Prognostic impact of Ki-67 in canine splenic hemangiosarcoma: A preliminary study.

Canine splenic hemangiosarcoma has a high metastatic rate and short survival time. Currently, the main prognostic parameters are tumor stage and therapy, while data on histologic parameters, such as grade and Ki-67 expression, are scarce. The aims of this study were to compare two methods of assessment of Ki-67, verify their prognostic impact, and define a threshold value based on survival. Thirty-one cases of histologically diagnosed canine splenic hemangiosarcoma, which were treated with splenectomy and had full staging and follow-up information, were collected. Three were stage I, 17 stage II, and 11 stage III. The mean mitotic count (MC) was 23.9 (standard deviation [SD]: 22.1) and the median was 15 (range, 1-93). Immunohistochemistry for Ki-67 was performed, the Ki-67 labeling index (Ki-67LI) was assessed as a percentage of positive neoplastic nuclei per ≥500 cell, and the Ki-67 count (KI-67C) was defined as the average number of positive nuclei using a 1 cm2 optical grid performed in 5, 40× fields. The mean Ki-67LI and Ki-67C were 56.4% (SD: 38.7) and 27.2 (SD: 12.9) and medians were 51% (range, 8.2-55.2) and 26 (range, 5.5-148), respectively. Using a cut-off of 56% and 9, respectively, Kaplan-Meier survival curves showed an association of overall survival with Ki-67LI and MC. In addition to clinical stage, Ki-67LI maintained its prognostic value on multivariate analysis, supporting the role of Ki-67LI as an independent prognostic parameter. Based on these results, we propose a diagnostically applicable cut-off value of 56% for Ki-67LI as a prognostic parameter for canine splenic hemangiosarcoma.

p53, ER, and Ki67 Expression in Canine Mammary Carcinomas and Correlation With Pathological Variables and Prognosis.

Using immunohistochemistry, 170 canine mammary carcinomas were evaluated for p53, ER (estrogen receptor), and Ki67. Of the 170 tumors, 89 were grade I (52.3%), 36 were grade II (21.2%), and 45 were grade III (26.4%). Eight cases (0.5%) were positive for p53 and 69/170 cases (40.5%) were positive for ER. Ki67 values were 24 ± 18% (mean ± SD). Using a cutoff value of 33.3% Ki67-positive neoplastic nuclei, 38/159 (23.8%) were classified as high proliferative and 121/159 (76.2%) as low proliferative. p53-positive cases had significantly higher Ki67 expression and higher histological grade. ER expression was not correlated with p53 expression but was significantly related to low Ki67 values and low histological grade. Moreover, ER-positive cases had significantly longer survival compared to ER-negative tumors, and ER expression had better correlation with tumor-related survival than histological grade. In summary, p53 accumulated in a small subset of canine mammary tumors and was associated with higher proliferative activity and higher histological grade. ER expression was confirmed as a differentiation marker associated with more favorable prognosis and biological behavior. The combined use of these 3 markers could be used in addition to histological grade to predict the biological behavior of canine mammary carcinomas.

Canine Mammary Carcinoma With Vacuolated Cytoplasm: Glycogen-Rich Carcinoma, a Histological Type Distinct From Lipid-Rich Carcinoma.

Lipid-rich carcinoma is a rare histotype of canine mammary tumors with cytoplasmic vacuolation. In humans, glycogen-rich carcinoma, secretory carcinoma, and myoepithelial neoplasms are included in the differential diagnosis for lipid-rich carcinoma. The aim of the study was to investigate the existence of histotypes other than lipid-rich in canine mammary carcinomas with vacuolated cytoplasm using a diagnostic algorithm based on histopathology, histochemistry, immunohistochemistry, and ultrastructure and to evaluate the molecular phenotype of these neoplasms. Ten mammary carcinomas were collected, histologically reviewed, and subjected to histochemistry (PAS, PAS with diastase, Alcian blue, Sudan III [1 case], and Congo red [1 case]); immunohistochemistry for CK19, CK5/6, CK14, p63, calponin, vimentin, ER, PR, and HER2; and transmission electron microscopy (TEM). Cytokeratin immunolabeling demonstrated the epithelial origin of all tumors. Sudan III and TEM confirmed the diagnosis of lipid-rich carcinoma in 8 tumors (one amyloid-producing). One tumor was reclassified as a glycogen-rich carcinoma based on PAS reactivity that was diastase-labile, and a second tumor was reclassified as a carcinoma-and-malignant myoepithelioma based on the differentiation markers. Lipid-rich carcinomas were basal-like (5/8), null-type (2/8), and luminal A phenotype (1/8). The glycogen-rich carcinoma was basal-like, while the carcinoma-and-malignant myoepithelioma was luminal A. Vacuolated morphology of neoplastic cells in canine mammary carcinoma can indicate either a neoplasm of luminal epithelial origin with cytoplasmic lipid or glycogen, or vacuolated neoplastic suprabasal myoepithelial cells. Glycogen-rich carcinoma is a novel histological type that should be considered in the differential diagnosis for canine mammary carcinomas with vacuolated cytoplasm.

