Reduction of phosphorylated signal transducers and activator of transcription-5 expression in feline mammary carcinoma.

Signal transducers and activators of transcription (STAT) is a family of transcription factors involved in various normal physiological cellular processes. Moreover, STATs have been recently identified as novel therapeutic targets for various human tumors. STAT3, STAT5a, and STAT6 have been suggested to be involved in tumorigenesis in human breast cancer. Owing to the similarity between feline mammary carcinomas (FMCs) and human breast cancers, these factors may play an important role in FMCs. However, studies on the expression of STATs in animal tumors are limited. Therefore, in this study, we aimed to characterize the expression of total STAT5 (tSTAT5) and phosphorylated STAT5 (pSTAT5) in FMCs, feline mammary adenomas, non-neoplastic proliferative mammary gland lesions, and normal feline mammary glands using immunohistochemistry. High expression of tSTAT5 was observed in the cytoplasm of all the samples assessed in this study. Moreover, high expression of tSTAT5 was observed in the nucleus; however, its levels varied depending on the lesion. The percentage of pSTAT5-nuclear positive cells varied among normal feline mammary glands (40.1 ± 25.1%), and non-neoplastic lesions, including mammary hyperplasia (43.2 ± 28.6%) and fibroadenomatous changes (18.0 ± 13.6%). Moreover, the percentage of pSTAT5-nuclear-positive cells in feline mammary adenomas was 24.5 ± 19.2%, which was significantly reduced in feline mammary carcinomas (2.4 ± 5.6%), regardless of histopathological subtype. This study suggests that decreased STAT5 activity may be involved in the development and malignant progression of feline mammary carcinomas.

Establishment of a novel canine soft tissue sarcoma cell line and comparison of its characteristics with other soft tissue sarcoma cell lines.

Soft tissue sarcoma (STS) is a relatively common tumor in dogs. However, very few canine STS cell lines are available. This study aimed to establish a new cell line, STS-YU1, derived from a recurrence of myxosarcoma in an 11-year-old mixed-breed dog. We examined STS-YU1 for in vitro cell proliferation, migration, anticancer drug sensitivity, transcriptome analysis using next-generation sequencing (RNA-seq), and in vivo tumorigenicity in mice and compared it with previously established STS cell lines, MUMA-G and A72. The cell proliferation and migration of STS-YU1 were higher than MUMA-G although MUMA-G only exhibited tumorigenicity in mice. STS-YU1 showed dose-dependent cytotoxicity to anticancer drugs, but with weak effects. RNA-seq analysis revealed the molecular phenotype of STS-YU1 was different from that of a previously reported cell line, A72. Hence, the use of STS-YU1 would help in efficient drug screening against canine STS in vitro.

Highly malignant endometrial stromal sarcoma in a cat.

An 11-year-old female Persian cat underwent ovariohysterectomy due to dilation of the uterine cavity with irregular thickening of the wall. Macroscopically, the middle and distal regions of the left uterine horn were swollen and the uterine wall was irregularly thickened due to the development of multiple coalescent, variably sized nodules. Microscopically, the nodules had originated in the endometrium and were composed of round to polygonal neoplastic cells arranged in dense sheets or ill-defined fascicles. The neoplastic cells had locally invaded the myometrium and reached the subserosa, with lymphovascular invasion. Immunohistochemically, the neoplastic cell population was partially positive for CD10, an established marker of endometrial stromal sarcoma (ESS) in humans, with focal and diffuse nuclear immunopositivity for oestrogen and progesterone receptors and immunonegativity for desmin and α-smooth muscle actin. Based on these findings, the uterine tumour was diagnosed as ESS and was considered to correspond morphologically to high-grade ESS in humans.

Uterine haemangiosarcoma in a Netherland Dwarf rabbit (Oryctolagus cuniculus Netherland dwarf).

A 7-year-old intact Netherlands Dwarf rabbit with bloody discharge from the vulva underwent ovariohysterectomy. Grossly, both sides of the uterus were enlarged. Histologically, the tumour had formed protruded from the myometrial wall toward the serosa and was composed of irregular small capillaries with irregularly shaped structures and bundled proliferation of spindle-shaped cells. No tumour cells infiltrated the endometrium. The tumour cells were positive for CD31, and histological and immunohistochemical staining confirmed the diagnosis of haemangiosarcoma. Vascular tumours in the uterus of animals are uncommon, and only one case has been reported in the uterus of rabbits.

