Fig. S1. Supplementary data for large scale genomic aberrations illustrated in Figure 2, including: A) the distribution of mapped read density of WGS; B) PCR confirmation of translocation and fusion junctions of a superamplicon; and C) PCR and RT-PCR amplification of MGAM, ZFAND3, and the ZFAND3-MGAM fusion gene. Fig. S2. The distribution of mapped read density of WES, supplementary data for Figure 3. Fig. S3. Differentially expressed genes and functions among complex, simple carcinomas and normal mammary glands, supplementary data for Figure 3. Fig. S4. IHC images of histone modifications in canine normal mammary glands at 400X magnification, supplementary data for Figure 4. Fig. S5. IHC images of histone modifications in simple carcinomas, complex carcinomas and normal mammary glands at 200X magnification, supplementary data for Figure 5. Fig. S6. Supplementary data for homology between canine MCs and human breast cancers described in Figure 6. These include differentially expressed genes between ER+ and ER- carcinomas at FDR ≤ 0.1 and an example of PAM50 clustering indicating canine and human tumor IDs.

Supplementary Table S3. Supplementary data for mutation discovery in canine MCs by WES and RNA-seq analyses, including read mapping information, mutated gene listed, base substitution type, etc.

Abstract Spontaneously occurring canine mammary cancer represents an excellent model of human breast cancer, but is greatly understudied. To better use this valuable resource, we performed whole-genome sequencing, whole-exome sequencing, RNA-seq, and/or high-density arrays on twelve canine mammary cancer cases, including seven simple carcinomas and four complex carcinomas. Canine simple carcinomas, which histologically match human breast carcinomas, harbor extensive genomic aberrations, many of which faithfully recapitulate key features of human breast cancer. Canine complex carcinomas, which are characterized by proliferation of both luminal and myoepithelial cells and are rare in human breast cancer, seem to lack genomic abnormalities. Instead, these tumors have about 35 chromatin-modification genes downregulated and are abnormally enriched with active histone modification H4-acetylation, whereas aberrantly depleted with repressive histone modification H3K9me3. Our findings indicate the likelihood that canine simple carcinomas arise from genomic aberrations, whereas complex carcinomas originate from epigenomic alterations, reinforcing their unique value. Canine complex carcinomas offer an ideal system to study myoepithelial cells, the second major cell lineage of the mammary gland. Canine simple carcinomas, which faithfully represent human breast carcinomas at the molecular level, provide indispensable models for basic and translational breast cancer research. Cancer Res; 74(18); 5045–56. ©2014 AACR.

PURPOSE:To perform a reconstructive blepharoplasty to obtain complete surgical excision of a darkly pigmented raised conjunctival-eyelid mass of the lower eyelid in a dog.ANIMAL STUDIED:A 7-year-old, female-spayed, Yorkshire Terrier was evaluated for a slowly progressive, dark brown-to-black raised mass of the lower left eyelid. Sampling of the mass via fine-needle aspirate or incisional biopsy was declined, and an excision of the mass with the goal to obtain complete margins and maintain normal eyelid function with cosmesis was elected.PROCEDURES:The lower palpebral conjunctival-eyelid pigmented mass was excised en bloc and the lower eyelid was reconstructed with a mucocutaneous subdermal plexus flap.RESULTS:Histopathology revealed a malignant dermal and conjunctivalmelanoma excised with complete margins (1-2 mm). Short-term complications included corneal ulceration and eschar formation, which resolved completely at the 1-month follow-up. Long-term complications included mild trichiasis with epiphora and porphyrin staining. Tumor recurrence was not observed during an 8-month follow-up period.CONCLUSIONS:The en bloc excision with mucocutaneous subdermal plexus flap was successful in obtaining complete surgical margins for a malignant conjunctival-eyelid melanoma. An excellent functional and cosmetic outcome was achieved without tumor recurrence during an 8-month follow-up period. A mucocutaneous subdermal plexus flap can be considered as a surgical option for malignant melanoma of the lower eyelid.

Supplementary Table S7. Supplementary data indicating altered genes and their function information.