Fibroblast-associated protein-α expression and BPV nucleic acid distribution in equine sarcoids.

Sarcoids are the most common cutaneous tumor of equids and are caused by bovine papillomavirus (BPV). Different clinical subtypes of sarcoids are well characterized clinically but not histologically, and it is not known whether viral activity influences the clinical or histological appearance of the tumors. The aim of this study was to verify whether the development of different clinical types of sarcoids or the presence of certain histological features were associated with BPV distribution within the tumor. The presence of BPV was assessed by polymerase chain reaction (PCR) and visualized in histological sections by chromogenic in situ hybridization (CISH) in 74 equine sarcoids. Furthermore, to better characterize the molecular features of neoplastic cells, immunohistochemistry for S100, smooth muscle actin-α (αSMA), and fibroblast-associated protein-α (FAPα) was performed. The presence of BPV was confirmed in all tissues examined by either or both PCR and CISH (72/74, 97% each). Of 70/74 CISH-positive cases, signal distribution appeared as either diffuse (61/70, 87%) or subepithelial (9/70, 13%); the latter was more frequently observed in the verrucous subtype. However, no statistically significant association was found between clinical subtypes and specific histological features or hybridization pattern. Moreover, CISH signal for BPV was not detected in the epidermis overlying sarcoids nor in the tissue surrounding the neoplasms. By immunohistochemistry, αSMA confirmed the myofibroblastic differentiation of neoplastic cells in 28/74 (38%) sarcoids. Using tissue microarrays, FAPα labelling was observed in neoplastic fibroblasts of all sarcoids, suggesting this marker as a potential candidate for the immunohistochemical diagnosis of sarcoids.

Equine Intestinal Lymphoma: Clinical-Pathological Features, Immunophenotype, and Survival.

Lymphoma is the most common intestinal neoplasm in horses, but its clinical-pathological features are poorly characterized. Primary intestinal lymphoma was diagnosed in 20 horses on biopsy samples and further confirmed by postmortem examination in 16 cases. Lymphoma was found in the small intestine in 12 of 20 (60%), in the colon in 5 of 20 (25%), and in both small and large intestines in 3 of 20 (15%) cases. Gross findings included thickening of the intestinal wall (45%), mural nodules or masses (30%), and both thickening and nodules (10%). Cases were classified according to the human World Health Organization classification as enteropathy-associated T-cell lymphoma (EATL) type 1 (40%), EATL type 2 (45%), and T-cell-rich large B-cell lymphoma (TCRLBCL) (15%). With respect to histologic grade, 70% of cases were grade 1 and 30% were grade 2. Of EATLs, the infiltrate was mucosal only (12%), mucosal and submucosal (53%), or transmural (35%). EATL1 was submucosal to transmural (2/8 and 6/8), EATL2 was mucosal to submucosal (3/9 and 6/9), and TCRLBCL was always transmural. Epitheliotropism was present in most EATLs and characterized by single-cell infiltrates within the epithelium in EATL1 and intraepithelial clusters or plaques in EATL2. Median survival was 25 days for EATL1, 90 days for EATL2, and 187.5 days for TCRLBCL; differences were not statistically significant. Of the EATLs, grade 1 had a median survival of 60 days and grade 2 had a median survival of 25 days; differences were not statistically significant.

p53 Expression in Canine Liposarcoma Correlates With Myxoid Variant and Higher Proliferative Activity.

