Histopathologic classification of 171 cases of canine and feline non-Hodgkin lymphoma according to the WHO.

A retrospective collection of 171 lymphoid neoplasms (123 dogs and 48 cats) was classified according to the Revised European-American Lymphoma (REAL) classification, adopted in 2002 by the World Health Organization (WHO), to evaluate the WHO system for categorization of canine and feline neoplasms. Microscopic examination was performed after standard hematoxylin-eosin staining and immunohistochemical labelling for B (CD79a) or T (CD3) cell phenotypes. B-cell lymphomas were prevalent in dogs and T-cell lymphomas in cats. B-Large cell lymphoma (B-LCL) frequently showed plasmacytoid differentiation; notably, two canine plasma cell tumours (PCT) expressed both CD79 and CD3. There were difficulties in differentiating B-lymphoblastic lymphoma (B-LBL) from Burkitt-type lymphoma. Furthermore, intestinal T-cell lymphoma (ITCL) exhibited a huge morphologic variability. Finally, multicentric mature small and thymic T-cell lymphomas were diagnosed, although these categories are not codified by the WHO classification.

Characterization of interstitial nephritis in pigs with naturally occurring postweaning multisystemic wasting syndrome.

Kidney samples with interstitial nephritis from 26 pigs affected by postweaning multisystemic wasting syndrome (PMWS) were selected. A histologic evaluation was carried out to describe the type of inflammation and its relationship with viral load, as assessed by in situ hybridization (ISH). Of 26 cases, 10 revealed a tubulointerstitial, lymphoplasmacytic nephritis, 11 an interstitial granulomatous nephritis, and 5 both types of inflammation (mixed type). In 4 cases of granulomatous inflammation, the pattern was not classically nodular, and a population of macrophages and lymphocytes was present (interstitial lymphohistiocytic nephritis). ISH confirmed the presence of porcine circovirus type 2 (PCV2) nucleic acid in all cases. The epithelium of the renal tubules was the most constantly ISH-positive structure. In tubulointerstitial nephritis, the higher the number of positive inflammatory cells, the more severe the inflammation. The ISH reaction was more heterogeneous and unpredictable in granulomatous nephritis, with some epithelioid and giant cells positive by ISH. To quantify macrophages distributed in the three patterns of nephritis, immunohistochemical methods using anti-major histocompatibility complex II (anti-MHC-II) and anti-lysozyme antibodies were undertaken, and semiquantitative evaluation was carried out. MHC-II was mainly expressed by lymphocytes in tubulointerstitial nephritis, but did not always stain macrophages in cases of granulomatous (including lymphohistiocytic) nephritis; the anti-lysozyme antibody revealed macrophages when present in tissues. The amount of PCV2 nucleic acid was not apparently associated with the pattern of inflammation (tubulointerstitial or granulomatous). PCV2 load seems to reflect the severity of the lymphoplasmacytic inflammation but not that of granulomatous and lympho histiocytic types.

Immunohistochemical MHC-II and interleukin 2-R (CD25) expression in lymph nodes of pigs with spontaneous postweaning multisystemic wasting syndrome (PMWS).

Immunohistochemical expression of immunocompetent cells bearing major histocompatibility complex (MHC-II) and interleukin 2-R (IL2-R) (CD25) molecules was performed on lymph nodes with spontaneous postweaning multisystemic wasting syndrome (PMWS). Control lymph nodes displayed intense diffuse immunoreactivity to MHC-II in both follicles and interfollicular areas. A marked reduction of follicular MHC-II immunoreactivity and inconsistent staining of histiocytes in interfollicular areas was observed in PMWS cases with a slight lymphoid depletion; in those cases with moderate to severe lymphoid depletion, there was a progressive decrease in MHC-II expression. In controls and in slightly depleted nodes, IL2-R was equally expressed in interfollicular tissue and in follicles, whereas in moderate and severe cases, it was detected in interfollicular remnants only. Immunohistochemical staining was scored semiquantitatively. The mean MHC-II score was significantly reduced in PMWS cases compared with controls (Spearman test), whereas there was no difference in the IL2-R score. The evident reduction of MHC-II immunoreactivity suggests an impairment in MHC-II linked antigen presenting cell expression.

Ten cases of feline mesothelioma: an immunohistochemical and ultrastructural study.

