Gabriele Ghisleni

p73 Overexpression increases VEGF and reduces thrombospondin-1 production: implications for tumor angiogenesis.

Tumor neovascularization is controlled by a balance between positive and negative effectors, whose production can be regulated by oncogenes and tumor suppressor genes. The aim of this study was to investigate whether the angiogenic potential of tumors could also be controlled by p73, a gene homologous to the tumor suppressor p53, whose involvement in tumor angiogenesis is known. We have studied the production of proangiogenic (VEGF, FGF-2, PIGF and PDGF) and antiangiogenic (TSP-1) factors in two p73 overexpressing clones obtained from the human ovarian carcinoma cells A2780. TSP-1 was downregulated in both clones compared to mock transfected cells, both at mRNA and protein level. Conversely, both clones showed an increased production of VEGF mRNA and protein. For both TSP-1 and VEGF, regulation of expression was partially due to modulation of the promoter activity, and was dependent on p53 status. Production of the other angiogenic factors FGF-2, PIGF and PDGF-B was also increased in p73 overexpressing clones. The two clones were more angiogenic than parental cells, as shown in vitro by their increased chemotactic activity for endothelial cells, and in vivo by the generation of more vascularized tumors. These findings suggest a potential role of p73 in tumor angiogenesis.

Wild rats as monitors of environmental lead contamination in the urban area of Milan, Italy.

Twenty-one wild brown rats (Rattus norvegicus) were collected from four areas of Milan (Italy). Main organs were harvested from each rat, fixed in 10% neutral buffer formalin, and embedded in paraffin. Organ sections were stained with hematoxylin and eosin, kidney sections were also stained with the Ziehl-Neelsen method. Graphite furnace atomic absorption spectrophotometry was performed on all kidney samples. The presence of inclusion bodies and karyocytomegaly was scored. Six adult rats out of 21 (28.6%) showed kariocytomegaly and intranuclear inclusion bodies of the renal proximal tubules, characteristic of lead toxicity. The nuclear inclusion bodies were always Ziehl-Neelsen positive. The six rats which showed the characteristic histological alterations ascribed to lead, had lead concentrations statistically higher than the three control rats (4 microg/g wet wt. versus 0.09 microg/g wet wt., P<0.001) and than the captured wild rats. Intranuclear inclusion bodies were found in rats with kidney lead concentrations greater than 0.6 microg/g wet weight. There were significant differences between the four areas of capture. The incidence of intranuclear inclusion bodies and karyocytomegaly in proximal tubular cells was higher in rats with high lead levels detected by spectrophotometry. The present results indicate a renal lead concentration of 0.6 microg/g wet weight as a threshold to detect histologic kidney changes due to lead absorption. Results demonstrate that the examination of adult free-living rats may be considered an appropriate method to obtain a rapid, sensitive, easy, cost effective estimate of lead contamination in urban areas. Animal models allow us to evaluate potential human health risk associated with environmental contamination.

Pulmonary carcinosarcoma in a cat.

A Domestic Shorthaired cat was presented with coughing and severe respiratory distress. Thoracic radiographs revealed a lobar mass and numerous additional cavitated intrapulmonary masses. The cat was euthanized and submitted for necropsy. Histological examination of the large mass revealed 2 distinct neoplastic components consisting of bronchial adenocarcinoma admixed with neoplastic areas composed of highly atypical undifferentiated spindle cells (sarcomatous component). Simultaneous expression of vimentin and cytokeratin by a subpopulation of neoplastic epithelial cells and by rare neoplastic spindle cells was identified. On the basis of histology and immunohistochemical results, a diagnosis of primary pulmonary carcinosarcoma with intrapulmonary epithelial metastases was made. Pulmonary carcinosarcoma is a well-known pathological entity in humans. It is a rare tumor in animals and has not been previously reported in cat.

