H2AFZ:A Novel Prognostic Marker in Canine Melanoma and a Predictive Marker for Resistance to CDK4/6 Inhibitor Treatment

Uncontrolled proliferation is a key feature of tumor progression and malignancy. This suggests that cell-cycle related factors could be exploited as cancer biomarkers and that pathways specifically involved in the cell cycle, such as the Rb-E2F pathway, could be targeted as an effective anti-tumor therapy. We investigated 34 formalin-fixed paraffin-embedded (FFPE) tissue samples of canine cutaneous melanocytoma, cutaneous melanoma, and oral melanoma. Corresponding clinical follow-up data were used to determine the prognostic value of the mRNA expression levels of several cell cycle regulated E2F target genes (E2F1, DHFR, CDC6, ATAD2, MCM2, H2AFZ, GINS2, and survivin/BIRC5). Moreover, using four canine melanoma cell lines, we explored the possibility of blocking the Rb-E2F pathway by using a CDK4/6 inhibitor (Palbociclib) as a potential anti-cancer therapy. We investigated the expression levels of the same E2F target gene transcripts before and after treatment to determine the potential utility of these molecules as predictive markers. The E2F target gene H2AFZ was expressed in 91.43% of the primary tumors and H2AFZ expression was significantly higher in cases with unfavorable clinical outcome. Among the other tested genes, survivin/BIRC5 showed as well-promising results as a prognostic marker in canine melanoma. Three of the four tested melanoma cell lines were sensitive to the CDK4/6 inhibitor. The resistant cell line displayed higher expression levels of H2AFZ before treatment compared to the CDK4/6 inhibitor-sensitive cell lines. The present results suggest that CDK4/6 inhibitors could potentially be used as a new anti-cancer treatment for canine melanoma and that H2AFZ could serve as a prognostic and predictive marker for patient selection.</p

Transcriptome Analysis of Canine Cutaneous Melanoma and Melanocytoma Reveals a Modulation of Genes Regulating Extracellular Matrix Metabolism and Cell Cycle

Interactions between tumor cells and tumor microenvironment are considered critical in carcinogenesis, tumor invasion and metastasis. To examine transcriptome changes and to explore the relationship with tumor microenvironment in canine cutaneous melanocytoma and melanoma, we extracted RNA from formalin-fixed, paraffin-embedded (FFPE) specimens and analyzed them by means of RNA-seq for transcriptional analysis. Melanocytoma and melanoma samples were compared to detect differential gene expressions and significant enriched pathways were explored to reveal functional relations between differentially expressed genes. The study demonstrated a differential expression of 60 genes in melanomas compared to melanocytomas. The differentially expressed genes cluster in the extracellular matrix-receptor interaction, protein digestion and absorption, focal adhesion and PI3K-Akt (phosphoinositide 3-kinase/protein kinase B) signaling pathways. Genes encoding for several collagen proteins were more commonly differentially expressed. Results of the RNA-seq were validated by qRT-PCR and protein expression of some target molecules was investigated by means of immunohistochemistry. We hypothesize that the developing melanoma actively promotes collagen metabolism and extracellular matrix remodeling as well as enhancing cell proliferation and survival contributing to disease progression and metastasis. In this study, we also detected unidentified genes in human melanoma expression studies and uncover new candidate drug targets for further testing in canine melanoma

Detection of Mycoplasma columbinasale in Cases of Respiratory Disease in Domestic Pigeons (Columba livia var. domestica)