Activation of the Akt signalling pathway as a prognostic indicator in canine soft tissue sarcoma.

Canine soft tissue sarcoma (STS) is relatively common in dogs and is the generic term for tumours that originate from mesenchymal cells. While histopathological grade and immunolabelling with Ki-67 have been used for estimating prognosis, additional indicators are needed for predicting prognosis. Aberrant cell signalling pathways may contribute to disease activity and, therefore, prognostic markers. However, their role in canine STS remains poorly understood. The aim of this study was to investigate expression of phosphorylated Akt (phospho-Akt) and phosphorylated S6 (phospho-S6) as potential prognostic indicators. Immunohistochemical labelling was conducted on clinical samples of canine STS (n = 67). We found that phospho-Akt expression was positively correlated with histopathological grade (P = 0.001) and Ki-67 index (P <0.01). There was no apparent relationship between the type of STS and the expression of phospho-Akt. The number of cases that expressed phospho-S6, which is the downstream molecule of the Akt signalling pathway, was higher in immunopositive phospho-Akt cases than in immunonegative phospho-Akt cases (P <0.0001). Furthermore, phospho-Akt expression was significantly higher in recurrent and metastatic cases. We also confirmed that phosphorylation of Akt occurred in conjunction with S6 phosphorylation in three canine STS cell lines. These results suggest that immunolabelling for phospho-Akt, phospho-S6 and Ki-67 could potentially be used as a prognostic indicator and therapeutic target in canine STS.

PARP deficiency causes hypersensitivity to Taxol through oxidative stress induced DNA damage.

Taxol is an antitumor drug derived from the bark of the Pacific Yew tree that inhibits microtubule disassembly, resulting in cell cycle arrest in late G2 and M phases. Additionally, Taxol increases cellular oxidative stress by generating reactive oxygen species. We hypothesized that the inhibition of specific DNA repair machinery/mechanisms would increase cellular sensitivity to the oxidative stress capacity of Taxol. Initial screening using Chinese hamster ovary (CHO) cell lines demonstrated that base excision repair deficiency, especially PARP deficiency, caused cellular Taxol hypersensitivity. Taxane diterpenes-containing Taxus yunnanensis extract also showed hypertoxicity in PARP deficient cells, which was consistent with other microtubule inhibitors like colcemid, vinblastine, and vincristine. Acute exposure of 50 nM Taxol treatment induced both significant cytotoxicity and M-phase arrest in PARP deficient cells, but caused neither significant cytotoxicity nor late G2-M cell cycle arrest in wild type cells. Acute exposure of 50 nM Taxol treatment induced oxidative stress and DNA damage. The antioxidant Ascorbic acid 2 glucoside partially reduced the cytotoxicity of Taxol in PARP deficient cell lines. Finally, the PARP inhibitor Olaparib increased cytotoxicity of Taxol in wild type CHO cells and two human cancer cell lines. Our study clearly demonstrates that cytotoxicity of Taxol would be enhanced by inhibiting PARP function as an enzyme implicated in DNA repair for oxidative stress.

Meningothelial hamartoma on the forehead of a young cat.

A 1 year and 2-months-old neutered male cat underwent surgical resection of a cutaneous nodule on the midline of the forehead that had been present since approximately 6 months of age. Histopathologically, the nodule was composed of interlacing collagenous fibres interspersed with varying numbers of spindloid cells with round to oval nuclei and moderate to abundant amounts of pale eosinophilic cytoplasm. Similar to meningothelial cells, the spindloid cells were immunopositive for vimentin, neuron-specific enolase, E-cadherin and somatostatin receptor 2. Based on these findings and the absence of nuclear atypia and mitotic figures, the nodule was diagnosed as meningothelial hamartoma. Although cases of cutaneous meningioma have been reported, this is the first report of meningothelial hamartoma in a domestic animal.

Feline uterine carcinosarcoma infiltrated with osteoclast-like giant cells.

A 12-year-old female Himalayan cat underwent an ovariohysterectomy to remove an intra-abdominal mass. Histologic examination using immunohistochemical staining revealed that the mass was comprised of epithelial and mesenchymal components. Within the lesion, multinucleated giant cells (MGCs) were observed diffusely. MGCs were positive for vimentin and Iba-1 and negative for cytokeratin AE1/AE3 and CD204. In addition, MGCs were negative for Ki-67, indicating nonneoplastic cells. Osteoclast-like MGC (OLMGC) phenotype with tartrate-resistant acid phosphatase positivity was also seen. These findings suggested that the uterine tumor was carcinosarcoma with OLMGCs. Uterine tumors in humans, such as leiomyosarcoma and carcinosarcoma, with OLMGC infiltration, are well-known pathologic entities; however, they are rare in animals and to our knowledge, have not been previously reported in cats.