Cytological features of canine haemangiopericytoma in fine needle aspiration biopsy

HAEMANGIOPERICYTOMA (HEP) is a mesenchymal neoplasm, which occurs in the dog (Mills and Nielsen 1967, Bostock and Dye 1980) and has also been rarely observed in other species. In human beings, HEP is a relatively uncommon soft tissue neoplasm (Tsuneyoshi and others 1984) which bears little resemblance to the canine disease (Hendrick and others 1998). Most HEPS have been reported in mature dogs aged between eight and 14 years. Females appear to be affected more often than males, and the boxer, springer spaniel, German shepherd dog, cocker spaniel and mixed breeds have been considered at higher risk (Mills and Nielsen 1967, Bostock and Dye 1980). Tumours develop as slowgrowing, often lobulated, masses in the subcutis, primarily on the extremities, that may adhere to deeper tissues, while the overlying epidermis is usually freely movable. Following surgery, canine HEP shows a variable rate of local recurrence (Mills and Nielsen 1967, Bostock and Dye 1980, Graves and others 1988, Postorino and others 1988), while distant metastases are rarely encountered (Richardson and others 1983, Postorino and others 1988, Handharyani and others 1999). Generally, the neoplastic cells are believed to originate from the pericytes surrounding blood vessels (Xu 1986), although the actual histogenesis is still uncertain. The diagnosis of canine HEP is based on histology and on the dominating presence of perivascular whorls of spindle cells which can be arranged in interlacing bundles (Hendrick and others 1998). Immunohistochemistry does not help in further differentiating these tumours from other spindle cell tumours due to the lack of specific markers (Perez and oth-

Diagnostic accuracy of brush cytology in canine chronic intranasal disease

BACKGROUND Most cases of canine chronic intranasal disease cannot be differentiated based on clinical examination alone, and biopsy is often required for a definitive diagnosis. Nonsurgical cytologic and histologic biopsy techniques represent desirable diagnostic approaches. OBJECTIVE The aim of this retrospective study was to determine the diagnostic accuracy of brush cytology in differentiating non-neoplastic and neoplastic diseases in dogs with chronic intranasal disease. METHODS Cytologic samples of lesions in dogs with chronic intranasal disease were obtained by brushing over a 12-year period. All dogs had complete physical examinations as well as radiographic, rhinoscopic, and cytologic evaluation. Histologic diagnosis, follow-up clinical information, or both were used as the gold standard, and dogs free of disease or with no progression of disease at 1 year were considered negative for neoplasia. Indicators of performance of brush cytology in detecting neoplasia were calculated and included sensitivity, specificity, positive and negative likelihood ratios, and diagnostic odds ratio. RESULTS Samples of nasal brushings from 138 dogs were evaluated. Of 62 cases of neoplastic disease, true-positive and false-negative diagnoses were made using cytologic evaluation in 44 (71.0%) and 18 (29.0%) cases, respectively. False-negative diagnoses of neoplasia were not attributed to low cellularity, but to the presence of inflammatory cells that masked neoplastic cells. Brush cytology had a sensitivity of 0.71, specificity of 0.99, positive likelihood ratio of 53.94, negative likelihood ratio of 0.29, and diagnostic odds ratio of 188.33. CONCLUSIONS Brush cytology has good diagnostic accuracy for chronic intranasal lesions in dogs.

Cytologic and immunocytochemical characterization of feline progressive histiocytosis.

BACKGROUND Feline Progressive Histiocytosis (FPH) is a cutaneous dendritic cell neoplasm characterized by slow progression and spread to internal organs in the terminal stage. FPH is often misdiagnosed as an inflammatory reaction and has not been fully characterized from a cytologic diagnostic perspective. OBJECTIVES The purpose of the study was to characterize the cytologic and immunocytochemical aspects useful for FPH diagnosis. METHODS Fine-needle aspiration cytologic samples of 5 cases of FPH confirmed by skin biopsy and necropsy were evaluated. Immunocytochemistry with antibodies recognizing CD1a, CD1c, CD3, CD11b, CD18, CD21, and MHCII was performed on air-dried, acetone-fixed smears. E-cadherin expression was assessed on paraffin-embedded skin biopsies. Transmission electron microscopy (TEM) was performed in one case. RESULTS Main cytologic findings on variably cellular samples were characterized by single to cohesive large, round to polygonal cells with intermediate to low N/C ratio, abundant clear homogeneous cytoplasm, and round to oval nuclei with rare bi- to multinucleated atypical cells, associated with low numbers of small lymphocytes and/or neutrophils. Neoplastic cells expressed CD1a, CD1c, CD11b, CD18, and MHCII. Anti-CD3 antibodies identified reactive T cells admixed with the neoplastic cells. E-cadherin expression was observed in all but one case. TEM failed to identify Birbeck granules in one case. CONCLUSIONS FPH is a distinctive neoplastic lesion composed of nonphagocytizing histiocytes variably admixed with neutrophils and small mature lymphocytes. Immunocytochemical analysis with CD1 is mandatory to confirm a dendritic cell origin. Immunocytochemistry and cytomorphology allowed the specific and rapid diagnosis of FPH on cytologic samples.