Canine liposarcoma is classified as well differentiated (WDL), dedifferentiated (DDL), myxoid (ML), and pleomorphic (PL). Overexpression of the protooncogene MDM2 has been reported in WDL and DDL, but little is known regarding the role of p53 in their tumorigenesis. The aim of this study was to assess p53 expression in canine liposarcoma and compare it with subtype, grade, mitotic count (MC), Ki67 labeling index (LI), and MDM2 expression. Forty-seven cases were included (13 WDL, 3 DDL, 7 ML, and 24 PL); 17 were MDM2-positive (13 WDL, 3DDL, and 1ML). Five were p53-positive (4 ML and 1 WDL) but DDL and PL were consistently negative. p53 expression correlated with higher Ki67-LI, higher MC, and myxoid histotype. No correlation was found with grade and MDM2 expression. Based on these results canine liposarcoma seems to embody a group of neoplasms whose subtypes, especially ML, may represent distinct diseases rather than morphological variants of the same entity.

PITUITARY PARS INTERMEDIA DYSFUNCTION (EQUINE CUSHING'S DISEASE) IN NONDOMESTIC EQUIDS AT MARWELL WILDLIFE: A CASE SERIES. ONE CHAPMAN'S ZEBRA ( EQUUS QUAGGA CHAPMANI) AND FIVE PRZEWALSKI's HORSES ( EQUUS FERUS PRZEWALSKII).

Pituitary pars intermedia dysfunction (PPID), also known as equine Cushing's disease, is widely reported in middle-aged to older domestic equids but to date reported in only one nondomestic equid, the onager ( Equus hemionus onager). This case series reports clinical, hematological, and pathological findings consistent with PPID in two further equid species: one Chapman's zebra ( Equus quagga chapmani) and five Przewalski's horses ( Equus ferus przewalskii). The case series reports basal adrenocorticotropic hormone (ACTH) testing as a method to diagnose and monitor PPID in zoological equids and the use of pergolide mesylate to reduce basal ACTH concentration and reduce clinical signs associated with PPID. Gross and histopathological examinations of the pituitary gland in four of these cases revealed either pars intermedia adenomas or adenomatous hyperplasia, similar to pathological findings in domestic equids affected by PPID. These findings suggest that clinicians working with nondomestic equids should be aware of this condition and consider screening for it routinely, particularly given that improvements in management and veterinary care for exotic animals are resulting in a more aged captive population. Early diagnosis and treatment of PPID may prevent the development of painful clinical sequelae and therefore improve the welfare of zoo equids.

Inhibition of the KCa3.1 Channel Alleviates Established Pulmonary Fibrosis in a Large Animal Model.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease of increasing prevalence marked by poor prognosis and limited treatment options. Ca2+-activated KCa3.1 potassium channels have been shown to play a key role in the aberrant activation and responses to injury in both epithelial cells and fibroblasts, both considered key drivers in the fibrotic process of IPF. Pharmacological inhibition of IPF-derived fibroblasts is able to somewhat prevent TGF-ß- and basic fibroblast growth factor-dependent profibrotic responses. In the current study, we investigated whether blockade of the KCa3.1 ion channel in vivo with a selective inhibitor, Senicapoc, was able to attenuate both histological and physiological outcomes of early fibrosis in our large animal (sheep) model for pulmonary fibrosis. We also determined whether treatment was targeting the profibrotic activity of sheep lung fibroblasts. Senicapoc was administered in established fibrosis, at 2 weeks after bleomycin instillation, and drug efficacy was assessed 4 weeks after treatment. Treatment with Senicapoc improved pre-established bleomycin-induced changes compared with vehicle control, leading to improved lung compliance, reduced extracellular matrix and collagen deposition, and a reduction in both α-smooth muscle actin expression and proliferating cells, both in vivo and in vitro. These studies show that inhibiting the KCa3.1 ion channel is able to attenuate the early fibrogenic phase of bleomycin-dependent fibrosis and inhibits profibrotic behavior of primary sheep lung fibroblasts. This supports the previous research conducted in human IPF-derived fibroblasts and suggests that inhibiting KCa3.1 signaling may provide a novel therapeutic approach for IPF.

Immune responses to vaccination and infection with Mycoplasma gallisepticum in turkeys.

