Different expression patterns of p63 and p73 in Felis catus papillomavirus type 2-associated feline Merkel cell carcinomas and other epidermal carcinomas.

Merkel cell carcinoma (MCC) is a cutaneous neuroendocrine tumor, and more than 90% of feline MCC cases test positive for Felis catus papillomavirus type 2 (FcaPV2). In the present study, basal cell markers p40, p63, and p73 and the stem cell marker SOX2 and cytokeratin 14 (CK14) were immunohistochemically examined in normal fetal, infant, and adult feline skin tissues. The expression of these proteins was examined in tumors positive for FcaPV2, including MCC, basal cell carcinoma (BCC), Bowenoid in situ carcinoma (BISC), and squamous cell carcinoma (SCC). Infant and adult feline skin tissues had mature Merkel cells, which were CK14-, CK18+, CK20+, SOX2+, synaptophysin+ and CD56+, while fetal skin tissue had no mature Merkel cells. MCC was immunopositive for p73, CK18, and SOX2 in 32/32 cases, and immunonegative for CK14 in 31/32 cases and for p40 and p63 in 32/32 cases. These results indicate that MCC exhibits different immunophenotypes from Merkel cells (p73-) and basal cells (p40+, p63+, and SOX2-). In contrast, all 3 BCCs, 1 BISC, and 2 SCCs were immunopositive for the basal cell markers p40, p63, and p73. The life cycle of papillomavirus is closely associated with the differentiation of infected basal cells, which requires the transcription factor p63. Changes in p63 expression in FcaPV2-positive MCC may be associated with unique cytokeratin expression patterns (CK14-, CK18+, and CK20+). Furthermore, SOX2 appears to be involved in Merkel cell differentiation in cats, similar to humans and mice.

Central nervous system mycobacteriosis caused by Mycobacterium genavense in degus (Octodon degus).

Degus (Octodon degus) that were kept at a breeding facility presented with neurological or respiratory symptoms and died. Necropsies were performed on 9 individuals, and no significant gross lesions were found. Histologically, spinal cord necrosis was observed in all 9 cases and granulomatous myelitis in 5 of the 9 cases. Locally extensive necrosis of the brain and encephalitis were observed in 7 of the 9 cases. Acid-fast bacteria were found in the spinal cords, brains, and lungs from all 9 cases. Immunohistochemically, Mycobacterium tuberculosis antigen was observed in the spinal cords, brains, and lungs from all 9 cases. Double-labeling immunofluorescence revealed M. tuberculosis antigen in IBA1- and myeloperoxidase-immunopositive cells. Extracted genomic DNA from 8 of the 9 cases was successfully amplified with the primers for Mycobacterium genavense ITS1 and hypothetical 21 kDa protein genes, and the polymerase chain reaction products were identified as M. genavense by DNA sequencing. This report highlights the susceptibility of degus to M. genavense infection in the central nervous system.

Genomic integration and expression of Felis catus papillomavirus type 2 oncogenes in feline Merkel cell carcinoma.

The involvement of Felis catus papillomavirus type 2 (FcaPV2) in feline Merkel cell carcinoma (MCC) has been previously hypothesized. In this study, the expression and localization of FcaPV2 oncogene mRNA, the integration of FcaPV2 genes, and p53 mutations in feline MCC were examined by RNAscope in situ hybridization (ISH), whole genome sequencing (WGS), and Sanger DNA sequencing, respectively. Furthermore, the morphological and molecular characteristics of FcaPV2-positive (FMX-MCC01) and FcaPV2-negative (AS-MCC01) MCC cell lines were compared in vitro and in vivo using immunofluorescence, ISH, xenotransplantation into mice, and immunohistochemistry. ISH for FcaPV2 E6/E7 detected viral RNA in 18/21 FcaPV2-positive MCC and not in 1/1 FcaPV2-negative MCC. WGS of 2 FcaPV2-positive cases revealed the integration of FcaPV2 genes in both cases. In cultured cells and xenograft tissues of FMX-MCC01, most cells were positive for E6/E7 by ISH and p16CDKN2A, a few cells were positive for the retinoblastoma protein (pRb), and all cells were negative for p53. In cultured cells and xenograft tissues of AS-MCC01, all cells were negative for p16CDKN2A, most cells were positive for pRb, and some cells were positive for p53. Missense mutations in p53 were identified in 8/10 FcaPV2-positive and 1/1 FcaPV2-negative MCC. These results suggest that the expression of integrated FcaPV2 oncogenes might be associated with reduced expression of the tumor suppressor proteins pRb and p53 and might contribute to the development of feline MCC. On the other hand, p53 mutations may be involved in both FcaPV2-positive and FcaPV2-negative MCC tumorigenesis.

