International Guidelines for Veterinary Tumor Pathology: A Call to Action.

Standardization of tumor assessment lays the foundation for validation of grading systems, permits reproducibility of oncologic studies among investigators, and increases confidence in the significance of study results. Currently, there is minimal methodological standardization for assessing tumors in veterinary medicine, with few attempts to validate published protocols and grading schemes. The current article attempts to address these shortcomings by providing standard guidelines for tumor assessment parameters and protocols for evaluating specific tumor types. More detailed information is available in the Supplemental Files, the intention of which is 2-fold: publication as part of this commentary, but more importantly, these will be available as "living documents" on a website (www.vetcancerprotocols.org), which will be updated as new information is presented in the peer-reviewed literature. Our hope is that veterinary pathologists will agree that this initiative is needed, and will contribute to and utilize this information for routine diagnostic work and oncologic studies. Journal editors and reviewers can utilize checklists to ensure publications include sufficient detail and standardized methods of tumor assessment. To maintain the relevance of the guidelines and protocols, it is critical that the information is periodically updated and revised as new studies are published and validated with the intent of providing a repository of this information. Our hope is that this initiative (a continuation of efforts published in this journal in 2011) will facilitate collaboration and reproducibility between pathologists and institutions, increase case numbers, and strengthen clinical research findings, thus ensuring continued progress in veterinary oncologic pathology and improving patient care.

Understanding the Efficiency of Splenic Hemangiosarcoma Diagnosis Using Monte Carlo Simulations.

Hemangiosarcoma is a common neoplasm of the spleen in older dogs. However, diagnosis is complicated by necrosis and hemorrhage, which can mimic a number of benign processes. Currently, there is no consensus about the number of sections pathologists should examine to rule out hemangiosarcoma. To answer this question, we examined 413 histopathologic sections from 50 cases of canine hemangiosarcoma (mean: 8.1 sections per case; range, 5-14). Each section had the presence or absence of hemangiosarcoma determined by 2 board-certified anatomic pathologists. Then, 100 Monte Carlo simulations were performed, randomly selecting sections from each case 10 000 times and the results averaged. These simulations suggest that examination of 5 sections from a spleen with hemangiosarcoma yields a 95.02% chance of diagnosing hemangiosarcoma, while examination of 10 sections yields a 98.59% chance of diagnosis when hemangiosarcoma is in fact present. The data emphasize the need to submit the entire spleen for histopathologic examination in suspected cases of hemangiosarcoma and suggest that 5 sections obtained by a trained individual are likely sufficient for diagnosis.

Laparoscopic access to the liver and application of laparoscopic microwave ablation in 2 dogs with liver neoplasia.

OBJECTIVE: To determine laparoscopic accessibility of liver lobes and access to the hilus and describe laparoscopic microwave ablation (LMWA) in 2 dogs with hepatic neoplasia. STUDY DESIGN: Experimental/descriptive case series. SAMPLE POPULATION: Six canine cadavers and 2 clinical dogs. METHODS: Cadavers in dorsal recumbency underwent laparoscopic assessment of the liver. A 17 gauge MWA probe was inserted to create an ablation zone at the most proximal aspect of the hilus. The distance from the center of each ablation zone to the most proximal aspect of the corresponding hilus was determined. Two dogs with hepatic neoplasia underwent LMWA. RESULTS: All lobes of the canine liver were accessible via laparoscopy. The median (interquartile range) distances from the ablation zones to the hilus for the caudate process, left lateral, left middle, quadrate, right lateral, and right middle lobes were 2.2 (1.2-2.6), 2.1 (1-4.4), 1.5 (1.4-3.7), 2, 1, 2.5 (1-4.1) cm, respectively. Histopathologic diagnoses treated by LMWA included metastatic hemangiosarcoma and primary hepatocellular carcinoma. Laparoscopic microwave ablation was technically feasible, and no complications from the procedure resulted. CONCLUSION: Laparoscopic access to the hilus of each liver lobe is possible via a ventrodorsal approach provided reverse Trendelenburg and lateral rotation is used, especially for the right lateral lobe. Laparoscopic microwave ablation is feasible in some dogs with hepatic neoplasia. The indications for and efficacy of LMWA for hepatic neoplasia in dogs requires additional investigation. CLINICAL SIGNIFICANCE: Laparoscopic access to all liver lobes and MWA of some neoplastic lesions is feasible in a canine pilot study.