Recent reports have highlighted a lower-than-expected prevalence of neoplasia in elephants and suggested mechanisms for cancer resistance. But despite infrequent reports in the literature, uterine neoplasia is common in managed Asian elephants (Elephas maximus). This study is an archival review of reproductive tract neoplasia in 80 adult female Asian elephant mortalities in managed care facilities in the United States from 1988 to 2019. Neoplasms occurred in 64/80 (80%) of cases. Most were in the uterus (63/64; 98%) with only a single case of ovarian neoplasia. Myometrial leiomyomas were present in 57/63 (90%) cases with uterine neoplasia. Uterine adenocarcinoma was present in 8/63 (13%) cases. Remaining cases included endometrial adenoma (2), focal carcinoma in situ in endometrial polyps (1), anaplastic carcinoma (1), endometrial hemangioma (1), primitive neuroectodermal tumor (PNET; 1), and angiosarcoma (1). One case with uterine adenocarcinoma had a separate pelvic mass histologically characterized as an anaplastic sarcoma. Distant metastases were documented in 5/8 (63%) cases of uterine adenocarcinoma, and in the uterine anaplastic carcinoma, PNET, and angiosarcoma. Four uterine adenocarcinomas and one carcinoma in situ were examined immunohistochemically for pan-cytokeratin, vimentin, and estrogen receptor. In all, neoplastic cells were pan-cytokeratin positive and vimentin negative, and in 2 cases were immunoreactive for estrogen receptor. Results show that female reproductive tract neoplasia, particularly of the uterus, is common in Asian elephants and is not limited to leiomyomas. Importantly, uterine neoplasms have the potential to impact fecundity and may represent obstacles to conservation in managed care.

Trichomonosis is an important cause of mortality in multiple avian species; however, there have been relatively few reports of this disease in owls. Two barn owls (Tyto alba) and four barred owls (Strix varia) submitted for diagnostic examination had lesions consistent with trichomonosis including caseous necrosis and inflammation in the oropharynx. Microscopically, these lesions were often associated with trichomonads and molecular testing, if obtainable, confirmed the presence of Trichomonas gallinae, the species most commonly associated with trichomonosis in birds. The T. gallinae genotype in one barn owl and two barred owls was identified as ITS-OBT-Tg-1 by sequence analysis. Columbids are the primary hosts for T. gallinae, and columbid remains found within the nest box of the barn owls were the likely source of infection. This study is the first to formally describe the strains and genetic variation of T. gallinae samples from clinical cases of trichomonosis in barn and barred owls in the eastern USA.

OBJECTIVETo evaluate the efficacy and safety of gastroscopy and biopsy of the proventriculus and ventriculus in pigeons (Columba livia).ANIMALS15 adult pigeons.PROCEDURESEach pigeon was anesthetized, and the upper gastrointestinal tract (from the cervical portion of the esophagus to the ventriculus) was endoscopically evaluated by use of a rigid endoscope inserted orally. Saline (0.9% NaCl) solution was orally infused to achieve lumen dilation and visibility. Two mucosal biopsy specimens were collected from each of the proventriculus and ventriculus, histologically evaluated, and graded for crush artifacts and depth. Pigeons were monitored for adverse effects for 3 to 6 days after the procedure, after which they were euthanized for necropsy.RESULTSGastroscopy via the oral approach provided excellent visibility of the lumen and mucosal surfaces of the proventriculus and cranial portion of the ventriculus and was safe provided that appropriate precautions were taken. Two intraoperative deaths occurred at the beginning of the study; following procedure refinement, no additional deaths occurred. No major adverse effects of the procedure were detected in the remaining 13 pigeons during the postoperative monitoring period or at necropsy. Diagnostic quality of proventriculus specimens was adequate for 10 of 13 pigeons. Eight of 13 ventriculus specimens were of inadequate quality, and only 3 were of adequate quality.CONCLUSIONS AND CLINICAL RELEVANCEGastroscopy was useful for evaluating the lumen and mucosal surface of the proventriculus and ventriculus in pigeons, and biopsy of those organs was safely performed with the appropriate technique. Further evaluation of these techniques is needed in birds with clinical disease and birds of other species.

