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Supplemental Material, Combined_supplemental_materials-Muscatello_et_al for HER2 Overexpression and Amplification in Feline Pulmonary Carcinoma by Luisa Vera Muscatello, Enrico Di Oto, Miriam Dignazzi, William J. Murphy, Ilaria Porcellato, Raffaella De Maria, Terje Raudsepp, Maria Pia Foschini, Monica Sforna, Cinzia Benazzi and Barbara Brunetti in Veterinary Pathology

Canine prostatic carcinoma is a relevant model for human prostatic carcinoma. Survivin is proposed as a biomarker of malignancy in human prostatic cancer. Sox9 is a stem cell marker required for prostate development and expressed in several adult tissues. The aims of the present study were to evaluate the patterns and expression levels of 2 putative stem cell markers, survivin and Sox9, in canine benign prostatic hyperplasia (BPH) and prostatic carcinoma to investigate their potential as stem cell markers. Immunohistochemistry with specific antibodies was performed on 3 samples of normal prostate gland, 18 samples of canine BPH, and 16 samples of prostatic carcinoma. The basal cell layer of normal and hyperplastic prostatic lobules had nuclear Sox9 immunolabeling and nuclear and rarely cytoplasmic survivin immunostaining, identifying them as potential stem cell markers. Significantly more frequent survivin and Sox9 expression (≥10% of nuclei) was observed in prostatic carcinoma as compared with BPH. The potential coexpression of survivin with Sox9, androgen receptor, and p63 was also investigated in selected BPH and prostatic carcinoma cases with immunofluorescence, and a partial colocalization was observed. Results indicate that Sox9 and survivin could be considered markers of stemness in canine prostate cells. Given its role in proliferation, cells in the basal cell layer with nuclear survivin expression are likely to be transit-amplifying cells that maintain some stem cell proprieties. © The Author(s) 2018.

Supplemental Material, DS1_VET_10.1177_0300985819868647 for P-Glycoprotein and Breast Cancer Resistance Protein in Canine Inflammatory and Noninflammatory Grade III Mammary Carcinomas by Michela Levi, Laura Peña, Angela Alonso-Díez, Barbara Brunetti, Luisa Vera Muscatello, Cinzia Benazzi, Maria Dolores Pérez-Alenza and Giuseppe Sarli in Veterinary Pathology

Vengono descritti sei casi di Ectopia cordis (anomalo posizionamento del cuore al di fuori della cavità toracica) nel bovino, cinque dei quali di tipo cervicale, uno sternale. La condizione, osservata in soggetti di più razze e di diverso genere, in due casi ha provocato natimortalità, in tre casi la morte nei primissimi giorni di vita, nell’ultimo vitello una sopravvivenza di un mese. Fra le specie animali, il bovino risulta quello col maggior numero di segnalazioni di Ectopia cordis, soprattutto del tipo cervicale, rappresentando, fra le altre cose, un potenziale modello per studi teratologici comparati con la medicina umana. La convinzione che i dati epidemiologici della patologia, al pari di altre malformazioni, soffrano di sottostima, motiva le sollecitazioni a veterinari ed allevatori a segnalare simili malattie malformative a centri di ricerca per favorirne gli studi. Fra le ipotesi eziologiche, infatti, non può essere taciuta quella genetico/ereditaria, fatto preoccupante nella generalizzata tendenza alla riduzione della variabilità genetica nelle razze bovine. Ectopia cordis is a congenital anomaly defined as an abnormal position of the heart outside the thorax. Based on the position of the heart, the ectopia is cervical, thoracic and abdominal. The first form has been observed most frequently in domestic animal, whereas the last two have been described mainly in man. The article reports 6 cases of Ectopia cordis in calves. In five cases the heart was situated in the cervical area, in one case outside the chest. Affected calves were both male and female, and of different breeds. Two calves were stillborn, three died during the first days of life whereas the last survived for one month. Among the different animals species Ectopia cordis seems to occur mostly in cattle. In this species the cervical Ectopia cordis accounts for the majority of the cases. Cervical Ectopia cordis is likely due to fibrous adhesions between the pericardium and the surrounding tissues that inhibit the migration of the heart into the thoracic cavity. This condition has been found in the subjects examined. Within the pericardial cavity, the apex of the heart was, generally, cranially oriented and the right ventricle was hypertrophic. Furthermore, the pulmonary trunk was connected with the aortic arch by a persistent arterial duct. Thoracic Ectopia cordis is due to a failure of the two embryonic sternal islands to come together, although it has also been hypothesized that the lack of fusion of the sternum is secondary to the ectopia of the heart itself. In any case, the heart protrude from the defective thorax and the pericardium is missing. It is conviction of the authors that, similarly to other congenital malformations, Ectopia cordis is underestimated. Therefore, the authors prompt practitioners and farmers to submit all cases of malformed calves to allow more in depth studies, as a lot of malformations may be caused by genetic defects.

