SOX-10 and TRP-1 expression in feline ocular and nonocular melanomas.

In felines, ocular and nonocular melanomas are uncommon tumors that represent a diagnostic challenge for pathologists, especially when amelanotic. To date, the immunohistochemical diagnostic panel in cats is based on specific melanocytic markers (Melan-A and PNL2) and a nonspecific but sensitive marker (S100). In human medicine, SOX-10 is reported to be a sensitive antibody for the detection of melanoma micrometastasis in the lymph node. TRP-1, an enzyme involved in melanogenesis, has recently been used in humans and dogs as a specific melanocyte marker. The aim of this study was to evaluate the cross-reactivity and the expression of SOX-10 and TRP-1 antibodies in feline normal tissue and melanocytic tumors. Thirty-one cases of ocular, cutaneous, and oral melanomas were retrospectively evaluated and confirmed by histopathological examination and by immunolabeling with Melan-A and/or PNL2. SOX-10 nuclear expression in normal tissues was localized in epidermal, subepidermal, hair bulb, and iridal stromal melanocytes and dermal nerves. In melanomas, nuclear expression of SOX-10 was detected in ocular (11/12; 92%), oral (6/7; 86%), and cutaneous sites (12/12; 100%). TRP-1 cytoplasmic immunolabeling in normal tissue was observed in epidermal and bulbar melanocytes and in the lining pigmented epithelium of the iris and in its stroma. Its expression was positively correlated to the degree of pigmentation in the tumor and was observed in 75% of ocular (9/12), 43% of oral (3/7), and 33% of cutaneous melanomas (4/12). This study demonstrated the cross-reactivity of SOX-10 and TRP-1 antibodies in feline non-neoplastic melanocytes and their expression in ocular and nonocular melanomas.

Vulvo-vaginal epithelial tumors in mares: A preliminary investigation on epithelial-mesenchymal transition and tumor-immune microenvironment.

Vulvo-vaginal epithelial tumors are uncommon in mares, and data on the epithelial-to-mesenchymal transition (EMT) and the tumor-immune microenvironment (TIME) are still lacking. This is a study investigating the equus caballus papillomavirus type 2 (EcPV2) infection state as well as the EMT process and the tumor microenvironment in vulvo-vaginal preneoplastic/ benign (8/22) or malignant (14/22) epithelial lesions in mares. To do this, histopathological, immunohistochemical, transcriptomic, in situ hybridization, and correlation analyses were carried out. Immunohistochemistry quantification showed that cytoplasmic E-cadherin and ß-catenin expression as well as nuclear ß-catenin expression were features of malignant lesions, while benign/preneoplastic lesions were mainly characterized by membranous E-cadherin and ß-catenin expression. Despite this, there were no differences between benign and malignant equine vulvo-vaginal lesions in the expression of downstream genes involved in the canonical and noncanonical wnt/ß-catenin pathways. In addition, malignant lesions were characterized by a lower number of cells with cytoplasmic cytokeratin expression as well as a slightly higher cytoplasmic vimentin immunolabeling. The TIME of malignant lesions was characterized by more numerous CD204+ M2-polarized macrophages. Altogether, our results support the hypothesis that some actors in TIME such as CD204+ M2-polarized macrophages may favor the EMT process in equine vulvo-vaginal malignant lesions providing new insights for future investigations in the field of equine EcPV2-induced genital neoplastic lesions.

Canine melanocytes: Immunohistochemical expression of melanocytic markers in different somatic areas.