Infection with Mycoplasma gallisepticum induces severe lymphoproliferative lesions in multiple sites along the respiratory tract in chickens and turkeys. These immunopathological responses have been well-characterized in chickens, but have not been studied closely in turkeys. The aim of the study described here was to examine the immune responses of turkeys after live vaccination and infection with M. gallisepticum. In a strain comparison study, the mean log10 antibody titre of birds exposed to an aerosol culture of M. gallisepticum strain Ap3AS was found to be significantly higher at day 14 than that of birds exposed to strain 100809/31. In a dose-response study, there was a significant difference in the mean log10 antibody titre between birds exposed to mycoplasma broth and birds exposed to the highest dose of strain Ap3AS at day 7 after exposure. Immunohistochemical analysis of the tracheal mucosa and the air sacs revealed similar patterns of distribution of CD4+ and CD8+ lymphocytes to those seen in the tracheal mucosa of chickens, implicating these cell types in the pathogenesis of respiratory mycoplasmosis in turkeys. Turkeys that had been vaccinated with M. gallisepticum GapA+ ts-11 had significantly higher antibody titres than unvaccinated birds at both 7 and 14 days after challenge with strain Ap3AS. Vaccination with GapA+ ts-11 protected against the lymphoproliferative response to infection with virulent M. gallisepticum in both the tracheal mucosa and the air sacs, suggesting that this strain may be a useful vaccine candidate for use in turkeys.

A novel segmental challenge model for bleomycin-induced pulmonary fibrosis in sheep.

BACKGROUND: Idiopathic Pulmonary fibrosis (IPF) is a fatal respiratory disease, characterized by a progressive fibrosis and worsening lung function. While the outcomes of recent clinical trials have resulted in therapies to slow the progression of the disease, there is still a need to develop alternative therapies, which are able to prevent fibrosis. AIM: This study uses a segmental lung infusion of bleomycin (BLM) to investigate pulmonary fibrosis in a physiologically relevant large animal species. METHODS: Two separate lung segments in eight sheep received two fortnightly challenges of either 3U or 30U BLM per segment, and a third segment received saline (control). Lung function was assessed using a wedged-bronchoscope procedure. Bronchoalveolar lavage fluid and lung tissue were assessed for inflammation, fibrosis and collagen content two weeks after the final dose of BLM. RESULTS: Instillation of both BLM doses resulted in prominent fibrosis in the treated lobes. More diffuse fibrosis and loss of alveolar airspace was observed in high-dose BLM-treated segments, while multifocal fibrosis was seen in low-dose BLM-treated segments. Extensive and disorganised collagen deposition occurred in the BLM-treated lobes, compared to controls. Significant loss of lung compliance was also observed in the BLM-treated lobes, which did not occur in controls. CONCLUSIONS: Fibrosis comparable to IPF was induced into isolated lung segments, without compromising the respiratory functioning of the animal. This model may have potential for investigating novel therapies for IPF by allowing direct comparison of multiple treatments with internal controls, and sampling and drug delivery that are clinically relevant.

Development of a Mycoplasma gallisepticum infection model in turkeys.

Mycoplasma gallisepticum causes chronic respiratory disease in chickens and is also highly pathogenic in turkeys. Several live attenuated M. gallisepticum vaccines are available for prevention of disease in chickens but they are considered to be either not safe or not efficacious in turkeys. The studies presented here aimed to develop a suitable infection model in turkeys, a prerequisite for development of a vaccine against M. gallisepticum for turkeys. Two wild-type Australian M. gallisepticum strains, Ap3AS and 100809/31, were used and their capacity to induce lesions was evaluated in 5-week-old to 6-week-old turkeys exposed to aerosols of these strains. Gross air sac lesion scores in the group exposed to Ap3AS were significantly greater than those in the group exposed to 100809/31 (P < 0.05). Histological tracheal lesion scores and tracheal mucosal thicknesses were significantly greater in birds exposed to either strain than in the unexposed birds (P < 0.05), but no significant differences were observed between the two infected groups. In a subsequent experiment, 6-week-old to 7-week-old turkeys were exposed to different doses of M. gallisepticum Ap3AS. Serology and M. gallisepticum re-isolation performed 14 days after infection showed that all birds exposed to Ap3AS were positive by rapid serum agglutination and by culture. Gross air sac lesion scores in the groups exposed to the highest dose, 8.17 × 10(8) colour-changing units Ap3AS/ml, as well as a 10-fold lower dose were significantly more severe than in the uninfected control group. Lesion scores and tracheal mucosal thicknesses were significantly greater in birds exposed to Ap3AS than in the unexposed birds (P < 0.05). However, no significant differences were seen in tracheal mucosal thicknesses or lesion scores between the groups exposed to the different doses of Ap3AS. This study has established a reliable challenge model for M. gallisepticum infection in turkeys, which will be useful for evaluation of potential M. gallisepticum vaccine candidates for this species.