Superinfection Exclusion of the Ruminant Pathogen Anaplasma marginale in Its Tick Vector Is Dependent on the Time between Exposures to the Strains.

UNLABELLED: The remarkable genetic diversity of vector-borne pathogens allows for the establishment of superinfection in the mammalian host. To have a long-term impact on population strain structure, the introduced strains must also be transmitted by a vector population that has been exposed to the existing primary strain. The sequential exposure of the vector to multiple strains frequently prevents establishment of the second strain, a phenomenon termed superinfection exclusion. As a consequence, superinfection exclusion may greatly limit genetic diversity in the host population, which is difficult to reconcile with the high degree of genetic diversity maintained among vector-borne pathogens. Using Anaplasma marginale, a tick-borne bacterial pathogen of ruminants, we hypothesized that superinfection exclusion is temporally dependent and that longer intervals between strain exposures allow successful acquisition and transmission of a superinfecting strain. To test this hypothesis, we sequentially exposed Dermacentor andersoni ticks to two readily tick-transmissible strains of A. marginale The tick feedings were either immediately sequential or 28 days apart. Ticks were allowed to transmission feed and were individually assessed to determine if they were infected with one or both strains. The second strain was excluded from the tick when the exposure interval was brief but not when it was prolonged. Midguts and salivary glands of individual ticks were superinfected and transmission of both strains occurred only when the exposure interval was prolonged. These findings indicate that superinfection exclusion is temporally dependent, which helps to account for the differences in pathogen strain structure in tropical compared to temperate regions. IMPORTANCE: Many vector-borne pathogens have marked genetic diversity, which influences pathogen traits such as transmissibility and virulence. The most successful strains are those that are preferentially transmitted by the vector. However, the factors that determine successful transmission of a particular strain are unknown. In the case of intracellular, bacterial, tick-borne pathogens, one potential factor is superinfection exclusion, in which colonization of ticks by the first strain of a pathogen it encounters prevents the transmission of a second strain. Using A. marginale, the most prevalent tick-borne pathogen of cattle worldwide, and its natural tick vector, we determined that superinfection exclusion occurs when the time between exposures to two strains is brief but not when it is prolonged. These findings suggest that superinfection exclusion may influence strain transmission in temperate regions, where tick activity is limited by season, but not in tropical regions, where ticks are active for long periods.

Keeping Pathologists in the Loop and an Adaptive F1-Score Threshold Method for Mitosis Detection in Canine Perivascular Wall Tumours.

Performing a mitosis count (MC) is the diagnostic task of histologically grading canine Soft Tissue Sarcoma (cSTS). However, mitosis count is subject to inter- and intra-observer variability. Deep learning models can offer a standardisation in the process of MC used to histologically grade canine Soft Tissue Sarcomas. Subsequently, the focus of this study was mitosis detection in canine Perivascular Wall Tumours (cPWTs). Generating mitosis annotations is a long and arduous process open to inter-observer variability. Therefore, by keeping pathologists in the loop, a two-step annotation process was performed where a pre-trained Faster R-CNN model was trained on initial annotations provided by veterinary pathologists. The pathologists reviewed the output false positive mitosis candidates and determined whether these were overlooked candidates, thus updating the dataset. Faster R-CNN was then trained on this updated dataset. An optimal decision threshold was applied to maximise the F1-score predetermined using the validation set and produced our best F1-score of 0.75, which is competitive with the state of the art in the canine mitosis domain.

Telangiectatic osteosarcoma in four dogs: Cytologic, histopathologic, cytochemical, and immunohistochemical findings.