Abstract Spontaneous canine mammary cancer represents an excellent model of human breast cancer but is greatly understudied. To better utilize this valuable resource, we performed the first comprehensive characterization of the genome, transcriptome and epigenome of 12 canine mammary cancer cases, including 7 simple carcinomas and 4 complex carcinomas (1). Simple carcinomas, which histologically match human breast carcinomas, harbor extensive genomic aberrations, many of which faithfully recapitulate key features of human breast cancer. Notably, these tumors closely cluster with human basal-like breast carcinomas from TCGA (2), but not with other intrinsic subtypes, in PAM50 classification with human breast carcinomas. Furthermore, these tumors match the basal-like cancer profile, including MYC amplification, PTEN deletion, BRCA1 mutation, etc. Our results indicate canine simple carcinomas could serve as a much-needed spontaneous cancer model of basal-like breast cancer by effectively bridging the gap between preclinical research and human trials, speeding up drug discovery for the worst subtype of breast cancer. Canine complex carcinomas, characterized by proliferation of both luminal and myoepithelial cells (rare in human breast cancer), appear to lack genomic abnormalities. Instead, these tumors have ~35 chromatin-modification genes downregulated, and are abnormally enriched with active histone modification H4-acetylation while depleted with repressive histone modification H3K9me3. Our findings indicate that these tumors likely originate from epigenomic alterations instead of genomic lesions. Canine complex carcinomas offer an ideal system to study myoepithelial cells, the second major yet significantly understudied cell lineage of the mammary gland. References: 1. Liu D, Xiong H, Ellis AE, Northrup NC, Rodriguez CO, Jr., O'Regan RM, et al. Molecular homology and difference between spontaneous canine mammary cancer and human breast cancer. Cancer research. 2014. 2. Cancer Genome Atlas N. Comprehensive molecular portraits of human breast tumours. Nature. 2012;490:61-70. Citation Format: Deli Liu, Huan Xiong, Angela E. Ellis, Nicole C. Northrup, Carlos O. Rodriguez Jr, Ruth M. O'Regan, Stephen Dalton, Shaying Zhao. Spontaneous canine mammary cancers: A much-needed model for human basal-like breast cancer and an ideal system to understand myoepithelial cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A07.

OBJECTIVE:To describe five cases of protozoal keratitis or conjunctivitis in dogs with chronic preexisting ocular surface disease treated with long-term immunosuppression.ANIMALS STUDIED:Five dogs that developed corneal or conjunctival mass lesions.PROCEDURES:The database of the Comparative Ocular Pathology Laboratory of Wisconsin was searched for canine cases diagnosed with corneal or conjunctival protozoal infection. Five cases were identified, and tissues were examined using routine and special histochemical stains: immunohistochemical labels for Neospora caninum, Toxoplasma gondii, and Leishmania spp., and tissue sample PCR for Leishmania spp., Trypanosoma cruzi, tissue coccidia (i.e., T. gondii/Sarcocystis/Neospora), piroplasms, trichomonads, and Acanthamoeba. Electron microscopy was performed for two cases, and serology for N. caninum and T. gondii was available for three cases.RESULTS:Preexisting ocular diseases included keratoconjunctivitis sicca and pigmentary keratitis (n = 4) and pyogranulomatous meibomian adenitis (n = 1). All dogs were treated with tacrolimus or cyclosporine for at least 1.2 years. Dogs were presented with fleshy corneal or conjunctival masses that were clinically suspected to be neoplastic (n = 4) or immune mediated (n = 1). Histologic examination revealed granulomatous inflammation with intralesional protozoal organisms. Amoeba (n = 2), T. gondii (n = 2), or Leishmania mexicana (n = 1) were identified using molecular techniques. Serological tests were negative.CONCLUSIONS:Protozoal keratitis and conjunctivitis without systemic involvement appears rare and may be associated with chronic preexisting ocular surface disease treated with long-term immunosuppression. Based upon clinical appearance, lesions could be confused with neoplasia. This is the first report of amoebic keratoconjunctivitis in dogs and of L. mexicana in dogs in the United States.