Benign Mixed Mammary Tumour with Hepatoid Gland Differentiation in a Dog.

A 10-year-old spayed female Shih Tzu underwent surgery to remove a tumour (8 mm diameter) in the right 4th mammary gland. Histopathologically, the tumour consisted of four different components: luminal epithelial cells, myoepithelial cells, cartilage and well-differentiated hepatoid gland-like cells. There were multiple nests composed predominantly of hepatoid gland-like tissue with a small number of tubules formed by luminal epithelial cells at the periphery, in which continuity between the two components was seen. Immunolabelling for cytokeratins (CK14, CK18 and CK19), p63 and α-smooth muscle actin clearly distinguished the neoplastic luminal epithelial, myoepithelial and hepatoid gland-like cells. The immunohistochemical phenotype of the hepatoid gland-like neoplastic cells was identical to that of normal hepatoid gland cells. Based on these findings, a diagnosis of benign mixed tumour of the mammary gland with differentiated hepatoid gland cells was made. To our knowledge, this is the first report of a canine mammary tumour with hepatoid gland differentiation.

Dysmegakaryopoiesis and Transient Mild Increase in Bone Marrow Blasts in Patients With Aplastic Anemia Treated With Eltrombopag May Be Signs of Hematologic Improvement and Not Portend Clonal Evolution.

OBJECTIVES: Eltrombopag, a thrombopoietin-receptor agonist, stimulates hematopoiesis in patients with acquired aplastic anemia (AA). Cytomorphologic changes in bone marrow after eltrombopag administration are still unclear. This study examined the effect of eltrombopag on cytomorphologic findings using data from prior phase 2 studies (E1201 and E1202). METHODS: Microscopic examinations were performed in 31 patients with AA (E1201 [n = 21], E1202 [n = 10]). The relationship between hematologic improvement and morphologic findings was also investigated. RESULTS: In 5 patients (E1201 [n = 3], E1202 [n = 2]), the bone marrow blast count increased after initiation of eltrombopag treatment compared with screening values. The blast count was less than 5%, and the increase in bone marrow blasts was transient in all 4 patients who had bone marrow examinations at follow-up. In 8 patients (E1201 [n = 5], E1202 [n = 3]), dysplastic forms of megakaryocytes were found in the bone marrow following treatment initiation. Dysmegakaryopoiesis of 10% or more was found in 3 patients. None of the patients revealed micromegakaryocytes. Ten patients showed an increase in bone marrow blasts and/or dysmegakaryopoiesis following treatment initiation. Nine of 10 patients showed hematologic improvement in 1 or more lineages. CONCLUSIONS: Dysmegakaryopoiesis without micromegakaryocytes and a transient increase of less than 5% in bone marrow blast count may be signs of hematologic improvement with eltrombopag for patients with AA.

Outcome of Localized Bile Duct Carcinoma in Six Dogs Treated with Liver Lobectomy.

The prognosis for bile duct carcinoma in dogs is generally believed to be poor. However, only a few studies have evaluated the postoperative outcomes in such cases. The objective of this case series was to describe the postoperative outcomes of localized intrahepatic bile duct carcinoma in dogs. The electronic medical records of 16 dogs with bile duct carcinoma were reviewed, and 6 dogs were included in the study. All cases were diagnosed as bile duct carcinoma using postoperative pathology, and five of them had already been diagnosed using preoperative core biopsy. The tumors in all of the dogs were confirmed as completely resected on histopathological examination. Two dogs received toceranib following the surgery. The median follow-up time was 693 days (range, 420-1386 days), with a median survival time of 894 days (range, 420-1386 days). Local recurrence or distant metastases were detected in two of the six dogs (33%) on 354 and 398 days following surgery, respectively. The median progression-free survival was 492 days (range, 354-1386 days). In conclusion, dogs with localized intrahepatic bile duct carcinoma had a good prognosis following complete surgical resection.

Extraskeletal osteosarcoma associated with two different types of synthetic fibers derived from a surgical swab in a dog.