In the cat only 10 cases of mesothelioma, mainly of the peritoneum, have been previously reported. This paper describes a further 10 cases, eight pleural and two peritoneal, in males and females aged 1-17 years. Histologically, five tumours were epithelial, three fibrosarcomatous and two biphasic. Immunohistochemical markers used in human pathology for the identification of mesotheliomas include vimentin, cytokeratin (CK) AE1/AE3, HBME-1, CK 5/6, calretinin, thrombomodulin, carcinoembryonic antigen (CEA), CD15, E-cadherin and desmin. All 10 feline mesotheliomas were positive for vimentin and CK AE1/AE3, six were positive for HBME-1, two for CK5/6, three for CEA and four for E-cadherin. All were negative for desmin and calretinin. Antibodies to thrombomodulin and CD15 failed to cross-react with feline tissues. Electron microscopy, performed in four cases, revealed microvillar structures, desmosomes and intracytoplasmic lumina, confirming its value as a diagnostic tool. The study showed that mesothelial marker antibodies commonly used in human patients can be used for the diagnosis of feline mesothelioma, preferably as a panel of antibodies rather than only one.

Deciduoid peritoneal mesothelioma in a dog.

Deciduoid mesothelioma is a rare variant of epithelial mesothelioma, up to now only described in human pathology, which bears remarkable cytomorphologic resemblance to the endometrium of pregnancy, termed decidua. A case of peritoneal mesothelioma with deciduoid features in a 10-year-old, female dog is reported. Multiple whitish-gray nodules (1-5 mm in diameter) in parietal peritoneum and mesentery were histologically composed of large, proliferating, polygonal or ovoid cells with an abundant eosinophilic, glassy cytoplasm. Immunohistochemical evaluation indicated that the neoplastic cells coexpressed cytokeratin and vimentin with strong and diffuse cytoplasmic staining, and ultrastructural analysis showed long and slender mesothelial-type microvilli; these findings confirmed the mesothelial origin of the tumor.

Cytological diagnosis of mandibular salivary gland adenocarcinoma in a dog.

A case of mandibular salivary gland adenocarcinoma in a 9-year-old female dog is described. Material collected by fine needle aspiration underwent cytological examination and after the diagnosis of salivary carcinoma the mass was surgically excised and then was processed for histological examination. The aim of this work is to describe the cytopathological features of this carcinoma and to emphasize the usefulness of fine needle aspiration technique, which is an effective, inexpensive and minimally invasive method of diagnosis that can be performed before incisional biopsy or even before surgical excision.

Apoptosis and proliferative activity in lymph node reaction in postweaning multisystemic wasting syndrome (PMWS).

Postweaning multisystemic wasting syndrome (PMWS) affects nursery and growing pigs, and is characterized by wasting, failure to thrive, pale skin, respiratory distress, diarrhoea and sometimes jaundice. Macroscopic findings are aspecific, but lymphocyte depletion in lymphoid tissues is one of the histological hallmarks [Vet. Q. 24 (2002) 109]. Spontaneous cases of PMWS were studied to evaluate proliferative activity and apoptosis as mechanisms involved in the pathogenesis of cell depletion in lymph nodes. The presence of Porcine Circovirus type 2 (PCV2) genome in the processed material was confirmed by in situ hybridization (ISH). The lymph node pattern of depletion was graded as initial, intermediate or final stage according to histological criteria in 10 superficial inguinal nodes from piglets with PMWS which died spontaneously or were slaughtered by euthanasia. The apoptotic and proliferative fraction were investigated by monoclonal antibody MIB1 immunohistochemistry and TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-nick end labeling) methods, respectively, and compared to three normal cases. One-way analysis of variance (ANOVA) comparison between the MIB1 index (number of positive cells per 100 nuclei) in controls and PMWS cases revealed a decrease of proliferation in both lymphoid and medulla-like tissues in the initial group (respectively, P=0.0017 and 0.024) but not in the intermediate (respectively, P=0.25 and 0.88) or final (respectively, P=0.47 and 0.81) cohorts. The apoptotic index (number of apoptosis/apoptotic bodies in 100 cells) revealed a statistically significant decrease only in the initial group (one-way ANOVA P=0.05). The proliferation/apoptosis ratio (MIB1/APO ratio) assessed to determine cell turnover disclosed a significant decrease of cell turnover from initial to final PMWS cases (Spearman's rank test: P=0.027). Decreased cell proliferation and not increased apoptosis seems to be the most important variable leading to cell depletion in PMWS lymphoid tissues.