Spontaneous canine head and neck squamous cell carcinoma (HNSCC) represents an excellent model of human HNSCC but is greatly understudied. To better understand and utilize this valuable resource, we performed a pilot study that represents its first genome-wide characterization by investigating 12 canine HNSCC cases, of which 9 are oral, via high density array comparative genomic hybridization and RNA-seq. The analyses reveal that these canine cancers recapitulate many molecular features of human HNSCC. These include analogous genomic copy number abnormality landscapes and sequence mutation patterns, recurrent alteration of known HNSCC genes and pathways (e.g., cell cycle, PI3K/AKT signaling), and comparably extensive heterogeneity. Amplification or overexpression of protein kinase genes, matrix metalloproteinase genes, and epithelial-mesenchymal transition genes TWIST1 and SNAI1 are also prominent in these canine tumors. This pilot study, along with a rapidly growing body of literature on canine cancer, reemphasizes the potential value of spontaneous canine cancers in HNSCC basic and translational research.

A 14-year-old neutered male Dachshund presented for the evaluation of oculus dexter (OD) third eyelid elevation ongoing for approximately 2 months. Complete ophthalmic examination revealed a large, nonpainful, well-demarcated, soft mass at the base of the right third eyelid causing elevation and mild hyperemia. The mass was freely moveable with the third eyelid, and no right globe deviation was noted. No other abnormalities were noted on physical examination, routine blood chemistry, complete blood count, serum T4, urinalysis, or urine cortisol/creatinine ratio. Ocular B-mode ultrasonography showed an anechoic, well-demarcated, homogenous, soft tissue mass at the base of the third eyelid with no orbital extension. A leiomyoma was diagnosed after multiple punch biopsies were obtained from the palpebral surface of the mass. The right third eyelid was excised surgically. Histopathology confirmed a completely excised, nodular, unencapsulated, expansile mass within the third eyelid. Positive smooth muscle actin and negative S-100 immunohistochemistry confirmed a leiomyoma. Bundles of normal smooth muscle were also present adjacent to the mass. The mass was compressing the adjacent lacrimal gland and associated with moderate dacryoadenitis. Twelve months postoperatively, the right globe position and motility remain normal with no evidence of mass regrowth. To the author's knowledge, this is the first reported case of a leiomyoma of the third eyelid in any species. In this case, the mass was completely excised and no regrowth has occurred twelve months after surgery. This case along with independently reviewed canine third eyelids clearly demonstrates the presence of smooth muscle within the canine third eyelid.

A four-year-old neutered male Labrador retriever presented to Portland Veterinary Specialists Ophthalmology Service for evaluation of a pigmented mass oculus sinister (OS) of approximately 4-month duration. Complete ophthalmic examination revealed a large, pigmented, raised, well-demarcated, epibulbar mass appearing to originate from the nasodorsal limbal region. The mass was smooth and roughly circular, extending approximately 4 mm into the sclera and 14 mm into the nasodorsal cornea. Gonioscopy directly under the mass was not possible due to mass size. The visible iridocorneal angle was normal. High-resolution B-scan ultrasound showed mass extension to Descemet's membrane and deep sclera, but no intraocular invasion. Penetrating sclerokeratoplasty was performed followed by autologous pinnal cartilage and conjunctival grafting to repair the corneoscleral defect (20 mm x 19 mm) and to restore globe integrity and function. Histopathology confirmed the mass to be a benign limbal melanoma with complete excision. The surgery site healed without complication, and the pinnal cartilage became fully incorporated into the globe. Twelve months postoperatively, the patient remains visual with a normal intraocular and fundic examination. The pinnal harvest site on the right ear healed without complication. To the authors' knowledge, this is the first reported case of corneoscleral grafting using autologous pinnal cartilage. This may represent a viable alternative to other corneoscleral grafting procedures for large defects and is an attractive treatment option due to lack of host rejection, readily available source of donor cartilage, and provision of tectonic support to the globe.