A 10-year-old spayed female Japanese Shiba Inu had an intraperitoneal mass that was excised surgically. The central area of the mass was composed of osteoblast-like neoplastic cells, osteoid, macrophages, and numerous fibers. The neoplastic cells showed nuclear atypia and many mitotic figures. Therefore, the central area of the mass was diagnosed as an extraskeletal osteosarcoma. The peripheral area of the mass was granuloma tissue with numerous fibers. The neoplastic and granuloma area included two types of fibers, which were identified as rayon and polyester by their morphological and staining characteristics. These fibers were consistent with those of commercial surgical swab, suggesting that the fibers may have been derived from retained surgical swabs at the time of ovariohysterectomy. Therefore, this lesion was considered an extraskeletal osteosarcoma associated with a retained surgical swab.

The immunohistochemical detection of peroxiredoxin 1 and 2 in canine spontaneous vascular endothelial tumors.

Peroxiredoxin (PRDX) is an antioxidant enzyme family with six isoforms (PRDX1-6). The main function of PRDXs is to decrease cellular oxidative stress by reducing reactive oxygen species, such as hydrogen peroxide, to H2O. Recently, it has been reported that PRDXs are overexpressed in various malignant tumors in humans, and are involved in the development, proliferation, and metastasis of tumors. However, studies on the expression of PRDXs in tumors of animals are limited. Therefore, in the present study, we immunohistochemically investigated the expression of PRDX1 and 2 in spontaneous canine hemangiosarcoma (HSA) and hemangioma (HA), as well as in selected normal tissue and granulation tissue, including newly formed blood vessels. Although there were some exceptions, immunolocalization of PRDX1 and 2 in normal canine tissues was similar to those in humans, rats, or mice. In granulation tissue, angiogenic endothelial cells were strongly positive for PRDX1 and 2, whereas quiescent endothelial cells in mature vessels were negative. Both PRDX1 and 2 were significantly highly expressed in HSA compared to HA. There were no significant differences in the expression of PRDX1 and 2 among the subtypes and primary sites of HSA. These results suggest that PRDX1 and 2 may be involved in the angiogenic phenotypes of endothelial cells in granulation tissue as well as in the behavior in the malignant endothelial tumors.

Lokivetmab improved pruritus in a dog with cutaneous epitheliotropic lymphoma.

A 13-year-old spayed female Cavalier King Charles Spaniel presented with chronic swelling and pruritus on the palmar aspect of the left forepaw and on the tail. Cutaneous epitheliotropic lymphoma (CEL) was diagnosed by histopathology and immunocytochemistry. Prednisolone was initially used alone as an alternative treatment for CEL. Despite long-term corticosteroid therapy, the patient's physiological (pruritus) and dermatological signs (alopecia, erythema, erosion, and ulceration with crust) progressed and showed no evidence of improvement. To address the worsening condition of pruritus, lokivetmab was started in combination with prednisolone. Once on lokivetmab, the pruritus steadily improved and was effective in resolving and maintaining remission. Further investigation on the critical role of IL-31 in the pruritus pathway of dogs with CEL is required.

Minimally invasive spinal surgery in a young cat with vertebral hypertrophy.

CASE SUMMARY: A 2-year-old neutered female Scottish Fold cat was presented with an 8-week history of progressive back pain, paraparesis and decrease of postural reactions in both pelvic limbs. MRI showed spinal cord compression from both ventral sides, which originated from the T4 vertebral body and pedicle. The lesion compressing the spinal cord had a bone-like density on CT, and endoscopic surgery was performed to excise it. Histopathological examination of the resected tissue showed no evidence of malignancy and the lesion was diagnosed as vertebral hypertrophy. After surgery, the neurological status of the cat gradually improved. The cat was ambulant at the follow-up evaluation 2 weeks after surgery. Six months later, hindlimb paresis had improved considerably, and no recurrence was observed on CT. RELEVANCE AND NOVEL INFORMATION: This is the first description of thoracic vertebral canal stenosis due to hypertrophy of a single vertebra in a young cat. Excision of the hypertrophic vertebra by endoscopic surgery is less invasive than open surgery and may give a good prognosis.

Development of synthetic microRNA-214 showing enhanced cytotoxicity and RNase resistance for treatment of canine hemangiosarcoma.