Gastrointestinal spindle cell tumours of the dog: histological and immunohistochemical study.

To assess the relevance of spindle cell tumours in the canine gastrointestinal (GI) tract and to classify them, a retrospective study was carried out on haematoxylin and eosin-stained sections from formalin-fixed paraffin wax-embedded samples of 105 primary GI tumours. Seventeen out of 105 (16%) GI tumours were mesenchymal, 48% were epithelial and 36% were round cell tumours. Spindle cell tumours were stained by Masson trichrome, Orcein-Van Gieson and labelled immunohistochemically (vimentin, desmin, smooth muscle actin, protein S100, glial fibrillar acid protein, CD117 and MIB-1) and the histological grade, mitotic index, nuclear size and cellular density were also assessed. The 17 gastrointestinal mesenchymal tumours were classified as 10 leiomyomas (10/10 positive for desmin and smooth muscle actin; 6/10 positive for vimentin) 2 leiomyosarcomas (2/2 positive for desmin, smooth muscle actin and vimentin) and 5 gastrointestinal stromal tumours (GISTs) (5/5 positive for CD117 and vimentin; 3/5 positive for smooth muscle actin). Canine GISTs appeared as densely packed spindle cell tumours, with a diffuse, strong, cytoplasmic immunopositivity for c-kit protein (CD117). GISTs, defined as CD117-positive spindle cell or epithelioid or pleomorphic neoplasms that presumably derive from interstitial cells of Cajal, are reported in recent medical studies as the most common mesenchymal tumours of the GI tract. Our data suggest that GISTs represent a significant portion of canine GI spindle cell tumours, which can be definitely distinguished from leiomyosarcomas only by their expression of CD117.

Histochemical and immunohistochemical characterization of canine mammary mucinous carcinoma.

Mucinous carcinoma is a rare mammary tumour, characterized by intracellular and extracellular mucin. It is still uncertain whether the origin of the mucin is epithelial, myoepithelial or fibroblastic. Eleven canine cases originally classified as mucinous carcinomas were reassessed and compared with myoepithelial nests of mixed tumours. All samples were examined (1) histochemically by the periodic acid-Schiff (PAS) and PAS-diastase methods, and with alcian blue (pH 2.5 and pH 1.0), mucicarmine and Grimelius silver stain, and (2) immunohistochemically for cytokeratin 19, vimentin, alpha-actin and chromogranin A. This examination revealed that only five of the 11 tumours were genuine mucinous carcinomas. In these five tumours the mucus-secreting cells showed cytoplasmic cytokeratin 19 positivity; the mucus showed PAS-diastase and mucicarmine positivity, and alcianophilia which was stronger at pH 2.5 than at 1.0. The remaining six cases were re-classified as mixed tumours because both mucus and mucus-producing cells shared the following similarities with myoepithelial nests of mixed tumours: vimentin and alpha-actin cytoplasmic positivity, PAS negativity, alcianophilia both at pH 2.5 and 1.0, and mucicarmine positivity.

Hypertrophy of intestinal smooth muscle in cats.

Pathological findings of four cats with severe and diffuse smooth muscle hypertrophy of the small intestine (MHSI) are reported and compared to those of five cats with segmental MHSI secondary to neoplastic obstruction and four controls. Histology demonstrated a constant association between idiopathic MHSI and submucosal fibrosis and chronic lymphoplasmacytic enteritis. Morphometry (gut diameter, thickness and area of muscular layers, number and density of smooth muscle nuclei) and MIB-1-immunolabelling showed that the thickness increase was mostly due to hypertrophy, but hyperplasia was also evident. Microbiology from ileal content samples was performed in two cats with primary MHSI, and Campylobacter spp. were isolated, which were also demonstrated by immunohistochemistry and ultrastructure. The association of chronic enteritis with idiopathic MHSI suggests that factors released in intestinal inflammation may also act as hypertrophy stimuli for smooth muscle cells.

Transitional cell carcinoma of the renal pelvis in two dogs.