Supplemental Material, DS1_VET_10.1177_0300985818794161 for Survivin and Sox9: Potential Stem Cell Markers in Canine Normal, Hyperplastic, and Neoplastic Canine Prostate by Laura Bongiovanni, Francesca Caposano, Mariarita Romanucci, Valeria Grieco, Daniela Malatesta, Chiara Brachelente, Marcella Massimini, Cinzia Benazzi, Rachel E. Thomas, and Leonardo Della Salda in Veterinary Pathology

Questo articolo espone le maggiori novità in merito all’oncologia istopatologica mammaria veterinaria prontamente applicabili alla diagnostica dei tumori mammari nel cane e nel gatto, concentrandosi in particolare su quelle acquisizioni utili nella pratica clinica. Verranno descritti gli aggiornamenti riguardanti la classificazione istologica morfologica, i nuovi metodi proposti per il grading istologico dei carcinomi della cagna e della gatta e, infine, le ultime acquisizioni riguardanti l’applicazione del sistema di classificazione fenotipica su base molecolare, già convalidato per il cancro al seno.

Introduction: In human breast cancer, phosphatidylinositol-3 kinase (PI3K) mutation is involved in tumour growth and resistance to hormone therapy. The aim of the present study was to standardize immunohistochemistry (IHC) by anti-PI3K antibody in canine mammary samples, and to evaluate the different immunohistochemical expression in tumour progression. Materials and Methods: To assess the anti-PI3K antibody (clone D-9) specificity a western blot (WB) analysis was performed in the salivary gland and in normal canine mammary tissue. Samples from three normal mammary glands (NMGs), five benign (B) and 13 malignant (M) tumours were available for IHC. Immunohistochemical labelling was interpreted independently by two authors and the expression was evaluated by a semiquantitative method (50%) and according to a labelling intensity scale from 0 to 3. Results: WB analysis revealed two major bands of expected molecular weight (83 and 50 kDa) of PI3K isoforms. PI3K expression was >50% in all samples. Low (0 and 1) and high (2 and 3) labelling intensity was present respectively in 1 and 2 NMGs, 1 and 4 B and 2 and 11 M tumours. Conclusions: In this investigation, the anti-PI3K antibody (clone D-9) results were specific for canine mammary tissue. However, since immunohistochemical assessment of PI3K expression showed low variation in both extension and intensity from NMG to M tumours, IHC may not be the best technique to investigate the role of PI3K in tumour progression.

Introduction: Canine smooth muscle tumours (SMTs) usually arise in the gastrointestinal and urogenital tracts, but rarely in soft tissues. While the criteria used to identify smooth muscle differentiation are well established, the distinction between leiomyomas and leiomyosarcomas is based on mitotic activity, cellular atypia and amount of necrosis, but reliable cut-off values are lacking. Materials and Methods: Cases of canine SMTs were collected retrospectively and examined histologically. Differentiation, cellular atypia, necrosis, mitotic count (MC) and MIB1-based labelling index (LI) were assessed and analyzed statistically. Results: Sixty-eight SMTs were collected from 67 dogs (male:female ratio 0.5). Twenty-seven SMTs were uterine/vaginal, 18 gastrointestinal, 10 soft tissue, nine urinary and four splenic. Twenty-five SMTs were leiomyomas and 25 were leiomyosarcomas. Eighteen SMTs were not classifiable based on veterinary criteria and were diagnosed as SMTs with uncertain malignant potential according to human criteria. MC ranged between 0 and 59 (mean 13.6; median 4) and LI between 0 and 30.1 (mean 14.6; median 4.7). Diagnosis was associated with gender (P !0.01), with leiomyomas more frequent in females and leiomyosarcomas in males, and with LI (P !0.01), which was O5 in 72% of leiomyosarcomas and _5 in 76% of leiomyomas. Conclusions: Relying on veterinary criteria, the differential diagnosis between leiomyomas and leiomyosarcomas was not possible in 18 cases that, despite being well-differentiated, had a slightly higher MC than leiomyomas and some degree of cellular atypia and necrosis. We suggest using the LI with a cut-off of 5 to distinguish leiomyomas from leiomyosarcomas when histology alone is not sufficient.