Structural and functional correlations in a large animal model of bleomycin-induced pulmonary fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a severe and progressive respiratory disease with poor prognosis. Despite the positive outcomes from recent clinical trials, there is still no cure for this disease. Pre-clinical animal models are currently largely limited to small animals which have a number of shortcomings. We have previously shown that fibrosis is induced in isolated sheep lung segments 14 days after bleomycin treatment. This study aimed to determine whether bleomycin-induced fibrosis and associated functional changes persisted over a seven-week period. METHODS: Two separate lung segments in nine sheep received two challenges two weeks apart of either, 3U bleomycin (BLM), or saline (control). Lung function in these segments was assessed by a wedged-bronchoscope procedure after bleomycin treatment. Lung tissue, and an ex vivo CT analysis were used to assess for the persistence of inflammation, fibrosis and collagen content in this model. RESULTS: Fibrotic changes persisted up to seven weeks in bleomycin-treated isolated lung segments (Pathology scores: bleomycin12.27 ± 0.07 vs. saline 4.90 ± 1.18, n = 9, p = 0.0003). Localization of bleomycin-induced injury and increased tissue density was confirmed by CT analysis (mean densitometric CT value: bleomycin -698 ± 2.95 Hounsfield units vs. saline -898 ± 2.5 Hounsfield units, p = 0.02). Masson's trichrome staining revealed increased connective tissue in bleomycin segments, compared to controls (% blue staining/total field area: 8.5 ± 0.8 vs. 2.1 ± 0.2 %, n = 9, p < 0.0001). bleomycin-treated segments were significantly less compliant from baseline at 7 weeks post treatment compared to control-treated segments (2.05 ± 0.88 vs. 4.97 ± 0.79 mL/cmH20, n = 9, p = 0.002). There was also a direct negative correlation between pathology scores and segmental compliance. CONCLUSIONS: We show that there is a correlation between fibrosis and correspondingly poor lung function which persist for up to seven weeks after bleomycin treatment in this large animal model of pulmonary fibrosis.

Ring 17 syndrome: first clinical report without intellectual disability.

Ring chromosomes are rare abnormalities caused by the fusion of the telomeric regions. Three-ring chromosome syndromes (Cr 20, Cr 17 and Cr 14) cause epilepsy with variable phenotypes. In ring 17 patients with mild phenotype, some authors have shown an epilepsy syndrome similar to that of ring 20. We report the first case of a girl with ring chromosome 17 and a normal neurological and general cognitive profile. She had had, from 9 years old, focal pharmacoresistant epilepsy associated with episodes of non-convulsive status epilepticus with mainly autonomic features. Cytogenetic analysis revealed an abnormal karyotype characterised by the presence of de novo ring chromosome 17 in 19% of metaphases. The array CGH (100 KB) did not show any genetic deletion. The clinical and epilepsy phenotype was, to a certain degree, similar to that of ring 20 syndrome.

Analysis of MDM2 and TP53 genes in canine liposarcoma.

Canine liposarcoma is an uncommon tumor that shares morphological similarities with its human counterpart. In dogs, the genetic features of this tumor are unknown and, based on immunohistochemical studies, amplification of the gene MDM2 and the mutation of TP53 are suspected. In this study 51 cases of primary liposarcomas were immunohistochemically stained for MDM2 and p53 and subjected to fluorescent in situ hybridization and next-generation sequencing to detect MDM2 amplification and TP53 mutations, respectively. MDM2 and p53 were expressed in 21 and 6 cases, respectively. MDM2 amplification and TP53 mutations were identified in 10 and 15 cases, respectively. Statistical analysis revealed an association of the myxoid subtype and the mitotic count with p53 expression and TP53 mutation. No association was found between MDM2 amplification and MDM2 expression or tumor subtype. These results suggest that despite morphological similarities, canine liposarcoma differs from its human counterpart, for which MDM2 amplification is diagnostic for well differentiated and de-differentiated variants, and TP53 mutations are more common in pleomorphic liposarcoma rather than the myxoid one as occur in our cases. Furthermore, canine myxoid liposarcoma likely represents a distinct disease rather than a mere morphological variant.