MicroRNA-214 (miR-214), a pivotal tumour-suppressive miRNA, is downregulated in canine hemangiosarcoma (HSA) cells. Although these tumour-suppressive miRNAs are potential therapeutic agents, their clinical efficacy may be limited because of their vulnerability to RNase-rich microenvironments and low in vivo transfection rates. We developed synthetic miR-214s with enhanced cytotoxicity, RNase resistance and quantity of miR-214 in/on cells. These synthetic miR-214s were synthesized by various chemical modifications (such as 4'-aminoethyl-2'-fluoro, 2'-fluoro, 2'-O-methyl, phosphorothioate and oligospermine modifications) of the wild-type mature miR-214 sequences. Transfection of HSA cells with synthetic miR-214 (miR-214 5AE) demonstrated significant growth suppressive effect and induced the strongest apoptotic response. Synthetic miR-214s (miR-214 5AE, miR-214 10AE and miR-214 OS) were much more stable than mature miR-214s in foetal bovine serum. Similar to mature miR-214, 5AE and OS suppressed the expression level of COP1 in HSA cells. The quantity of synthetic miR-214s in/on cells was higher than that of mature miR-214. In conclusion, we developed a clinically applicable, synthetic miR-214 5AE that regulates the COP1 protein expression similar to that mediated by mature miR-214. Additionally, miR-214 5AE confers better cytotoxicity, nuclease resistance and transfection rate than mature miR-214. Thus, miR-214 5AE could potentially be a novel miRNA-based chemotherapeutic agent that could improve the prognosis of HSA. Its in vivo effects on canine HSA need to be examined in future.

Primary malignant peripheral nerve sheath tumors arising from the spinal canal invading the abdominal cavity in a dog.

A 9-year-old neutered male Wire Fox Terrier presented with an 1-month history of hindlimb paresis. Magnetic resonance imaging revealed a contrast-enhanced mass at the level of the L2 vertebral canal. The dog became paraplegic with no deep perception of the hindlimbs, and the mass was surgically removed. The histopathological diagnosis was of a malignant peripheral nerve sheath tumor (MPNST). The dog suffered a relapse of right hindlimb ataxia at 225 days after the surgery. The dog died 434 days after the surgery. Necropsy found a large mass in the abdominal cavity invading from the L2-nerve. This is the first report of MPNST invading the abdominal cavity through the nerve root.

Electron Scattering in Conventional Cell Flask Experiments and Dose Distribution Dependency.

Electron beam therapy (EBT) is commonly used for treating superficial and subdermal tumors. Previous cellular radiosensitivity research using EBT may be underestimating the contribution from flask wall scattering and the corresponding dose distribution. Single cell suspensions of Chinese hamster ovary (CHO) cells were plated on flasks and irradiated with 3, 4, 7, 9, and 18 MeV energy electron beams from two different institutions, and the spatial locations of surviving colonies were recorded. Gafchromic film dosimetry and Monte Carlo simulations were carried out to determine the spatial electron scattering contribution from the flask walls. Low electron irradiation resulted in an uneven surviving colony distribution concentrated near the periphery of the flasks, while spatial colony formation was statistically uniform at energies above 7 MeV. Our data demonstrates that without proper dosimetric corrections, studies using low energy electrons can lead to misinterpretations of energy dependent cellular radiosensitivity in culture vessels, and radiotherapeutic applications.

Trimer form of tumor necrosis factor-related apoptosis inducing ligand induces apoptosis in canine cell lines derived from mammary tumors.

We evaluated the cytotoxic effect of isoleucine-zipper tumor necrosis factor-related apoptosis inducing ligand (izTRAIL) against cell lines, B101592, Cha, and C090115, derived from canine mammary gland tumors. These cells were derived from three dogs diagnosed with mammary adenoma or carcinoma. All three cells were positive for vimentin, while B101592 and C090115 were positive for cytokeratin (CK) AE1/AE3 and CK CAM5.2. Treatment with izTRAIL decreased the viability of the three cell lines. The proportion of annexin V+/propidium iodide- cells increased in all three cell lines after treatment with izTRAIL. Additionally, cell cycle analysis revealed that izTRAIL treatment increased the number of cells in sub-G1 phase. Moreover, izTRAIL treatment activated caspase-8 and caspase-3 and enhanced the levels of cleaved poly (ADP-ribose) polymerase. The cytotoxic effect of izTRAIL was mitigated upon co-treatment with caspase-8 or caspase-3 inhibitor. These results indicated that izTRAIL induces apoptosis in cell lines derived from canine mammary tumor, which was also previously reported in canine hemangiosarcoma cell lines. This suggested that canine tumor cells have conserved TRAIL receptors. This study will provide the basis for further studies on TRAIL receptors and TRAIL-related molecules.