A 25-year-old, female eclectus parrot (Eclectus roratus) presented for dyspnea 3 weeks after anesthesia and surgery for egg yolk coelomitis. Radiography, computed tomography, and tracheoscopy revealed multiple tracheal strictures spanning a length of 2.6 cm in the mid to distal trachea. Histopathologic examination revealed mild fibrosis, inflammation, and hyperplasia consistent with acquired tracheal strictures. Tracheal resection was not considered possible because of the length of the affected trachea. The strictures were resected endoscopically, and repeated balloon dilation under fluoroscopic guidance over the course of 10 months resulted in immediate but unsustained improvement. Computed tomography was used to measure the stenotic area. A 4 × 36-mm, custom-made, nitinol wire stent was inserted into the trachea under fluoroscopic guidance. After stent placement, intermittent episodes of mild to moderate dyspnea continued, and these responded to nebulization with a combination of saline, acetylcysteine, and dexamethasone. Multiple attempts to wean the patient off nebulization therapy and to switch to a corticosteroid-free combination were unsuccessful. The parrot eventually developed complications, was euthanatized, and necropsy was performed. Histologically, the tracheal mucosa had widespread erosion to ulceration, with accumulation of intraluminal exudate and bacteria, severe degeneration of skeletal muscle and tracheal rings, prominent fibrosis, and mild to moderate, submucosal inflammation. Clinicopathologic findings in this case suggested tracheomalacia, which has not been previously described in birds. Custom-made tracheal stents can be used for severe tracheal stenosis in birds when tracheal resection and anastomosis is not possible. Complications of tracheal stent placement in birds may include tracheitis and tracheomalacia. To our knowledge, this is the first report of tracheal stent placement in an avian species.

Porocephalus crotali is a pentastomid parasite that uses crotaline snakes as definitive hosts and a variety of rodents as intermediate hosts. A study of definitive and intermediate pentastome hosts on Cumberland Island, Georgia, revealed high prevalence of P. crotali infection in crotalid snakes as well as several mammalian species. Despite the presence of numerous nymphs in some animals, clinical signs of disease were not observed. In intermediate hosts, the liver, mesentery, and reproductive organs were most commonly infected. No gross evidence of tissue damage was noted in association with the numerous encysted nymphal pentastomes, and histopathology demonstrated minimal reaction to the encysted nymphs. Partial 18S rRNA gene sequences confirmed the parasites were P. crotali. In contrast to many previous reports in rodents, the prevalence on this barrier island was high, and this is the first report of Virginia opossums (Didelphis virginiana) and any insectivore species as intermediate hosts. Although generally not considered pathogenic, the long-term consequences of high nymph intensities on individuals deserve attention.

Vacuolar myelinopathy (VM) is a neurologic disease primarily found in birds that occurs when wildlife ingest submerged aquatic vegetation colonized by an uncharacterized toxin-producing cyanobacterium (hereafter “UCB” for “uncharacterized cyanobacterium”). Turtles are among the closest extant relatives of birds and many species directly and/or indirectly consume aquatic vegetation. However, it is unknown whether turtles can develop VM. We conducted a feeding trial to determine whether painted turtles (Chrysemys picta) would develop VM after feeding on Hydrilla (Hydrilla verticillata), colonized by the UCB (Hydrilla is the most common “host” of UCB). We hypothesized turtles fed Hydrilla colonized by the UCB would exhibit neurologic impairment and vacuolation of nervous tissues, whereas turtles fed Hydrilla free of the UCB would not. The ability of Hydrilla colonized by the UCB to cause VM (hereafter, “toxicity”) was verified by feeding it to domestic chickens (Gallus gallus domesticus) or necropsy of field collected American coots (Fulica americana) captured at the site of Hydrilla collections. We randomly assigned ten wild-caught turtles into toxic or non-toxic Hydrilla feeding groups and delivered the diets for up to 97 days. Between days 82 and 89, all turtles fed toxic Hydrilla displayed physical and/or neurologic impairment. Histologic examination of the brain and spinal cord revealed vacuolations in all treatment turtles. None of the control turtles exhibited neurologic impairment or had detectable brain or spinal cord vacuolations. This is the first evidence that freshwater turtles can become neurologically impaired and develop vacuolations after consuming toxic Hydrilla colonized with the UCB. The southeastern United States, where outbreaks of VM occur regularly and where vegetation colonized by the UCB is common, is also a global hotspot of freshwater turtle diversity. Our results suggest that further investigations into the effect of the putative UCB toxin on wild turtles in situ are warranted.

Supplementary Table S2. Supplementary data for CNA discovery in canine MCs by aCGH and WGS analyses, including CNA result summary, amplified/deleted gene list and chimeric sequence reads related to the superamplicon.