Keeping Pathologists in the Loop and an Adaptive F1-Score Threshold Method for Mitosis Detection in Canine Perivascular Wall Tumours.

Performing a mitosis count (MC) is the diagnostic task of histologically grading canine Soft Tissue Sarcoma (cSTS). However, mitosis count is subject to inter- and intra-observer variability. Deep learning models can offer a standardisation in the process of MC used to histologically grade canine Soft Tissue Sarcomas. Subsequently, the focus of this study was mitosis detection in canine Perivascular Wall Tumours (cPWTs). Generating mitosis annotations is a long and arduous process open to inter-observer variability. Therefore, by keeping pathologists in the loop, a two-step annotation process was performed where a pre-trained Faster R-CNN model was trained on initial annotations provided by veterinary pathologists. The pathologists reviewed the output false positive mitosis candidates and determined whether these were overlooked candidates, thus updating the dataset. Faster R-CNN was then trained on this updated dataset. An optimal decision threshold was applied to maximise the F1-score predetermined using the validation set and produced our best F1-score of 0.75, which is competitive with the state of the art in the canine mitosis domain.

Whole body computed tomographic characteristics of skeletal and cardiac muscular metastatic neoplasia in dogs and cats.

Muscular metastatic neoplasia has been reported to be rare in domestic animals, however previous studies were based primarily on necropsy findings. The purpose of this retrospective study was to describe whole body computed tomography (CT) characteristics of confirmed muscular metastases in a cohort of dogs and cats presented for oncology evaluation. Medical records of 1201 oncology patients were reviewed. Included animals underwent pre and postcontrast whole body CT, and CT-guided tru-cut biopsy or fine needle aspiration of one or more metastatic lesions. Twenty-one dogs and six cats met inclusion criteria, representing 2.08% of all canine oncology patients and 3.1% of all feline oncology patients. Mean age was 9.6 years. Postcontrast CT characteristics included well-demarcated, oval-to-round lesions with varying enhancement patterns: ring enhancing (n = 16), heterogeneously enhancing (n = 8), or homogeneously enhancing (n = 5). Five animals showed concurrent and varying nodular patterns. In seven cases (five dogs and two cats), one single muscular nodule was observed. In 20 cases, two or more lesions were observed. In two cases, cardiac hypodense nodules were observed in the postcontrast CT, while appearing isodense in the precontrast study. Necropsy confirmed neoplasia in both of them. Locations of muscular metastases included epaxial/paraspinal muscles of the cervical, thoracic, and lumbar spine (n = 18), superficial muscles of the thoracic wall (n = 13), scapular/shoulder region (n = 3), hind limb (n = 3), and abdominal wall muscles (n = 1). Findings supported the use of pre and postcontrast whole body CT for oncologic staging in dogs and cats, especially for primary tumors characterized by a high metastatic rate.

Validation of p53 Immunohistochemistry (PAb240 Clone) in Canine Tumors with Next-Generation Sequencing (NGS) Analysis.

In human medicine, p53 immunohistochemistry (IHC) is a common method that is used for the identification of tumors with TP53 mutations. In veterinary medicine, several studies have performed IHC for p53 in canine tumors, but it is not known how well it actually predicts the mutation. The aim of this study was to estimate the accuracy of the IHC method for p53 (clone PAb240) using a lab-developed NGS panel to analyze TP53 mutations in a subset of malignant tumors in dogs. A total of 176 tumors were analyzed with IHC and then 41 were subjected to NGS analysis; among them, 15 were IHC positive and 26 were negative, and 16 out of 41 (39%) were found to be inadequate for NGS analysis. Excluding the non-evaluable cases at NGS, of the remaining eight IHC-positive cases, six were mutants and two were wild-type. Among the 17 IHC-negative cases, 13 were wild type, and 4 were mutants. The sensitivity was 60%, specificity was 86.7%, and the accuracy was 76%. These results suggest that when using IHC for p53 with this specific antibody to predict mutation, up to 25% wrong predictions can be expected.

Detection of Necrosis in Digitised Whole-Slide Images for Better Grading of Canine Soft-Tissue Sarcomas Using Machine-Learning.