BACKGROUND: Melanoblasts originate in the neural crest from where they migrate to peripheral tissues and differentiate into melanocytes. Alteration during melanocyte development and life can cause different diseases, ranging from pigmentary disorders and decreased visual and auditory functions, to tumours such as melanoma. Location and phenotypical features of melanocytes have been characterised in different species, yet data on dogs are lacking. OBJECTIVE: This study investigates the expression of melanocytic markers Melan A, PNL2, TRP1, TRP2, SOX-10 and MITF in melanocytes of selected cutaneous and mucosal surfaces of dogs. ANIMALS: At necropsy, samples from five dogs were harvested from oral mucosa, mucocutaneous junction, eyelid, nose and haired skin (abdomen, back, pinna, head). MATERIALS AND METHODS: Immunohistochemical and immunofluorescence analyses were performed to assess marker expression. RESULTS: Results showed variable expression of melanocytic markers in different anatomical sites, particularly within epidermis of haired skin and dermal melanocytes. Melan A and SOX-10 were the most specific and sensitive melanocytic markers. PNL2 was less sensitive, while TRP1 and TRP2 were seldomly expressed by intraepidermal melanocytes in haired skin. MITF had a good sensitivity, yet the expression often was weak. CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate a variable expression of melanocytic markers in different sites, suggesting the presence of subpopulations of melanocytes. These preliminary results pave the way to understanding the pathogenetic mechanisms involved in degenerative melanocytic disorders and melanoma. Furthermore, the possible different expression of melanocyte markers in different anatomical sites could influence their sensitivity and specificity when used for diagnostic purposes.

FoxP3, CTLA-4, and IDO in Canine Melanocytic Tumors.

Despite promising immunotherapy strategies in human melanoma, there are few studies on the immune environment of canine melanocytic tumors. In humans, the activation of immunosuppressive cell subpopulations, such as regulatory T cells (Tregs) that express forkhead box protein P3 (FoxP3), the engagement of immunosuppressive surface receptors like cytotoxic T lymphocyte antigen (CTLA-4), and the secretion of molecules inhibiting lymphocyte activation, such as indoleamine-pyrrole 2,3-dioxygenase (IDO), are recognized as immunoescape mechanisms that allow tumor growth and progression. The aim of our study was to investigate the expression of these immunosuppression markers in canine melanocytic tumors and to postulate their possible role in melanoma biology and progression. Fifty-five formalin-fixed, paraffin-embedded canine melanocytic tumors (25 oral melanomas; 20 cutaneous melanomas; 10 cutaneous melanocytomas) were selected to investigate the expression of FoxP3, CTLA-4, and IDO by immunohistochemistry and RT-qPCR (real-time quantitative polymerase chain reaction). All of the tested markers showed high gene and protein expression in oral melanomas and were differently expressed in cutaneous melanomas when compared to their benign counterpart. IDO expression was associated with an increased hazard of death both in univariable and multivariable analyses (P < .05). FoxP3 protein expression >6.9 cells/HPF (high-power field) was an independent predictor of death (P < .05). CTLA-4 gene and protein expressions were associated with a worse prognosis, but only in the univariable analysis (P < .05). FoxP3, CTLA-4, and IDO likely play a role in canine melanoma immunoescape. Their expression, if supported by future studies, could represent a prognostic tool in canine melanoma and pave the way to future immunotherapeutic approaches in dogs.

Investigation of the Epithelial to Mesenchymal Transition (EMT) Process in Equine Papillomavirus-2 (EcPV-2)-Positive Penile Squamous Cell Carcinomas.

Equine penile squamous cell carcinoma (epSCC) is the most frequent tumor of the external male genitalia, representing 67.5% of equine genital cancers. epSCC is associated with papilloma virus (PV) infection and has been recently proposed as a model for human PV-induced squamous cell carcinomas. It has already been suggested that epSCC might undergo epithelial-to-mesenchymal transition (EMT). This work aims to investigate in detail this process and the possible role of PV oncoproteins in epSCC. For this purpose, 18 penile SCCs were retrospectively selected and tested for both EcPV2 presence and oncoproteins (EcPV2 E6 and EcPV2 E7) expression. Moreover, immunohistochemical EMT characterization was carried out by analyzing the main epithelial markers (E-cadherin, ß-catenin, and pan-cytokeratin AE3/AE1), the main mesenchymal markers (N-cadherin and vimentin), and the main EMT-related transcription factors (TWIST-1, ZEB-1). PCR analysis was positive for EcPV2 in 16 out of 18 samples. EMT was investigated in epSCC positive for EcPV2. The immunohistochemistry results suggested the presence of EMT processes in the neoplastic cells at the tumor invasive front. Moreover, the significant upregulation of RANKL, together with BCATN1, LEF1, and FOSL1 genes, might suggest a canonical Wnt pathway activation, similarly to what is reported in human penile squamous cell carcinomas.

