diagnosis of exclusion in dogs.

Feline Large Granular Lymphocyte (LGL) Lymphoma with Secondary Leukemia: Primary Intestinal Origin with Predominance of a CD3/CD8αα Phenotype

Clinicopathologic and immunophenotypic characteristics of large granular lymphocyte (LGL) neoplasia in 21 cats were examined. All cats were domestic short (19) or long hair (2) with a mean age of 9.3 years at diagnosis. Increased peripheral blood LGL counts were present in 18/21 cats. Neutrophilia (12/21 cats) and increased serum liver enzymes (7/12), total and direct bilirubin (7/13), BUN (5/14), and creatinine (2/14) were observed. Cats usually presented with advanced disease and none survived longer than 84 days (mean 18.8 days) postdiagnosis. Cytologically, LGLs had a mature (6/21), immature (13/21), or mixed (2/21) morphology. Necropsy lesions consisted of neoplastic lymphoid infiltrates in the jejunum, ileum, and duodenum in decreasing order of frequency. In the small intestine, mucosal ulceration (9/13) and epitheliotropism of neoplastic cells (9/13) were common. Neoplastic infiltrates were also present in the mesenteric lymph nodes (13/13), liver (12/13), spleen (8/13), kidneys (5/ 7), and bone marrow (5/7). A T cell phenotype (CD3∊+) characterized LGL neoplasia in 19/21 cases. A CD8αα+ cytotoxic/suppressor phenotype was present in 12/19 T cell tumors, 2 had a CD4+CD8αα phenotype, 3 had a CD4-CD8- phenotype, and 2 were CD4+ helper T cells. CD8β chain expression was not detected in any instance. In two cats, a B or T cell origin could not be established. CD103 was expressed by 11 of 19 (58%) of the lymphomas tested. The immunophenotypic features shared by neoplastic LGLs in the cat and feline intestinal intraepithelial lymphocytes (IELs) support a small intestinal IEL origin for feline LGL lymphoma.

Predictors of long-term survival in dogs with high-grade multicentric lymphoma

OBJECTIVE To determine factors predicting survival in dogs with high-grade multicentric lymphoma. Design-Retrospective cohort study. Animals-127 dogs with high-grade multicentric lymphoma evaluated at 4 veterinary hospitals from 2000 to 2009. PROCEDURES Records were reviewed to identify dogs with completely staged high-grade multicentric lymphoma treated with chemotherapy. Data collected included signalment, history, hematologic findings, tumor characteristics, treatment, and outcome. Long-term survival was defined as surviving > 2 years after diagnosis. Variables were analyzed for associations with dogs living > 2 years. RESULTS Among the 127 enrolled dogs, 13 (10%) survived > 2 years with a median survival time of 914 days (range, 740 to 2,058 days). Survival rates at 3, 4, and 5 years were 4%, 3%, and 1 %, respectively. At diagnosis, 11 of the 13 long-term survivors had a body weight ≥ 10 kg, PCV ≥ 35%, absence of ionized hypercalcemia, centroblastic lymphoma, immunophenotype B, absence of bone marrow involvement, and lymphoma stages I through IV and were not previously treated with corticosteroids. The same combination of factors was present in 26 of 114 (23%) dogs surviving ≤ 2 years, yielding a negative predictive value of 97.8% for long-term survivors. Four of the 6 long-term survivors that died during the study died of another cancer; 3 of them had osteosarcoma. CONCLUSIONS AND CLINICAL RELEVANCE Absence of the aforementioned combination of variables at diagnosis may help identify dogs with lymphoma that will not survive > 2 years. Other types of neoplasia, in particular osteosarcoma, may develop in long-term-surviving dogs.

Extra hepatic expression of the acute phase protein alpha 1-acid glycoprotein in normal bovine tissues.

Combined quantitative (real-time) polymerase chain reaction and immunohistochemistry were used to evaluate the expression of the minor acute phase protein alpha 1-acid glycoprotein (AGP, orosomucoid) in bovine extrahepatic tissues. AGP was produced mainly in the salivary glands and spleen, whereas minor expression was detected in all other tissues sampled, including lung, lymph nodes, uterus, ovary, kidney and tongue. The findings were consistent with immunohistochemical results. In view of the immunomodulatory and direct antibacterial activity of AGP, its expression in the salivary glands may signal an involvement in the regulation of the local immunity, even in non-pathological conditions.

Characterization of the diffuse mucosal associated lymphoid tissue of feline small intestine.