Immunohistochemical study of mixed germ cell sex cord stromal tumours in 13 canine testes.

Mixed germ cell sex cord stromal tumours (MGSCTs) are composed of seminiferous tubules, filled with admixed neoplastic Sertoli cells (SCs) and germ cells (GCs). The aim of the present study was to describe 13 canine testicular MGSCTs and to investigate the histochemical features and the immunophenotype of the neoplastic GCs and SCs. Neoplastic SCs were always diffusely labelled for vimentin (VIM), neuron specific enolase (NSE), inhibin (INH)-α and anti-Müllerian hormone (AMH). Cytokeratins AE1/AE3 (CK) and desmin (DES) were expressed in 6/13 and 8/13 cases, respectively. Neoplastic GCs were labelled for placental alkaline phosphatase (PLAP) in 7/13 cases and for CD117 (KIT) in 8/13 cases, while 10 cases were stained uniformly by periodic acid-Schiff (PAS). Immature canine SCs are known to express CK, DES, INH-α and AMH, while immature GCs are stained by PAS and express PLAP and KIT. This GC phenotype also distinguishes between classical and spermatocytic seminoma, with the latter being negative for these markers. The results of the present study show that both neoplastic SCs and GCs in MGSCTs have a de-differentiated phenotype.

Primary angiocentric/angioinvasive T-cell lymphoma of the tympanic bulla in a feline leukaemia virus-positive cat

Case summary A 5-year-old neutered female feline leukaemia virus (FeLV)-positive domestic shorthair cat with a 5 month history of otitis media was referred for head tilt, stertor and dyspnoea. Computed tomography scan revealed soft tissue opacities inside the right tympanic bulla, with bone remodelling, and concurrent nasopharyngeal and intracranial invasion. Endoscopically guided bioptic samples were collected from the nasopharynx and middle ear. Histology revealed dense sheets of round, large, neoplastic cells, often surrounding or invading vascular walls. Neoplastic cells expressed CD3, FeLV p27 and gp70 antigens. A middle ear angiocentric/angioinvasive T-cell lymphoma was diagnosed. After improvement of clinical conditions following radiation therapy, the cat died unexpectedly. At necropsy, hepatic and splenic spread was detected. Relevance and novel information Primary middle ear tumours are rare and their diagnosis is often delayed as clinical signs mimic more common otological conditions. Multiple bioptic specimens are pivotal for a definitive diagnosis. The young age of the cat, serology and immunohistochemistry revealed a possible transforming role of FeLV.

Intra‐abdominal fluid aspirate from a dog

A 12‐year‐old, neutered female, Siberian husky, was presented with a 6‐months history of progressive abdominal distension, anorexia, and weight loss. The dog appeared normal on physical examination except for marked abdominal distension. A fluid wave was balloted strongly suggesting an abdominal effusion. Ultrasound examination confirmed this clinical finding. Results of the CBC included mild nonregenerative anemia, with an RBC count of 4.9 × 106/µL (reference interval 5.5–8.5 × 106/µL), hemoglobin concentration of 12 g/dL (reference interval 12–18 g/dL), HCT of 36% (reference interval 37–55%), and reticulocytes <60,000/µL. No abnormalities in serum chemistry were detected. Diagn. Cytopathol. 2016;44:119–120.