E-Cadherin Expression in Canine Melanocytic Tumors: Histological, Immunohistochemical, and Survival Analysis.

E-cadherin, a glycoprotein involved in cell-cell adhesion, has a pivotal role in epithelial-mesenchymal transition, a process through which neoplastic epithelial cells develop an invasive phenotype. In human cutaneous melanomas, decreased E-cadherin expression is associated with shorter survival and increased Breslow thickness, whereas in the dog its role is poorly understood. Tumor thickness and modified Clark level were recently proposed as useful features to assess canine melanocytic tumors, but no studies investigated their association with E-cadherin expression. We performed immunohistochemistry on 77 formalin-fixed, paraffin-embedded primary canine melanocytic tumors. A 3-tier and a 2-tier classification system for assessing E-cadherin expression were tested, with the latter being more informative for the assessment of canine melanocytic tumors. E-cadherin expression was lower in cutaneous melanomas than melanocytomas, as well as in amelanotic tumors compared to pigmented tumors. In amelanotic melanomas, absent E-cadherin expression was associated with an unfavorable outcome, suggesting a potential use of this marker in defining the prognosis of amelanotic melanomas. E-cadherin expression was lower in tumors with greater tumor thickness and modified Clark level ≥IV, suggesting its possible utility in identifying the most invasive tumors. The expression of E-cadherin in oral melanomas was heterogeneous, but was associated with pigmentation and clinical outcome; thus, E-cadherin evaluation could be advantageous to detect the most aggressive neoplasms. However, cutaneous melanomas without E-cadherin expression frequently had a favorable clinical outcome. Hence, its importance as prognostic factor should be carefully considered depending on the tumor origin.

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.

FoxP3 and IDO in Canine Melanocytic Tumors.

Human melanoma is one of the deadliest forms of cancer, with poor prognosis and high resistance to chemotherapy and radiotherapy. The discovery of immunosuppressive mechanisms in the human melanoma microenvironment led to the use of new prognostic markers and to the development of immunotherapies targeting immune checkpoint molecules. Immunoescape mechanisms in canine melanoma have not yet been investigated, and no such immunotherapy has been tested. The aim of this study was to provide preliminary data on the expression of transcription factor forkhead box protein P3 (FoxP3) and indoleamine 2,3-dioxygenase (IDO) in primary canine melanocytic tumors and to investigate their prognostic role. Formalin-fixed, paraffin-embedded samples from 74 canine melanocytic tumors (26 oral melanomas, 23 cutaneous melanomas, and 25 cutaneous melanocytomas) were retrospectively evaluated by immunohistochemistry to explore the expression of FoxP3 and IDO. An increased risk of death due to melanoma was associated with a higher number of FoxP3+ cells per high-power field (FoxP3+/HPF), a higher percentage of CD3+ cells that were also FoxP3+ infiltrating and surrounding the tumor (%FoxP3), and a higher number of IDO+ cells/HPF (IDO+/HPF). A prognostic value for FoxP3 and IDO is suggested by our study, with optimal cutoffs of 14.7 FoxP3+ cells/HPF, 6.1 IDO+ cells/HPF, and 12.5% FoxP3+ cells. Both markers were also associated with tumor type. Multivariable analysis identified IDO+/HPF ( P < .001) as an independent prognostic marker. Even though stratification by diagnosis caused a loss of significance, results from the present study suggest a prognostic role for IDO and FoxP3, possibly related to the establishment of an immunosuppressive microenvironment.

Feline herpesvirus ulcerative dermatitis: an atypical case?