In 2017, respiratory disease and low mortality were reported in domestic flying pigeons (Columba livia var. domestica) trained as hunting live bait in a breeding farm in Umbria, Italy. Clinically, open beak breathing, dropped wings, and pharyngeal and laryngeal hyperaemia were observed. Three birds were submitted for necropsy. Gross pathological evaluation revealed in all cases diffuses hyperaemia of the tracheal mucosa in association with mild emaciation and multiorgan congestion. Microscopically, diffuse epithelial hyperplasia of the trachea (n = 3) and diffuse lymphocytic infiltration of the lamina propria (n = 3) were observed. No lesions were reported in other organs. Based on reported clinical signs and lesions, Mycoplasma spp. were suspected, and molecular detection was performed on tracheal specimens leading to the identification of Mycoplasma columbinasale. Immunohistochemistry was subsequently performed to localize the microorganism within tissue lesions. Immunohistochemistry confirmed the presence of Mycoplasma species on the tracheal epithelial cells of all birds. Following tylosin administration, complete resolution of the clinical condition and lack of recurrence of clinical signs were reported in the breeding farm. These findings suggest that M. columbinasale could potentially have a role in the respiratory disease and low mortality in domestic pigeons

Occupational health and safety risk management related to chemicals use. The Xylem Lowara case.

reservedIl rischio chimico, normato dal D.lgs 81/08, è uno dei rischi più diffusi all’interno dei luoghi di lavoro. In particolare, ad esso sono legati diversi pericoli per la salute e per l’ambiente, incendi o esplosioni. Una corretta gestione di esso comporta la buona riuscita di un ambiente sicuro che non risulti dannoso per i lavoratori e per l’ambiente. La tesi verte sulla gestione delle sostanze chimiche nell’area produttiva MM1 all’interno dell’azienda Xylem Lowara situata a Montecchio Maggiore in provincia di Vicenza. Nello specifico, l'obiettivo di questa tesi è focalizzato sullo stoccaggio e contenimento delle sostanze pericolose e la trattazione di esse nel caso di sversamento accidentale attraverso appositi kit. Complessivamente il progetto è stato attuato attraverso la collaborazione con i capi reparto, il personale dell’azienda e in particolare Responsabile Servizio Prevenzione e Protezione (RSPP) aziendale e Responsabile Servizio Prevenzione e Protezione della parte produttiva. La tematica è stata affrontata inizialmente tramite l’accurata revisione delle schede di sicurezza delle sostanze chimiche presenti all’ interno dell’azienda. In questa fase, sono state applicate delle migliorie nel database per renderne più fruibile la ricerca. Allo stesso tempo, è avvenuto un aggiornamento delle schede non più coerenti con la policy aziendale. Successivamente, in base alla necessità dei vari reparti, si è disposto un piano di sostituzione dei vari bacini di contenimento sottodimensionati basato sul D.Lgs. 2000 n. 258. Infine, negli ambienti adibiti allo stoccaggio di sostanze liquide pericolose si sono posizionati dei kit di pronto intervento ambientale utilizzabili dal personale addestrato. L’ applicazione di questa metodologia ha permesso di ottenere risultati rilevanti tramite il raggiungimento di misure di sicurezza che hanno portato, ove possibile, a processi lavorativi più sicuri all’ interno dell’ambiente di lavoro.Chemical risk, regulated by the Legislative Decree 81/08, is one of the most prevalent risks in the workplace. In particular, several health and environmental risks, fires or explosions are linked to it. A proper management of it involves achieving a safe and non-harmful environment for workers and the environment. This thesis focuses on the management of chemicals in the MM1 production area of the company Xylem Lowara, located in Montecchio Maggiore in the province of Vicenza. Specifically, the focus of this thesis is on the storage and containment of dangerous substances and their management through special kits in case of accidental spills. Overall, the project was carried out thanks to the collaboration with the department managers, the staff of the company and in particular with the company Prevention and Protection Service Manager (RSPP) and the Prevention and Protection Service Manager of the production part. The issue has been addressed initially through a thorough review of the safety data sheets of the chemicals present in the company. At this stage, improvements to the database have been implemented to make it more usable for research. At the same time, an update was made to the sheets what were no longer consistent with the company policy. Subsequently, based on the needs of the various departments, a replacement plan has been prepared for the various undersized containment basins according to Legislative Decree No. 258 of 2000. Finally, emergency environmental intervention kits, to be used by trained personnel, have been placed in the rooms used for the storage of hazardous liquid substances. The application of this methodology has led to relevant results through the achievement of safety measures that have led, where possible, to safer work processes within the work environment