Telangiectatic osteosarcoma is a rare variant of osteosarcoma histologically and clinically similar to hemangiosarcoma (HSA). This case series describes the imaging and cytologic features of four histologically confirmed telangiectatic osteosarcomas, including the use of cytochemical stains. Alkaline phosphatase (ALP) was applied to Wright-Giemsa-stained cytology slides, and Factor VIII immunohistochemistry was evaluated. Cytologic characteristics included atypical mesenchymal cells with evidence of acute and chronic hemorrhage. Telangiectatic osteosarcoma cases had positive ALP cytochemical staining, while control HSA cases were negative. Factor VIII immunohistochemistry was negative in telangiectatic osteosarcoma and positive in HSA. Cytologic diagnosis of telangiectatic osteosarcoma with positive ALP cytochemical staining can help differentiate this neoplasm from HSA.

Automated Nuclear Morphometry: A Deep Learning Approach for Prognostication in Canine Pulmonary Carcinoma to Enhance Reproducibility.

The integration of deep learning-based tools into diagnostic workflows is increasingly prevalent due to their efficiency and reproducibility in various settings. We investigated the utility of automated nuclear morphometry for assessing nuclear pleomorphism (NP), a criterion of malignancy in the current grading system in canine pulmonary carcinoma (cPC), and its prognostic implications. We developed a deep learning-based algorithm for evaluating NP (variation in size, i.e., anisokaryosis and/or shape) using a segmentation model. Its performance was evaluated on 46 cPC cases with comprehensive follow-up data regarding its accuracy in nuclear segmentation and its prognostic ability. Its assessment of NP was compared to manual morphometry and established prognostic tests (pathologists' NP estimates (n = 11), mitotic count, histological grading, and TNM-stage). The standard deviation (SD) of the nuclear area, indicative of anisokaryosis, exhibited good discriminatory ability for tumor-specific survival, with an area under the curve (AUC) of 0.80 and a hazard ratio (HR) of 3.38. The algorithm achieved values comparable to manual morphometry. In contrast, the pathologists' estimates of anisokaryosis resulted in HR values ranging from 0.86 to 34.8, with slight inter-observer reproducibility (k = 0.204). Other conventional tests had no significant prognostic value in our study cohort. Fully automated morphometry promises a time-efficient and reproducible assessment of NP with a high prognostic value. Further refinement of the algorithm, particularly to address undersegmentation, and application to a larger study population are required.

Multiple biliary duct hamartomas in a cat resulting in a hepatic mass: A case report.

A 13-year-old, male neutered domestic short-haired cat was diagnosed with multiple biliary duct hamartomas after liver lobectomy for a suspected malignant hepatic mass. Distinguishing ultrasonographic findings included a lobular, mostly well-defined, heterogeneous, predominantly hyperechoic, left hepatic mass. Computed tomography (CT) confirmed the presence of a lobular, well-defined, fluid to soft tissue attenuating, heterogeneously hypoenhancing left divisional hepatic mass. Grossly, a large left sided multilobular pale pink gelatinous hepatic mass was surgically excised. Histopathologically, the mass was composed of irregular cystic spaces lined by cuboidal epithelium and separated by mature regular fibrous tissue. Three months following surgery there was no evidence of recurrence or progression of disease on repeat abdominal ultrasound (AUS).

Detection of Necrosis in Digitised Whole-Slide Images for Better Grading of Canine Soft-Tissue Sarcomas Using Machine-Learning.

The definitive diagnosis of canine soft-tissue sarcomas (STSs) is based on histological assessment of formalin-fixed tissues. Assessment of parameters, such as degree of differentiation, necrosis score and mitotic score, give rise to a final tumour grade, which is important in determining prognosis and subsequent treatment modalities. However, grading discrepancies are reported to occur in human and canine STSs, which can result in complications regarding treatment plans. The introduction of digital pathology has the potential to help improve STS grading via automated determination of the presence and extent of necrosis. The detected necrotic regions can be factored in the grading scheme or excluded before analysing the remaining tissue. Here we describe a method to detect tumour necrosis in histopathological whole-slide images (WSIs) of STSs using machine learning. Annotated areas of necrosis were extracted from WSIs and the patches containing necrotic tissue fed into a pre-trained DenseNet161 convolutional neural network (CNN) for training, testing and validation. The proposed CNN architecture reported favourable results, with an overall validation accuracy of 92.7% for necrosis detection which represents the number of correctly classified data instances over the total number of data instances. The proposed method, when vigorously validated represents a promising tool to assist pathologists in evaluating necrosis in canine STS tumours, by increasing efficiency, accuracy and reducing inter-rater variation.