BACKGROUND: Feline herpesvirus ulcerative dermatitis is an uncommon skin disease in cats, with a predominantly facial distribution characterized by massive infiltration of eosinophils and, occasionally, predominant neutrophils. OBJECTIVE: To describe the clinical and histopathological features of a putative atypical case of feline herpesvirus dermatitis. ANIMAL: A 10-month-old, intact male, European cat was presented with chronic monolateral ulcerative dermatitis with adherent crusts on the left pinna. The lesion had been present for six months and worsened after the administration of corticosteroids. METHODS: Clinical and histopathological examination, immunohistochemistry, nested PCR and transmission electron microscopy (TEM). RESULTS: Histological examination of skin biopsies showed multifocal ulcerative and necrotic lesions, involving the superficial and deep dermis covered by thick haemorrhagic and serocellular crusts. The superficial, medium and deep dermis was heavily infiltrated with mast cells and plasma cells, with a lower number of neutrophils and eosinophils. In the nuclei of some cells in the deep dermis, whose histotype was unrecognizable with routine haematoxylin and eosin stain, intranuclear eosinophilic inclusion bodies were noticed. Nested PCR and TEM supported the hypothesis of FeHV-1-induced dermatitis. CONCLUSIONS AND CLINICAL IMPORTANCE: This case is noteworthy for the infrequent location on the pinna and the atypical histopathological features of the lesion, with a predominant infiltration of mast cells and plasma cells. Our findings suggest that herpesvirus dermatitis should be listed as a differential diagnosis in case of ulcerative dermatitis when the location and histological features are atypical.

Canine cutaneous melanocytic tumours: significance of ß-catenin and survivin immunohistochemical expression.

BACKGROUND: Recent investigations have highlighted the controversial role of Wnt/ß-catenin pathway activation in human cutaneous melanoma. Survivin has been proposed as a valid prognostic marker for invasive and metastatic melanomas and lymph node melanoma metastasis in human cutaneous melanoma and is a promising therapeutic target. HYPOTHESIS/OBJECTIVES: Our aim was to investigate the immunohistochemical expression of survivin and ß-catenin in canine cutaneous melanocytic tumours, in order to understand their prognostic significance. METHODS: Twenty-one melanocytic tumours (10 melanocytomas and 11 melanomas) were investigated by immunohistochemistry using specific anti-survivin and anti-ß-catenin antibodies. A semi-quantitative method was used to analyse the results; ß-catenin immunolabelling in neoplastic cells was evaluated as cytoplasmic, membranous or nuclear. The number of survivin-positive cells was counted within ~1000 neoplastic cells. Results were related to histopathological features, evaluated in haematoxylin- and eosin-stained slides, and to the clinical data obtained through a telephone survey with referring veterinarians. RESULTS: Despite a low level of expression in the majority of cases, ß-catenin was found to be correlated strongly with malignant behaviour (P < 0.01). An overexpression of nuclear survivin was statistically related to histological features of malignancy, presence of metastasis and death related to melanoma spread (P < 0.01). CONCLUSIONS AND CLINICAL IMPORTANCE: The low nuclear ß-catenin expression, mainly found in metastatic cases, would indicate that ß-catenin activation may have only limited importance in the development or progression of canine cutaneous melanoma. The correlation of nuclear survivin expression with malignancy would indicate that survivin is possibly a useful prognostic marker and therapeutic target in canine melanoma patients.

Feline eosinophilic dermatoses: a retrospective immunohistochemical and ultrastructural study of extracellular matrix remodelling.

BACKGROUND: Feline eosinophilic dermatoses (FEDs) are common diseases of cats with an unknown pathogenesis. They are histologically characterized by an eosinophilic infiltration and often by the presence of flame figures (FFs) and/or areas of loss of tissue architecture, here termed necrotic foci (NF). It has been postulated that an alteration in the degradation of the extracellular matrix could be responsible for these histological features. Matrix metalloproteinases (MMPs) are a group of proteases that are fundamental in extracellular matrix remodelling. HYPOTHESIS/OBJECTIVES: The aim of the study was to investigate retrospectively the expression of a subgroup of MMPs, in particular MMP-2 and MMP-9 gelatinases, in FEDs. The expression of one of their inhibitors, TIMP-2, was also investigated in order to establish the role of these molecules in the pathogenesis of FEDs. The ultrastructural characteristics of extracellular matrix in FFs and NF were subsequently assessed. METHODS: Fifty-one formalin-fixed, paraffin-embedded specimens from cutaneous and mucosal biopsies diagnosed as FEDs were investigated immunohistochemically. Two selected samples were processed for electron microscopy. RESULTS: This study revealed an increased expression of MMP-2 in NF and a decreased expression of this gelatinase in FFs. An imbalance between MMP-2 and TIMP-2 was evident using immunohistochemistry. No significative results were observed for MMP-9 expression. Electron microscopy confirmed the lack of normal collagen fibres in NF, whereas in FFs only occasional, amorphous material was observed among normal collagen fibres. CONCLUSIONS AND CLINICAL IMPORTANCE: Our study suggests that an imbalance in the expression of matrix metalloproteinases could be responsible for different morphological findings in FEDs. Further studies are needed to assess the role of matrix metalloproteinases in the pathogenesis of FEDs.