MiRNAs in Canine and Human Osteosarcoma: A Highlight Review on Comparative Biomolecular Aspects

Osteosarcoma (OS) is the most frequent primary malignant tumor of bone in humans and animals. Comparative oncology is a field of study that examines the cancer risk and tumor progression across the species. The canine model is ideally suited for translational cancer research. The biological and clinical characteristics of human and canine OS are common to hypothesize as that several living and environmental common conditions shared between the two species can influence some etiopathogenetic mechanisms, for which the canine species represents an important model of comparison with the human species. In the canine and human species, osteosarcoma is the tumor of bone with the highest frequency, with a value of about 80–85% (in respect to all other bone tumors), a high degree of invasiveness, and a high rate of metastasis and malignancy. Humans and dogs have many genetic and biomolecular similarities such as alterations in the expression of p53 and in some types of microRNAs that our working group has already described previously in several separate works. In this paper, we report and collect new comparative biomolecular features of osteosarcoma in dogs and humans, which may represent an innovative update on the biomolecular profile of this tumor

The contribution of stem cells to epidermal and hair follicle tumours in the dog

Background -  Although cutaneous stem cells have been implicated in skin tumourigenesis in humans, no studies have been conducted to elucidate the presence and the possible role of stem cells in hair follicle tumours in the dog. Hypothesis -  Stem cell markers are expressed in canine epidermal and follicular tumours and can be used to better understand the biology and origin of these tumours. Animals and Methods -  In the present study, normal skin sections and 44 follicular tumours were retrospectively investigated for the immunohistochemical expression of keratin15 (K15) and nestin. In addition, 30 squamous cell carcinomas were evaluated for K15 expression. Results -  In normal skin, K15 and nestin were expressed in the outer root sheath cells of the isthmic portion of the hair follicle (bulge region), and K15 expression was also scattered in the basal cell layer of the epidermis. Infundibular keratinizing acanthomas, pilomatricomas and squamous cell carcinomas were mostly negative for K15, trichoblastomas were moderately to strongly positive, tricholemmomas were either negative or strongly positive, and trichoepitheliomas had heterogeneous staining. Nestin expression was generally faint in all follicular tumours. Conclusions and clinical importance -  Our results show that K15 can be a reliable marker for investigating the role of stem cells in hair follicle tumours of the dog, while nestin was judged to be a nonoptimal marker. Furthermore, our study suggests that hair follicle stem cells are present in the bulge region of hair follicles and could possibly play a role in tumourigenesis of canine tumours originating from this portion of the follicle, namely trichoblastomas, tricholemmomas and trichoepitheliomas. The loss of K15 expression in squamous cell carcinomas compared with normal skin suggests that this event could be important in the malignant transformation

The contribution of stem cells to epidermal and hair follicle tumours in the dog

Background -  Although cutaneous stem cells have been implicated in skin tumourigenesis in humans, no studies have been conducted to elucidate the presence and the possible role of stem cells in hair follicle tumours in the dog. Hypothesis -  Stem cell markers are expressed in canine epidermal and follicular tumours and can be used to better understand the biology and origin of these tumours. Animals and Methods -  In the present study, normal skin sections and 44 follicular tumours were retrospectively investigated for the immunohistochemical expression of keratin15 (K15) and nestin. In addition, 30 squamous cell carcinomas were evaluated for K15 expression. Results -  In normal skin, K15 and nestin were expressed in the outer root sheath cells of the isthmic portion of the hair follicle (bulge region), and K15 expression was also scattered in the basal cell layer of the epidermis. Infundibular keratinizing acanthomas, pilomatricomas and squamous cell carcinomas were mostly negative for K15, trichoblastomas were moderately to strongly positive, tricholemmomas were either negative or strongly positive, and trichoepitheliomas had heterogeneous staining. Nestin expression was generally faint in all follicular tumours. Conclusions and clinical importance -  Our results show that K15 can be a reliable marker for investigating the role of stem cells in hair follicle tumours of the dog, while nestin was judged to be a nonoptimal marker. Furthermore, our study suggests that hair follicle stem cells are present in the bulge region of hair follicles and could possibly play a role in tumourigenesis of canine tumours originating from this portion of the follicle, namely trichoblastomas, tricholemmomas and trichoepitheliomas. The loss of K15 expression in squamous cell carcinomas compared with normal skin suggests that this event could be important in the malignant transformation