Involvement of Felis catus papillomavirus type 2 in the tumorigenesis of feline Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a cutaneous neuroendocrine tumor. We recently demonstrated that cats with MCC often have other proliferative cutaneous lesions, such as squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). Based on this finding, we hypothesize that Felis catus papillomavirus (FcaPV) is involved in the development of MCC in cats, similar to SCC and BCC. To investigate this hypothesis, the presence of FcaPV nucleic acid and immunoreactivity for tumor suppressor proteins were examined in 21 feline MCC cases. Polymerase chain reaction using FcaPV type-specific primers detected FcaPV2 DNA in 20/21 samples of MCC. The complete FcaPV2 sequence was characterized in one case. In situ hybridization for FcaPV2 E7 revealed punctate nuclear signals within tumor cells in 19/21 MCC. Increased immunoreactivity for p16CDKN2A protein and decreased immunoreactivity for retinoblastoma (pRb) and p53 proteins were observed in 20/21 MCC. These results suggest that feline MCC cases are infected with FcaPV2 and the subsequent inhibition of pRb and p53 induced by integrated viral oncogenes is associated with feline MCC tumorigenesis, similar to other PV-induced proliferative cutaneous lesions. On the other hand, the single case of FcaPV2-negative MCC showed strong p53 immunoreactivity, suggesting mutations in p53 caused by cancer inducers other than FcaPV2 infection in this case. The present study suggests FcaPV2 as a cause of feline MCC.

Comparative In Vitro and In Vivo Studies on Feline, Canine, and Human Merkel Cell Carcinoma.

Merkel cell carcinoma (MCC) is an aggressive cutaneous neuroendocrine tumor, and most human MCC cases are infected by Merkel cell polyomavirus (MCPyV). However, the underlying pathogeneses of MCC in animals remain unclear. In the present study, newly established cell lines from feline and canine MCC, a MCPyV-positive human MCC cell line, and MCC tissues from 25 cats and 1 dog were examined and compared pathologically. Feline and canine MCCs were composed of tumor cells arranged in trabeculae and solid packets. Twenty out of 25 feline MCC cases (80%) had other proliferative cutaneous lesions, such as carcinoma in situ and squamous cell carcinoma. Among the 25 feline MCC cases, tumor cells were immunopositive for cytokeratins (CKs), including CK5/6 (4/25 cases, 16%), CK7 (5, 20%), CK18 (25, 100%), CK19 (20, 80%), and CK20 (20, 80%). The tumor cells of feline MCC were also immunopositive for synaptophysin (24/25, 96%) and CD56 (22/25, 88%). The tumor cells of canine MCC were immunopositive for CK18, CK19, CK20, and synaptophysin. Cultured feline and canine MCC cells grew in adherent monolayers and exhibited diffuse cytoplasmic immunoreactivity for CKs, whereas human MCC cells grew in suspension and exhibited dot-like cytoplasmic immunoreactivity for CKs. Differences in the distribution of CKs between human and animal MCC may be attributed to cell adhesion propensities. MCPyV genes and antigen were not detected in feline or canine MCC, suggesting a different etiology from human MCC.

Immunophenotyping of Nonneoplastic and Neoplastic Histiocytes in Cats and Characterization of a Novel Cell Line Derived From Feline Progressive Histiocytosis.