Establishment of Primary Cell Cultures from Canine Oral Melanomas via Fine-Needle Aspiration: A Novel Tool for Tumorigenesis and Cancer Progression Studies.

Oral melanomas are the most common oral malignancies in dogs and are characterized by an aggressive nature, invasiveness, and poor prognosis. With biological and genetic similarities to human oral melanomas, they serve as a valuable spontaneous comparative model. Primary cell cultures are widely used in human medicine and, more recently, in veterinary medicine to study tumorigenesis, cancer progression, and innovative therapeutic approaches. This study aims to establish two- and three-dimensional primary cell lines from oral canine melanomas using fine-needle aspiration as a minimally invasive sampling method. For this study, samples were collected from six dogs, represented by four primary oral melanomas and five lymph nodal metastases. The cells were digested to obtain single-cell suspensions, seeded in flasks, or processed with Matrigel® to form organoids. The cell cultures were characterized through flow cytometry using antibodies against Melan-A, PNL2, and Sox-10. This technique offers a minimally invasive means to obtain cell samples, particularly beneficial for patients that are ineligible for surgical procedures, and enables the establishment of in vitro models crucial for comparative studies in mucosal melanoma oncology. To the best of our knowledge, this is the first work establishing neoplastic primary cell cultures via fine-needle aspiration in dogs.

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 keratin 15 (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.

Epithelial-to-mesenchymal transition: immunohistochemical investigation of related molecules in canine cutaneous epithelial tumours.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is a multistep process, important in tumour invasion and metastasis, characterized by loss of epithelial markers, redistribution of ß-catenin and gain of mesenchymal markers. HYPOSTHESIS/OBJECTIVES: Our aim was to investigate the immunohistochemical aberrant expression of cytokeratin, vimentin, survivin and heat shock protein 72 (Hsp72) in canine cutaneous epithelial tumours, to understand the association of expression of these molecules with features of malignancy and their role in the EMT phenotype. METHODS: Ten canine squamous cell carcinomas (SCCs; one with lymph node metastasis), 30 canine hair follicle tumours (six pilomatricomas, eight infundibular keratinizing acanthomas, six trichoepitheliomas and 10 trichoblastomas) and five normal skin samples were investigated by immunohistochemistry using specific anti-vimentin, -cytokeratin, -survivin and -Hsp72 antibodies. A semi-quantitative method was used to analyse the results, as follows: 0 to <5%; ≥ 5 to <10%; ≥ 10 to <25%; and ≥ 25% of positive cells. Immunofluorescence was performed to investigate survivin-vimentin and survivin-Hsp72 colocalization in selected SCCs. Results - In malignant hair follicle tumours and SCCs, a reduced intensity of cytokeratin and increased survivin and Hsp72 expression were observed. In SCCs, loss of cytokeratin expression and vimentin immunolabelling, suggestive of the EMT phenotype, were evident in <5% of neoplastic cells in the front of tumour invasion. In the same areas, strong nuclear survivin and cytoplasmic Hsp72 staining was evident, often colocalizing. Only a few neoplastic cells in the front of tumour invasion showed vimentin-survivin colocalization. CONCLUSIONS AND CLINICAL IMPORTANCE: A possible simultaneous involvement of survivin and Hsp72 in tumour invasion and the multistep process of EMT of cutaneous epithelial tumours of dogs is suggested.

Genetic Characterization of a Novel Equus caballus Papillomavirus Isolated from a Thoroughbred Mare.