Tumor Thickness and Modified Clark Level in Canine Cutaneous Melanocytic Tumors

Breslow thickness and Clark level are prognostic factors for human cutaneous melanomas. Breslow thickness is measured with an ocular micrometer from the top of the granular layer of the epidermis to the deepest invasive cell across the broad base of the tumor, while Clark level is based on the anatomical level of invasion through the layers of the dermis. Because of the anatomical differences between humans and dogs, we evaluated the tumor thickness and a modified Clark level in 77 canine primary cutaneous melanocytic tumors. Tumor thickness (using both a traditional and a more convenient system) and modified Clark level were measured and associated with histological diagnosis and clinical outcome. Tumor thickness was a prognostic factor, being greater in animals with shorter overall survival and disease-free time. Cutoffs of 0.95 cm and 0.75 cm defined a higher hazard for an unfavorable outcome and to develop recurrence/metastasis, respectively. Because of an excellent agreement between the 2 methods, it was concluded that tumor thickness could be measured with a ruler when an ocular micrometer is not available. Modified Clark level was not found to be relevant for prognosis. However, we suggest that both tumor thickness and a modified Clark level can be valid additional parameters when histological diagnosis is uncertain. Further studies, including a wider sample population, would be worthwhile to confirm the prognostic significance of these 2 parameters

H2AFZ: A Novel Prognostic Marker in Canine Melanoma and a Predictive Marker for Resistance to CDK4/6 Inhibitor Treatment

Uncontrolled proliferation is a key feature of tumor progression and malignancy. This suggests that cell-cycle related factors could be exploited as cancer biomarkers and that pathways specifically involved in the cell cycle, such as the Rb-E2F pathway, could be targeted as an effective anti-tumor therapy. We investigated 34 formalin-fixed paraffin-embedded (FFPE) tissue samples of canine cutaneous melanocytoma, cutaneous melanoma, and oral melanoma. Corresponding clinical follow-up data were used to determine the prognostic value of the mRNA expression levels of several cell cycle regulated E2F target genes (E2F1, DHFR, CDC6, ATAD2, MCM2, H2AFZ, GINS2, and survivin/BIRC5). Moreover, using four canine melanoma cell lines, we explored the possibility of blocking the Rb-E2F pathway by using a CDK4/6 inhibitor (Palbociclib) as a potential anti-cancer therapy. We investigated the expression levels of the same E2F target gene transcripts before and after treatment to determine the potential utility of these molecules as predictive markers. The E2F target gene H2AFZ was expressed in 91.43% of the primary tumors and H2AFZ expression was significantly higher in cases with unfavorable clinical outcome. Among the other tested genes, survivin/BIRC5 showed as well-promising results as a prognostic marker in canine melanoma. Three of the four tested melanoma cell lines were sensitive to the CDK4/6 inhibitor. The resistant cell line displayed higher expression levels of H2AFZ before treatment compared to the CDK4/6 inhibitor-sensitive cell lines. The present results suggest that CDK4/6 inhibitors could potentially be used as a new anti-cancer treatment for canine melanoma and that H2AFZ could serve as a prognostic and predictive marker for patient selection