Mitosis domain generalization in histopathology images - The MIDOG challenge.

The density of mitotic figures (MF) within tumor tissue is known to be highly correlated with tumor proliferation and thus is an important marker in tumor grading. Recognition of MF by pathologists is subject to a strong inter-rater bias, limiting its prognostic value. State-of-the-art deep learning methods can support experts but have been observed to strongly deteriorate when applied in a different clinical environment. The variability caused by using different whole slide scanners has been identified as one decisive component in the underlying domain shift. The goal of the MICCAI MIDOG 2021 challenge was the creation of scanner-agnostic MF detection algorithms. The challenge used a training set of 200 cases, split across four scanning systems. As test set, an additional 100 cases split across four scanning systems, including two previously unseen scanners, were provided. In this paper, we evaluate and compare the approaches that were submitted to the challenge and identify methodological factors contributing to better performance. The winning algorithm yielded an F1 score of 0.748 (CI95: 0.704-0.781), exceeding the performance of six experts on the same task.

Comparative genomics and transcriptomics of trait-gene association.

BACKGROUND: The Order Rickettsiales includes important tick-borne pathogens, from Rickettsia rickettsii, which causes Rocky Mountain spotted fever, to Anaplasma marginale, the most prevalent vector-borne pathogen of cattle. Although most pathogens in this Order are transmitted by arthropod vectors, little is known about the microbial determinants of transmission. A. marginale provides unique tools for studying the determinants of transmission, with multiple strain sequences available that display distinct and reproducible transmission phenotypes. The closed core A. marginale genome suggests that any phenotypic differences are due to single nucleotide polymorphisms (SNPs). We combined DNA/RNA comparative genomic approaches using strains with different tick transmission phenotypes and identified genes that segregate with transmissibility. RESULTS: Comparison of seven strains with different transmission phenotypes generated a list of SNPs affecting 18 genes and nine promoters. Transcriptional analysis found two candidate genes downstream from promoter SNPs that were differentially transcribed. To corroborate the comparative genomics approach we used three RNA-seq platforms to analyze the transcriptomes from two A. marginale strains with different transmission phenotypes. RNA-seq analysis confirmed the comparative genomics data and found 10 additional genes whose transcription between strains with distinct transmission efficiencies was significantly different. Six regions of the genome that contained no annotation were found to be transcriptionally active, and two of these newly identified transcripts were differentially transcribed. CONCLUSIONS: This approach identified 30 genes and two novel transcripts potentially involved in tick transmission. We describe the transcriptome of an obligate intracellular bacterium in depth, while employing massive parallel sequencing to dissect an important trait in bacterial pathogenesis.

RNA in situ hybridisation as a molecular diagnostic technique targeting IBA-1 and CD204 in canine histiocytic sarcoma.

BACKGROUND: Canine histiocytic sarcoma (HS) is an aggressive cancer with morphologically variable features; therefore, obtaining a definitive diagnosis can be challenging. Two proteins, IBA-1, ionised calcium-binding adapter molecule 1, and CD204, a macrophage scavenger receptor, have been shown to be specific immunohistochemical markers helpful in distinguishing HS from other tumour types with similar morphological features. OBJECTIVES: This study was performed to demonstrate the use of RNA in situ hybridisation (ISH) technology allowing single-molecule RNA visualisation in formalin-fixed paraffin-embedded (FFPE) tissues as a molecular tool for the diagnosis of canine HS. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis for IBA-1 and CD204 were performed to correlate gene expression and protein expression of these two markers in the histiocytic sarcoma DH82 cell line. RNA-ISH for IBA-1 and CD204 was performed on the DH82 cell line to validate the RNA-ISH probes. RNA-ISH and immunohistochemistry (IHC) were performed in clinical HS FFPE samples to demonstrate mRNA and protein expression of IBA-1 and CD204. FFPE archived samples of canine round cell tumours, melanoma and anaplastic sarcoma were used as negative controls. RESULTS: RNA-ISH and IHC showed moderate to strong expression for IBA-1 and CD204 in the neoplastic cells in both the canine DH82 cell line and the archived canine HS samples. RNA-ISH and IHC showed scattered positive staining in the control tumours samples, consistent with macrophagic infiltration. CONCLUSION: RNA-ISH for CD204 and IBA-1 appeared to have a high specificity and sensitivity in our samples and may be an additional valuable diagnostic technique in identifying HS.