The Epiphysitis is an inflammation of the growth plate, frequently detected in young beef at the level of the distal metaphyses in the long bones. Etiopathogenesis of the epiphysis inflamation in young beef bulls is still unclear. In literature, this pathological process is associated with rapid growing and high weight increase, physical activity and traumatic injuries, inadequate enviromental condictions, toxicosis, excess or deficiency in diet (alteration Ca/P ratio, deficiency in microelements, hypoparatyroidism), ereditary or genetic causes. This case report shows the radiological and pathological findings performed on 36 limbs of 18 young bulls fattening. The bulls had an average weight of 580 +/- 65 kg and the average age was 17 +/- 4 months. Fully slatted floor pens house on 10 bulls were used. On clinical examination the animals presented swelling of the metatarsophalangeal joint which was not painful or warm, no limb hypertermia, rapid weight loss, difficulty in keeping the standing position. Animals treated with antibiotic and/or antinflammatory medication were not included. X-Ray of the limbs were performed for all the animals, both for the clinically normal and lame, at the level of the distal metaphyses in the metatarsophalangeal joint. Pathological examinations were performed following the slaughter of the animals. Four different stages of inflammation in the distal epiphysis of the metatarsus were found in this report: Grade 0, no lesions in the growth plate; Grade 1, hyperemic areas localized on the growth plate and bone; Grade 2, multifocal lesions with thickening of the growth plate associated with the presence of whitish material of fibrous appearance; Grade 3, severe and diffuse thickening of the growth plate with bone tissue degeneration associated with the presence of whitish material of fibrous appearance. The breeding of fattening bulls on slatted floor may be a negative factor in the development of this pathology. The rapid gain weight and the hard slatted floor may induce traumatic or micro-traumatic joint lesions especially on the growth plate, causing chronic inflamation and bone tissue degeneration.

The onset of multidrug resistance can be related to PGP and BCRP expression, already described in canine neoplasia (2,3). Medical therapeutic approach for canine mast cell tumors (MCT) includes conventional chemotherapy and tyrosine kinase inhibitors (TKIs). TKIs are able to block disregulated KIT activity (1), whose immunohistochemical (IHC) pattern has been related to the biological behaviour of MCT (4). A case of a non-responsive to treatment MCT is described below and IHC expression of PGP, BCRP and KIT were examined, before and after treatment. A 7 years old, female spayed, mixed-breed, dog was referred for a cutaneous carpal mass and ipsilateral prescapular lymphadenomegaly. Lesions were removed and the final diagnosis was cutaneous MCT (Patnaik’s Grade 2, Kiupel’s low grade) with lymphnode metastases. The owner refused adjuvant chemotherapy and 6 months later, 3 new mast cell tumors in the surgical scar region were diagnosed. A Vinblastine + Prednisone protocol was initiated. After three doses, the disease progressed and tumors were excised: final histologic diagnosis was cutaneous MCT (Patnaik’s Grade 2, Kiupel’s high grade), infiltrating subcutaneous MCT and MCT lymphnode metastasis. Masitinib, a TKIs, was added to the chemotherapeutic protocol. After 2 months, the dog developed new MCTs and a rescue protocol with Lomustine was initiated. Due to the disease progression, the dog was euthanized 285 days after diagnosis. A retrospective IHC exam with PGP, BCRP, KIT was performed and evaluated with already described methods (4,5,6). Samples collected before chemotherapy did not show PGP (0%cell/HPF) and BCRP (10%cell/HPF); KIT staining was pattern III (diffuse cytoplasmic) in all the samples. The increase in PGP expression, seen in MCT after the first treatment, is likely to be related with the onset of PGP-mediated chemoresistance, being Vinblastine a substrate of PGP (5). These chemotherapeutic drugs, instead, are not BCRP substrates (7), however, the BCRP expression has been associated with biological aggressiveness (7), present in this case. Pattern III of KIT, seen in all metastases, is related to worse prognosis (4). The increased expression of PGP, BCRP and KIT in this case could be related to a chemoresistant and malignant neoplastic phenotype. 1) Hahn, Drug Update: Masitinib. In: Bonagura & Twedt, Elsevier, Kirk’s Current Veterinary Therapy XV, 360-362; 2014. 2) Ginn et al., Vet Path 33(5):533-41; 1996. 3) Nowak et al., In vivo 23:705-710; 2009. 4) Kiupel et al., Vet Path 41:371–377; 2004. 5) Petterino et al., Veterinaria 18(2):35-39; 2004. 6) Diestra et al., J Pathol 198:213–219; 2002. 7) Nakanishi et al., CJC 31(2),73-99; 2012