The definitive diagnosis of canine soft-tissue sarcomas (STSs) is based on histological assessment of formalin-fixed tissues. Assessment of parameters, such as degree of differentiation, necrosis score and mitotic score, give rise to a final tumour grade, which is important in determining prognosis and subsequent treatment modalities. However, grading discrepancies are reported to occur in human and canine STSs, which can result in complications regarding treatment plans. The introduction of digital pathology has the potential to help improve STS grading via automated determination of the presence and extent of necrosis. The detected necrotic regions can be factored in the grading scheme or excluded before analysing the remaining tissue. Here we describe a method to detect tumour necrosis in histopathological whole-slide images (WSIs) of STSs using machine learning. Annotated areas of necrosis were extracted from WSIs and the patches containing necrotic tissue fed into a pre-trained DenseNet161 convolutional neural network (CNN) for training, testing and validation. The proposed CNN architecture reported favourable results, with an overall validation accuracy of 92.7% for necrosis detection which represents the number of correctly classified data instances over the total number of data instances. The proposed method, when vigorously validated represents a promising tool to assist pathologists in evaluating necrosis in canine STS tumours, by increasing efficiency, accuracy and reducing inter-rater variation.

Porcine Lawsonia intracellularis Ileitis in Italy and Its Association with Porcine Circovirus Type 2 (PCV2) Infection.

The objective of this study was to employ a diagnostic algorithm, which involves detecting positive farms by stool PCR followed by PCR and histology/immunohistochemistry on ileum samples, for diagnosing Lawsonia intracellularis proliferative enteritis in Northern Italy. The primary aim was to examine the relationship between the gold standard of L. intracellularis diagnostics, namely histology and immunohistochemistry, and PCR in acute and chronic cases of L. intracellularis enteritides. An additional goal was to investigate the coinfection of L. intracellularis with porcine circovirus type 2 (PCV2). Twenty-eight ileum samples, including four from acute cases and 24 from chronic cases, were collected. PCR yielded positive results in 19 cases (four acute and 15 chronic cases). In comparison, immunohistochemistry was positive in 16 cases (four acute and 12 chronic cases), with an observed agreement of 89%. The findings suggest that performing the two tests in series can increase the specificity of the causal diagnosis. PCR may be used as a screening tool to identify the presence of the microorganism, and only positive cases will be examined by histology and immunohistochemistry to confirm the causative role of L. intracellularis. Co-infection with PCV2 was demonstrate in two out of four acute cases and in two out of 24 chronic cases, providing further evidence to support the hypothesis that when the infection starts with ubiquitous pathogens such as L. intracellularis, it may boost the possibility of PCV2 replication, especially in acute cases. As a result, this may trigger a transition from subclinical to clinical forms of PCV2 disease.

SGCD Missense Variant in a Lagotto Romagnolo Dog with Autosomal Recessively Inherited Limb-Girdle Muscular Dystrophy.

An 8-month-old female Lagotto Romagnolo dog was presented for a 1-month history of an initial severe reluctance to move, rapidly progressing to a marked stiff gait and progressive muscular weakness and evolving to tetraparesis, which persuaded the owner to request euthanasia. A primary muscle pathology was supported by necropsy and histopathological findings. Macroscopically, the muscles were moderately atrophic, except for the diaphragm and the neck muscles, which were markedly thickened. Histologically, all the skeletal muscles examined showed atrophy, hypertrophy, necrosis with calcification of the fibers, and mild fibrosis and inflammation. On immunohistochemistry, all three dystrophin domains and sarcoglycan proteins were absent. On Western blot analysis, no band was present for delta sarcoglycan. We sequenced the genome of the affected dog and compared the data to more than 900 control genomes of different dog breeds. Genetic analysis revealed a homozygous private protein-changing variant in the SGCD gene encoding delta- sarcoglycan in the affected dog. The variant was predicted to induce a SGCD:p.(Leu242Pro) change in the protein. In silico tools predicted the change to be deleterious. Other 770 Lagotto Romagnolo dogs were genotyped for the variant and all found to be homozygous wild type. Based on current knowledge of gene function in other mammalian species, including humans, hamsters, and dogs, we propose the SGCD missense variant as the causative variant of the observed form of muscular dystrophy in the index case. The absence of the variant allele in the Lagotto Romagnolo breeding population indicates a rare allele that has appeared recently.