Transitional cell carcinoma (TCC) of the renal pelvis was found in two dogs, a 7-year-old male English Setter and a 11-year-old female Shetland shepherd. Affected dogs were presented for clinical examination without any specific symptoms but haematuria in case 1 and occurrence of whitish material in the urine of case 2; neoplastic disorders were discovered with ultrasonographic investigation and fine needle aspiration biopsy. Histopathological examination was carried out after nephrectomy and ureterectomy of the affected kidney of both dogs, and confirmed the diagnosis of non-invasive and low grade TCC in case 1 and of infiltrating TCC in case 2. The clinical, gross, cytological and histopathological features of these rare tumours originating from transitional epithelium of the renal pelvis are reported.

Multiparametric survival analysis of histological stage and proliferative activity in feline mammary carcinomas.

Mammary tumours are among the most frequent malignant neoplasms in the cat and determination of prognosis on histological grounds alone can be unsatisfactory because it does not always correspond to the clinical behaviour of the neoplastic disease. The aim of this two-year post-mastectomy survival study is to relate the histological stage or invasiveness (the most commonly used histological parameter to grade malignancy) to several parameters assessing the proliferative activity-mitotic index, MIB1 index, and AgNOR index. Invasiveness was graded as local and vascular invasion whilst values of the parameters expressing proliferative activity, all quantified by image analysis, have been classified into low and high proliferative activity groups according to their median values, (0.719 for mitotic index, 12.11 for MIB1 index, and 3.19 for AgNOR index). For each group, mean survival (months+/-SD) was calculated. Histological stage (local invasion 21.83+/-7.83 months, blood vessels and/or lymphatics invasion 13.38+/-8.99,P<0.01), mitotic index (low 22.43+/-88.78, high 12.37+/-7.49,P<0.001), and AgNOR index (low 21.86+/-10.68, high 13.82+/-7.11,P<0.05) revealed a significant association with survival in univariate analysis and had an independent prognostic value in multiparametric survival test (P<0.001).

Cell proliferation patterns in canine infundibular keratinizing acanthoma and well differentiated squamous cell carcinoma of the skin.

The aim of the present study was to investigate if the evaluation of cell proliferation of the well differentiated squamous cell carcinoma (WDSCC) and infundibular keratinizing acanthoma (IKA) could be useful in the differential diagnosis between these two tumours. Eighteen IKAs and ten WDSCCs were selected for this study. Two different methods were used to assess the activity of cell proliferation: MIB1 immunohistochemical detection and AgNOR proteins silver staining. The quantification of proliferative parameters was performed by means of an image analyzer and expressed as MIB1 index and AgNOR area (MNORA). Both MIBI immunohistochemical and AgNOR histochemical patterns were different in WDSCC and IKA; moreover analysis of variance showed a significant difference for both parameters employed (MIB1 index, MNORA) between WDSCC and IKA (P<0.003 for MIB1 index; P<0.0001 for AgNOR area). The results show that canine WDSCC and IKA have a different proliferative behaviour and the assessment of cell proliferation can be considered as a useful adjunctive tool to the histopathological investigation in the differential diagnosis of these tumours.

Central nervous system cryptococcoma in a cat.

This report describes the gross, histopathological, immunohistochemical and electron microscopic findings in a 4-year-old cat with systemic Cryptococcus neoformans infection. A 1-cm diameter pontine mass, pinpoint lesions in the cerebellum and in the right kidney were the main macroscopic findings. A presumptive diagnosis of cerebral neoplasia with metastasis was formulated. Light microscopy revealed a huge number of yeasts surrounded by a pyogranulomatous inflammatory reaction in the cerebral parenchyma and, to a lesser extent, in the kidney, while the meninges were not involved. The positive mucicarmine stain that coloured the capsule of the yeasts was indicative of cryptococcal infection. Immunohistochemistry confirmed the presence of C. neoformans var. grubii (C. neoformans serotype A). Electron microscopy revealed yeasts with a massive fibrillar capsule and lamellar cell wall free in the cerebral tissue and within macrophages.

Sertoli cell tumour with Call-Exner-like bodies in a dog.