Deep learning for necrosis detection using canine perivascular wall tumour whole slide images.

Necrosis seen in histopathology Whole Slide Images is a major criterion that contributes towards scoring tumour grade which then determines treatment options. However conventional manual assessment suffers from inter-operator reproducibility impacting grading precision. To address this, automatic necrosis detection using AI may be used to assess necrosis for final scoring that contributes towards the final clinical grade. Using deep learning AI, we describe a novel approach for automating necrosis detection in Whole Slide Images, tested on a canine Soft Tissue Sarcoma (cSTS) data set consisting of canine Perivascular Wall Tumours (cPWTs). A patch-based deep learning approach was developed where different variations of training a DenseNet-161 Convolutional Neural Network architecture were investigated as well as a stacking ensemble. An optimised DenseNet-161 with post-processing produced a hold-out test F1-score of 0.708 demonstrating state-of-the-art performance. This represents a novel first-time automated necrosis detection method in the cSTS domain as well specifically in detecting necrosis in cPWTs demonstrating a significant step forward in reproducible and reliable necrosis assessment for improving the precision of tumour grading.

Superinfection as a driver of genomic diversification in antigenically variant pathogens.

A new pathogen strain can penetrate an immune host population only if it can escape immunity generated against the original strain. This model is best understood with influenza viruses, in which genetic drift creates antigenically distinct strains that can spread through host populations despite the presence of immunity against previous strains. Whether this selection model for new strains applies to complex pathogens responsible for endemic persistent infections, such as anaplasmosis, relapsing fever, and sleeping sickness, remains untested. These complex pathogens undergo rapid within-host antigenic variation by using sets of chromosomally encoded variants. Consequently, immunity is developed against a large repertoire of variants, dramatically changing the scope of genetic change needed for a new strain to evade existing immunity and establish coexisting infection, termed strain superinfection. Here, we show that the diversity in the alleles encoding antigenic variants between strains of a highly antigenically variant pathogen was equal to the diversity within strains, reflecting equivalent selection for variants to overcome immunity at the host population level as within an individual host. This diversity among strains resulted in expression of nonoverlapping variants that allowed a new strain to evade immunity and establish superinfection. Furthermore, we demonstrated that a single distinct allele allows strain superinfection. These results indicate that there is strong selective pressure to increase the diversity of the variant repertoire beyond what is needed for persistence within an individual host and provide an explanation, competition at the host population level, for the large genomic commitment to variant gene families in persistent pathogens.

SDHB and SDHA Immunohistochemistry in Canine Pheochromocytomas.

Pheochromocytomas (PCs) are tumors arising from the chromaffin cells of the adrenal glands and are the most common tumors of the adrenal medulla in animals. In people, these are highly correlated to inherited gene mutations in the succinate dehydrogenase (SDH) pathway; however, to date, little work has been done on the genetic basis of these tumors in animals. In humans, immunohistochemistry has proven valuable as a screening technique for SDH mutations. Human PCs that lack succinate dehydrogenase B (SDHB) immunoreactivity have a high rate of mutation in the SDH family of genes, while human PCs lacking succinate dehydrogenase A (SDHA) immunoreactivity have mutations in the SDHA gene. To determine if these results are similar for dogs, we performed SDHA and SDHB immunohistochemistry on 35 canine formalin-fixed, paraffin-embedded (FFPE) PCs. Interestingly, there was a loss of immunoreactivity for both SDHA and SDHB in four samples (11%), suggesting a mutation in SDHx including SDHA. An additional 25 (71%) lacked immunoreactivity for SDHB, while retaining SDHA immunoreactivity. These data suggest that 29 out of the 35 (82%) may have an SDH family mutation other than SDHA. Further work is needed to determine if canine SDH immunohistochemistry on PCs correlates to genetic mutations that are similar to human PCs.