Papillomaviruses (PVs) are small, non-enveloped viruses, ubiquitous across the animal kingdom. PVs induce diverse forms of infection, such as cutaneous papillomas, genital papillomatosis, and carcinomas. During a survey on the fertility status of a mare, a novel Equus caballus PV (EcPV) has been identified using Next Generation Sequencing, and it was further confirmed with genome-walking PCR and Sanger sequencing. The complete circular genome 7607 bp long shares 67% average percentage of identity with EcPV9, EcPV2, EcPV1, and EcPV6, justifying a new classification as Equus caballus PV 10 (EcPV10). All EcPV genes are conserved in EcPV10, and phylogenetic analysis indicates that EcPV10 is closely related to EcPV9 and EcPV2, genus Dyoiota 1. A preliminary EcPV10 genoprevalence study, carried out on 216 horses using Real Time PCRs, suggested a low incidence of this isolate (3.7%) compared to EcPVs of the same genus such as EcPV2 and EcPV9 in the same horse population. We hypothesize a transmission mechanism different from the one observed in the closely related EcPV9 and EcPV2 that particularly infect Thoroughbreds. This horse breed is usually submitted to natural mating, thus indicating a possible sexual diffusion. No differences were detected for breeds in terms of susceptibility to EcPV10. Further studies are needed to investigate the molecular mechanisms behind the host and EcPV10 infection to explain the reduced viral spread.

A case of generalised cutaneous apocrine cystomatosis in a Pekingese dog.

Clinical, histological and immunohistochemical examination of a 13-year-old male client-owned Pekingese dog revealed an uncommon presentation of apocrine cutaneous cystomatosis. This is a rare non-neoplastic condition of uncertain cause, characterised by multiple cystically dilated apocrine sweat glands. We aimed to describe the features of this unusual case of generalised cutaneous apocrine cystomatosis in the dog, which can be useful to distinguish it from multifocal benign cystic apocrine tumours.

Tumor-Associated Macrophages in Canine Oral and Cutaneous Melanomas and Melanocytomas: Phenotypic and Prognostic Assessment.

The tumor microenvironment is a complex system, where neoplastic cells interact with immune and stromal cells. Tumor-associated macrophages (TAMs) are considered among the most numerically and biologically noteworthy cellular components in tumors and the attention on this cellular population has been growing during the last decade, both for its prognostic role and as a potential future therapeutic target. Melanoma, particularly the oral form, despite being one of the most immunogenic tumors, bears a poor prognosis in dogs and humans, due to its highly aggressive biological behavior and limited therapeutic options. The aims of this study are to characterize and quantify TAMs (using CD163, CD204, Iba1, and MAC387) in canine melanocytic tumors and to evaluate the association of these markers with diagnosis, histologic prognostic features, presence of metastases, and outcome, and to provide preliminary data for possible future therapies targeting TAMs. Seventy-two melanocytic tumors (27 oral melanomas, 25 cutaneous melanomas, 14 cutaneous melanocytomas, and 6 oral melanocytomas) were retrospectively selected and submitted to immunohistochemistry and double immunofluorescence. Double immunolabeling revealed that most CD163+ and CD204+cells co-expressed Iba1, which labeled also dendritic cells. Iba1 was instead rarely co-expressed with MAC387. Nevertheless, the expression of macrophagic markers showed a mild to moderate association among the four markers, except for CD204 and MAC387. The number of CD163+, CD204+, and MAC387+ cells was significantly higher in oral melanomas compared to oral melanocytomas (p < 0.001; p < 0.05 and p < 0.01, respectively), whereas Iba1 was differentially expressed in cutaneous melanomas and melanocytomas (p < 0.05). Moreover, CD163, IBA1 and MAC387 expression was associated with nuclear atypia and mitotic count. The number of CD163+cells was associated with the presence of metastases and tumor-related death in oral melanocytic tumors (p < 0.05 and p = 0.001, respectively).

Detection of Equus Caballus Papillomavirus Type-2 in Asymptomatic Italian Horses.