Histiocytic proliferative diseases are rare in cats, and their pathogenesis is poorly understood. In the present study, 25 cases of histiocytic sarcoma (HS) and 6 of feline progressive histiocytosis (FPH) were examined, and survival times were recorded in 19 cases. The immunophenotypes of tumor cells in these cases as well as of nonneoplastic feline histiocytes were characterized using formalin-fixed, paraffin-embedded tissues. An FPH cell line (AS-FPH01) and xenotransplant mouse model of FPH were also established. The median survival time of HS (150 days) was significantly shorter than that of FPH (470 days). Immunohistochemically, nonneoplastic histiocytes were immunopositive for various combinations of Iba-1, HLA-DR, E-cadherin, CD204, CD163, CD208, and MAC387. By immunohistochemistry, dermal interstitial dendritic cells (iDCs) and macrophages were CD204+/E-cadherin-, while epidermal Langerhans cells (LCs) were CD204-/E-cadherin+. Neoplastic cells of 4 FPH and 18 HS were CD204+/E-cadherin- (iDC/macrophage immunophenotype), while 2 FPH and 2 HS were CD204-/E-cadherin+ (LC immunophenotype), and 5 HS were CD204+/E-cadherin+ (LC-like cell immunophenotype). Furthermore, immunohistochemical and western blot analyses of AS-FPH01 cells derived from E-cadherin-negative FPH revealed that cultured cells were immunopositive for both CD204 and E-cadherin in vitro and in vivo. These results indicate that the neoplastic cells of feline HS and FPH were variably positive for iDC/macrophage and LC markers, and their immunophenotype changed in different microenvironments. The novel cell line established in the present study may serve as an experimental model of FPH that will enable further molecular and therapeutic studies on this disease.

Malignant Leydig cell tumor in dogs: two cases and a review of the literature.

Malignant Leydig cell tumor (MLCT) is a rare testicular tumor in dogs. We report herein 2 dogs with MLCT and cutaneous metastasis. Grossly, marked enlargement and distortion of the involved testes were noted; on cut surface, the parenchyma was completely replaced by neoplastic tissue. In addition, these tumors had extensive necrosis and hemorrhage. Case 1 had a rapidly growing cutaneous mass in the left angle of the mouth; the lesion was well-circumscribed and had an indistinct lobular pattern. Case 2 had multiple cutaneous masses in the dorsal neck region, the thoracic back region, and the right hindlimb. Microscopically, the tumor lobules were composed of oval-to-polyhedral cells with eosinophilic cytoplasm and resembled testicular tumors. By immunohistochemistry, the neoplastic cells in both the testicular and cutaneous tumors were positive for inhibin-alpha and melan A. The mitotic counts of the primary tumors from cases 1 and 2 were 21 and 11 per 10 high-power fields, respectively. Based on these findings, the cases were diagnosed as MLCT with cutaneous metastasis. Ki-67 expression in the neoplastic cells of the 2 cases was higher than in benign Leydig cell tumors. Our findings may be helpful for the diagnosis of canine MLCT.

A retrospective study of disease incidence in African pygmy hedgehogs (Atelerix albiventris).

The African pygmy hedgehog (Atelerix albiventris) is becoming a popular pet in Japan. The aim of this study was to determine the incidence of various diseases in African pygmy hedgehogs. We histologically investigated 105 samples from 100 privately-owned pet African pygmy hedgehogs that were submitted to two laboratories (North Lab and Patho Labo) between 2012 and 2017. Tissues submitted for this study were taken from female reproductive organs (33 cases; 31.43%), skin (20 cases; 19.05%), and the oral mucosa (19 cases; 18.1%). The most common histological diagnoses included endometrial stromal nodules identified as benign uterine neoplasia (14 cases; 13.33%); endometrial polyps identified as non-neoplastic polyps (7 cases; 6.67%), gingival hyperplasia and chronic suppurative inflammation in the oral mucosa (11 cases; 10.48%), fibrosarcomas in the skin (8 cases; 7.62%), and mammary tumors (8 cases; 7.62%). In this study, lymphoma and oral squamous cell carcinoma were less common than in the previous reports. The present study revealed the disease prevalence in captive African pygmy hedghogs that were histopathologically examined.