Questo studio riguarda 21 gatti riferiti all’Ospedale Didattico Veterinario del Dipartimento di Scienze Mediche Veterinarie dell’Università di Bologna dal 2000 al 2013 con anamnesi, segni neurologici, dati laboratoristici e di diagnostica per immagini riferibili complessivamente ad un coinvolgimento progressivo multifocale/diffuso del SNC e compatibili con la diagnosi di FIP. Quasi tutti i gatti (20/21) avevano un’età inferiore ai cinque anni; la maggior parte era di sesso femminile (13/21). Quattro casi (19%) provenivano da colonie feline. Dai soggetti è stato eseguito un campionamento multiorganico per l’istologia; da sezioni trasversali di SNC intracranico sono state registrate le lesioni elementari, che erano le seguenti: leptomeningite piogranulomatosa (71,4%), corioidite piogranulomatosa (66,7%), manicotti perivascolari (61,9%), iperemia dei vasi meningei (47,6%), necrosi fibrinoide della parete dei vasi (42,8%), ependimite piogranulomatosa (61,9%), periventricolite (33,3%), emorragie (33,3%), degenerazione neuronale e/o focolai malacici (33,3%), trombosi (14,3%), essudato intraventricolare (14,3%), iperemia dei plessi corioidei (4,7%), edema vasogenico (4,7%). In due casi il midollo spinale cervicale mostrava segni di sofferenza neuronale e leucomalacia, subependimite e leptomeningite piogranulomatosa. In tre casi erano presenti anche lesioni oculari (panoftalmite piogranulomatosa). I reperti istopatologici si sono confermati analoghi a quelli riportati in bibliografia; rimane aperta la discussione relativa alla possibilità che nella casistica le lesioni piogranulomatose meningee siano più gravi in alcuni distretti, quali il tronco cerebrale ed il telencefalo.

Canine prostatic carcinoma (PCa) is considered a relevant model for studying advanced, hormone refractory PCa in men. It has been proposed that cancer contains a minor population of cells that can selfrenew while simultaneously giving rise to tumour cells (cancer stem cells). Survivin is an acknowledged cancer therapy-resistance factor overexpressed in several tumour types, proposed as a valid cancer biomarker for human prostatic cancer for an early screening for malignancy. Sox9 is a stem cell marker expressed in several adult tissues, required for prostate development. Accumulating evidence indicates that it contributes to the development of human PCa. No studies have been published concerning the immunolocalization of survivin and Sox9 in canine prostatic hyperplasia (BPH) and neoplasia.AIM: to evaluate the patterns and levels of expression of survivin and Sox9 in canine BPH and PCa, in order to correlate their expression with malignant histological features. immunhistochemistry with specific antibodies in a set of canine BPH and PCa. Survivin nuclear and rare cytoplasmic immunostaining were present among the basal/reserve cell layer of normal and hyperplastic prostatic lobules. An increase of survivin expression was observed in PCas compared with BPHs. 6/11 PCas showed rare positive nuclei, mainly among the “basaloid” neoplastic cells in the areas with a tubular-papillary arrangement. In the areas with a solid pattern the cytoplasmic immunostaining was more diffuse. Sox9 expression was absent in normal prostatic glands and in all BPHs. 6/9 cases of PCa were highly positive. Based on the role of survivin as a stem cell marker and the main role in proliferation of nuclear survivin, the positive cells among basal cell layer in normal and HBP cases could represent transit amplifying cells maintaining some stem cell proprieties. The increased survivin expression in Pcas would indicates the molecule as a valid prognostic marker. The absence of expression of Sox9 in the normal gland and all the BPHs suggests that Sox9 is not a stem cell marker of canine adult prostatic stem cells. The high expression observed in PCas clearly suggests an important role of the molecule in canine prostatic carcinogenesis and malignant progression. Further studies should be done in order to confirm this hypothesis.