Equine Papillomavirus 2 (EcPV2) is responsible for squamous cell carcinomas (eSCCs) of external genitalia of both male and female horses. However, few studies report the EcPV2 prevalence among healthy horses. Currently, the lack of these data does not permit identifying at-risk populations and, thus, developing screening protocols aimed at the early detection of the infection, as for humans. The aim of our study was to estimate the genoprevalence of EcPV2 in clinically healthy horses in Italy and to evaluate their innate immune response. For this purpose, penile and vulvar swabs of 234 healthy horses were collected through sampling with sterile cytobrushes. Nucleic acids were isolated and EcPV2-L1 presence (DNA) and gene expression (RNA) were checked by RT-qPCR. Our results showed EcPV2-L1 DNA presence in 30.3% of the samples and L1 expression in 48% of the positive samples. No statistically significant differences were found in genoprevalence in relation to sex, age, and origin, while, concerning breeds, the Thoroughbred had the highest risk of infection. Concerning specifically the mares, 40.2% of them resulted in being positive for EcPV2; our findings show a major positivity in pluriparous (p = 0.0111) and mares subjected to natural reproduction (p = 0.0037). Moreover, samples expressing L1 showed an increased expression of IL1B (p = 0.0139) and IL12p40 (p = 0.0133) and a decreased expression of RANKL (p = 0.0229) and TGFB (p = 0.0177). This finding suggests the presence of an effective immune response, which could explain the low incidence of SCCs in positive horses, despite a high EcPV2 genoprevalence (30%).

Oxidative stress in the pathogenesis of canine zinc-responsive dermatosis.

Zinc deficiency causes skin diseases both in humans and in animals. The underlying pathogenic mechanisms remain unclear, but a growing body of evidence indicates a role for zinc in skin protection against free radical-induced oxidative damage. The immunohistochemical expression of heat shock proteins (HSPs; Hsp27, Hsp72, Hsp73 and Hsp90), Cu/Zn superoxide dismutase (SOD), metallothionein (MT), Ki-67 antigen and active caspase-3 were evaluated in normal canine skin and in samples from eight dogs with zinc-responsive dermatosis. All investigated HSPs showed intense cytoplasmic immunostaining in the affected epidermis. Focal nuclear positivity of Hsp72 was also detected in keratinocytes. Although Cu/Zn SOD expression was similar to that observed in normal skin, MT immunoreactivity occurred in both the cytoplasm and the nucleus of basal cells in normal skin but was absent from the affected epidermis. Caspase-3 activation was also absent in the involved epidermis, which revealed a high Ki-67 index (a 3.5- to 9-fold increase compared with normal skin). These results support the hypothesis that cellular response to stress, particularly oxidative stress, is involved in the pathogenesis of skin lesions in canine zinc-responsive dermatosis. The lack of MT immunoreactivity in the affected epidermis may be indicative of low zinc levels, thus resulting in vulnerability to oxidative damage. In contrast, high expression levels of HSPs in skin during zinc deficiency may confer protection against a variety of dangerous stimuli, contributing to inhibition of apoptosis and to cell cycle regulation of proliferating keratinocytes.

Tumor-infiltrating lymphocytes (TILs) in feline melanocytic tumors: A preliminary investigation.

The presence and the role of tumor-infiltrating lymphocytes (TILs) in different types of tumors, but particularly in melanoma, has become more and more investigated during the last decade, both in human and veterinary medicine. Melanocytic tumors are quite rare in cats, with diffuse iris melanoma being the most commonly diagnosed in this species. The aim of this study was to characterize the lymphocytic infiltration in feline melanocytic tumors and to analyze their association with the histological features of malignancy recognized in these tumors, as well as with the expression of the most commonly used immunohistochemical markers. Thirty-eight feline melanocytic tumors were retrospectively selected; histological and immunohistochemical characterization of the tumors (histologic criteria of malignancy; S100, Melan A, and PNL2 expression) and of TILs (presence/absence, density, distribution, and grade; CD3, CD20 expression) were performed and associations between them tested. Results showed that TILs grade increased with cellular pleomorphism (P < 0.05) and, within the group of cutaneous melanocytic tumors, also with the mitotic count (P < 0.05). On the other hand, TILs grade was inversely associated with the percentage of neoplastic cells positive for Melan A (P < 0.05) and PNL2 (P < 0.05). Both CD3+ and CD20+ lymphocytes increased significantly with TILs grade and in association with mitotic count, when stratified in low/high quantity. This preliminary study suggests that TILs in feline melanoma may be associated with histologic features of malignancy and loss of melanocytic-specific markers, such as Melan A and PNL2. Further studies, with a larger cohort and follow-up information, are recommended.