An histological diagnosis of Sertoli cell tumour and concurrent seminoma was formulated after complete resection of the right testicle in a 8-year-old Doberman. The unusual histological feature of abundant eosinophilic material with a extracellular location forming the Call-Exner-like bodies is reported.

Immunohistochemical characterisation of the lymph node reaction in pig post-weaning multisystemic wasting syndrome (PMWS).

The superficial inguinal lymph nodes of 10 piglets which had died spontaneously of post-weaning multisystemic wasting syndrome (PMWS), in which the porcine circovirus type II (PCV-II) genome was revealed by PCR, were submitted to immunohistochemical investigation for CD4, CD8, IgM, MAC387, S-100 protein, vimentin and F-VIII-RA and compared with three normal cases. The lymph node reaction was graded as initial, intermediate and end stage according to histological criteria. In the initial and intermediate stages, absence of follicles and depletion of lymphocytes were evident. Associated with this was a reduction in numbers of interfollicular dendritic cells and interdigitating cells and a reduction/absence of B cells and mainly CD4+ T lymphocytes. In the end stage the reduced expression of high endothelial venules and the prevalence of the stromal component of the lymph node was prominent, as well as the above changes. It is concluded that more than one mechanism is involved in the immunosuppressive ability of PCV-II: reduction of the antigen presenting ability and reduction of B cells and CD4+ T cell function.

Cell proliferation and apoptosis in the pathogenesis of oesophagogastric lesions in pigs.

Recent studies have stressed the importance of epithelial hyperproliferation in the pathogenesis of early lesions (parakeratotic hyperkeratosis) of the porcine gastric pars oesophagea (PO). In this study, immunohistochemical staining with Ki67 (clone MIB1) and AgNOR proteins silver staining were used to evaluate, by means of image analysis, cell proliferation in normal and parakeratotic (parakeratotic hyperkeratosis) epithelia of the PO. Apoptotic activity was also assessed with the TUNEL assay and compared with cell proliferative parameters. Early lesions of the PO were characterised by a significant increase in epithelial proliferative activity while there was no difference in the apoptotic activity between normal and parakeratotic epithelia. Our data confirm the hyperproliferative nature of epithelial changes preceding degeneration and erosion/ulcer of the PO and suggest that an underlying feature of gastric ulcers in pigs is an imbalance between cell proliferation and programmed cell death.

Evaluating mitotic activity in canine and feline solid tumors: standardizing the parameter.

Three different methods for evaluating mitotic activity (mitotic count, mitoses/area, mitotic index) were applied to different types of canine and feline solid tumors to determine the method that is most objective and correlates best with other parameters of cell proliferation. Mitotic activity was evaluated on toluidine blue stained histological sections. Slides stained with histochemical (AgNOR proteins) and immunohistochemical (MIB1, PCNA) markers of cell proliferation were available for each case. Quantitation of mitotic activity and cell proliferation parameters was performed with an image analyzer. Mitotic activity assessment was compared with cell proliferation indices and its ability to discriminate tumors grouped on histologically based criteria including the histological type, malignant or benign characteristics, and grade. A significant correlation by linear regression analysis with other parameters assessing cell proliferation revealed that mitotic index correlated 1000% and mitoses/area and mitotic count correlated 40% of the time. In discriminating the proliferative activity of tumors grouped by histological criteria, mitotic index and mitotic count revealed 1000% concordance with the other parameters of cell proliferation, while mitoses/areas showed 80% concordance.

Protein degradation and apoptotic death in lymphocytes during Fiv infection: activation of the ubiquitin-proteasome proteolytic system.

The movement of a cell through the sequential phases of apoptosis is accompanied by a progressive decrease in cell size with loss in protein mass. In lymphocytes from Hiv-infected persons, protein loss during apoptosis is due to increased protein degradation rather than decreased synthesis. To identify and characterize the proteolytic enzymes or enzyme systems involved in this process, we studied several features of protein turnover in lymphocytes from peripheral blood and lymph nodes during the natural and experimental infection by feline immunodeficiency virus (Fiv). This animal model allowed us to integrate in vivo results with in vitro observations of protein damage. Here we report that protein breakdown in apoptotic cells is concomitant with the activation of the ATP and ubiquitin-dependent multicatalytic system (proteasome). We suggest that proteasome activation is part of the proteolytic cascade in the execution phases of apoptosis in AIDS.