A 10-month-old domestic shorthair intact male cat was evaluated due to multiple skin lacerations located in the regions of the dorsum, thorax and head. On physical examination the skin was hyperextensible, fragile and pendulous on the ventral region of the abdomen. During the clinical examination, several cutaneous samples were taken using a skin punch. The obtained material was used for histological and electron microscopy investigations. The patient died spontaneously after 1 month and was submitted to necropsy, which revealed the rupture of the vena cava. The results of electron microscopy, together with the data of clinical, necropsy and histological examination confirmed the suspicion of cutaneous asthenia.

Preliminary pathological results on chondrodysplastic dwarfism in Tyrolean Grey cattle due to deletion in the EVC2 gene C Benazzi*, KE Dittmer†, KG Thompson†, C Drögemüller‡, A Gentile*, LV Muscatello*, L Murgiano‡, C Piffer#, M Bolcato* and B Brunetti* *Department of Veterinary Medical Sciences, University of Bologna, 40064 Ozzano dell’Emilia, Italy. Email: cinzia.benazzi@unibo.it †Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Private Bag 11222, Palmerston North 4442, New Zealand ‡Institute of Genetics, Vetsuisse Faculty, University of Bern, 3001 Bern, Switzerland. #Gesundheitbezirk Bozen, Laura Conti Strasse 4, 39100 Bozen, Italy. During July–November 2013 breeders reported the birth of Italian Tyrolean Grey calves with abnormally short limbs. Seven calves, aged 2–5 months, were referred to the Department of Veterinary Medical Sciences, Bologna, Italy. Whole genome resequencing of an affected calf detected a single candidate causal mutation in the Ellis van Creveld syndrome 2 (EVC2) gene. All animals had difficulties in assuming or maintaining a quadrupedal stance, with deterioration in this ability with increased growth. The limbs were disproportionately short and bulky, variably rotated and arched in a dumbbell-like position. At necropsy, the limbs (in particular the femur and humerus) were rotated and significantly shortened. Histologically the growth plates of long bones and vertebrae were irregular and prematurely closed. The reserve zone showed variable thickness at the expense of proliferative and hypertrophic zones, which were disorganised with multifocal loss of the normal columnar arrangement. In one calf reduced development of the heart valves was noted. In three female calves the genital tracts appeared fully mature, with numerous follicle-like structures on the ovaries. The uteri had multiple polypoid structures.

Introduction: The hernias of intervertebral discs are a common and debilitating disease in dogs usually hesitate in surgery. This study was aimed to score, based on a modified histological grading system, the samples taken in vivo, and associate these with related clinical data. Materials and Methods: Surgery herniated disc material was collected from 29 dogs. The samples were processed and stained by H&E, histochemistry using Gruber, and by immunohistochemistry using FVIII. For all samples, at least 3 months follow up was available. Results: Inflammation was added as a parameter in the grading system and scored 0 when not present and 1 when present. All cases with inflammatory cells showed simultaneously presence of fibrosis, mineralization and neoangeogenesis. The amount of proteoglycans of the extracellular matrix decreased proportionally with the increase of cells chondrocyte proliferation. Discussion and Conclusion: According to the present results, most of the samples were scored as grade 3 in the modified grading system going from grade 1 to 5. This data can be partially explained by the variability of histological material, this being taken in vivo and in most cases consists only of the nucleus pulposus. Separating and applying the scoring for the annulus fibrosus and for the pulpy nucleus, based on the collected material, may give more precise scores.