Abstract Characterization of the feline intestinal mucosal associated lymphoid tissue (MALT) will facilitate investigation of intestinal disease in the cat and promote the cat as an animal model for a range of human diseases which involve the intestinal lymphoid tissue. This includes inflammatory bowel disease, viral and non-viral associated intestinal lymphomas and immunodeficiency associated syndromes. Morphologic and phenotypic characterization of the normal small intestinal diffuse MALT in 22 SPF cats was performed using flow cytometry and cytology on isolated intestinal leukocytes from the intra-epithelial and lamina proprial compartments, as well as immunohistology on tissues from the feline duodenum, jejunum and ileum. The intra-epithelial compartment (IEC) was dominated by lymphocytes (>85%) which frequently contained intracytoplasmic granules. The most striking findings in the IEC were the elevated percentages of CD8α+ lymphocytes (40%), presumed to express CD8αα chains, and CD4−/CD8− (double negative) lymphocytes (44%), and the consistent presence of a minor subpopulation of CD3+/CD11d+ IELs (6%). Small percentages of CD4+ lymphocytes (10%) were observed such that the IEL CD4:CD8 ratio (0.25) was low. The LPC also contained a majority of T cells and few plasma cells. However, this compartment had reduced percentages of CD8α+ lymphocytes (28%) and increased percentages of CD4+ lymphocytes (27%) relative to the IEC. However, the LPL CD4:CD8 ratio (1.0) remained low compared with the ratio in peripheral blood. In feline MALT, MHC class II expression was lower than in other peripheral lymphoid compartments. The results of this study provide important reference values for future investigations involving feline intestinal lymphocytes and demonstrates that the leukocyte distribution and phenotypic characteristics of the feline diffuse MALT appear largely similar to the murine, rat and human counterparts.

Identification of suitable endogenous controls and differentially expressed microRNAs in canine fresh-frozen and FFPE lymphoma samples

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.

Pancytopenia in a cat with visceral leishmaniasis

A 4-year-old, domestic shorthair, female spayed cat was presented for decreased appetite and depression. Severe pancytopenia with erythrocyte autoagglutination was found. The cat was seronegative for feline immunodeficiency and leukemia viruses. Immune-mediated hemolytic anemia was suspected but no response to treatment with a blood transfusion, enrofloxacin, and prednisone was observed. Blood and bone marrow smears obtained 11 days later contained Leishmania amastigotes in the cytoplasm of neutrophils and macrophages, respectively. Serologic and PCR testing of peripheral blood confirmed infection with Leishmania infantum. Despite treatment, the cat worsened clinically and was euthanized. At necropsy, visceral dissemination of the parasite was confirmed. The findings in this case indicate that visceral leishmaniasis should be considered as a differential diagnoses in cats with pancytopenia in areas endemic for Leishmania. In addition, amastigotes may be observed in peripheral blood neutrophils.

Evaluation of inflammation and immunity in cats with spontaneous parvovirus infection: Consequences of recombinant feline interferon-ω administration

Abstract Administration of recombinant feline interferon-ω (rFeIFN) has been proposed for the prophylaxis of canine and feline parvovirosis. In the present study, the influence of the administration of rFeIFN on blood markers of inflammation (α-globulins, α1-acid glycoprotein) and immune system activation (γ-globulins, IgG, IgM, specific anti-feline parvovirus IgG or IgM) was evaluated in a cattery developing an outbreak of feline panleukopenia due to feline parvovirus (FPV) infection few days after initial administration of rFeIFN. Kittens (n =23) were injected with rFeIFN (1MU/kg subcutaneoulsy, once a day for 3 days) and their blood parameters were compared with those of 17 untreated cats. Cats that survived the outbreak were vaccinated and re-sampled 1 month after the last rFeIFN administration. Time of emergence of clinical signs and survival rate were not significantly different between the two groups. Controls and treated cats surviving the infection had high levels of γ-globulins, total- and anti-FPV specific IgGs, likely due to passive transfer of maternal immunity. Compared to controls, treated kittens had lower levels of α1-globulins and higher mean values of γ-globulins and immunoglobulins. Data from samples collected after vaccination revealed a higher level of γ-globulins, total- and anti-FPV specific IgGs in treated kittens, compared with controls, suggesting that rFeIFN stimulates antibody production. Based on this results, rFeIFN should be administered to the queen, to increase passive maternal immunity, or to kittens before introduction in a potentially contaminated environment.