Primary Hyperparathyroidism in a Quarter Horse Mare Associated With a Chief Cell Adenoma.

Primary hyperparathyroidism is rare in large animal species, and little is known regarding its pathophysiology, endocrine and electrolyte derangements, diagnosis, medical management, and prognosis. This report describes the clinicopathologic diagnosis of a parathyroid (PT) gland chief cell adenoma in a 12-year-old Quarter Horse mare, including PT hormone (PTH) and electrolyte disarrangements associated with the neoplasia, the surgical removal of the adenoma, and medical management of the case. This report also describes for the first time the use PTH immunohistochemistry to confirm the nature of this neoplasia in a horse.

Feasibility and efficacy of ultrasonographic and laparoscopic guidance for microwave ablation of clinically normal canine ovaries.

OBJECTIVE: To determine the optimal energy profile for and to assess the feasibility and efficacy of ultrasonographic and laparoscopic guidance for microwave ablation (MWA) of clinically normal canine ovaries. SAMPLE: 44 extirpated ovaries from 22 healthy dogs. PROCEDURES: In the first of 2 trials, 13 dogs underwent oophorectomy by routine laparotomy. Extirpated ovaries underwent MWA at 45 W for 60 (n = 11) or 90 (12) seconds; 3 ovaries did not undergo MWA and served as histologic controls. Ovaries were histologically evaluated for cell viability. Ovaries without viable cells were categorized as completely ablated. Histologic results were used to identify the optimal MWA protocol for use in the subsequent trial. In the second trial, the ovaries of 9 dogs underwent MWA at 45 W for 90 seconds in situ. Ultrasonographic guidance for MWA was deemed unfeasible after evaluation of 1 ovary. The remaining 17 ovaries underwent MWA with laparoscopic guidance, after which routine laparoscopic oophorectomy was performed. Completeness of ablation was histologically assessed for all ovaries. RESULTS: 2 ovaries were excluded from the trial 1 analysis because of equivocal cell viability. Six of 11 ovaries and 10 of 10 ovaries that underwent MWA for 60 and 90 seconds, respectively, were completely ablated. In trial 2, laparoscopic-guided MWA resulted in complete ablation for 12 of 17 ovaries. Dissection of the ovarian bursa for MWA probe placement facilitated complete ablation. CONCLUSIONS AND CLINICAL RELEVANCE: Laparoscopic-guided MWA at 45 W for 90 seconds was feasible, safe, and effective for complete ablation of clinically normal ovaries in dogs.

Conservation in the face of diversity: multistrain analysis of an intracellular bacterium.

BACKGROUND: With the recent completion of numerous sequenced bacterial genomes, notable advances have been made in understanding the level of conservation between various species. However, relatively little is known about the genomic diversity among strains. We determined the complete genome sequence of the Florida strain of Anaplasma marginale, and near complete (>96%) sequences for an additional three strains, for comparative analysis with the previously fully sequenced St. Maries strain genome. RESULTS: These comparisons revealed that A. marginale has a closed-core genome with few highly plastic regions, which include the msp2 and msp3 genes, as well as the aaap locus. Comparison of the Florida and St. Maries genome sequences found that SNPs comprise 0.8% of the longer Florida genome, with 33.5% of the total SNPs between all five strains present in at least two strains and 3.0% of SNPs present in all strains except Florida. Comparison of genomes from three strains of Mycobacterium tuberculosis, Bacillus anthracis, and Nessieria meningiditis, as well as four Chlamydophila pneumoniae strains found that 98.8%-100% of SNPs are unique to each strain, suggesting A. marginale, with 76.0%, has an intermediate level of strain-specific SNPs. Comparison of genomes from other organisms revealed variation in diversity that did not segregate with the environmental niche the bacterium occupies, ranging from 0.00% to 8.00% of the larger pairwise-compared genome. CONCLUSION: Analysis of multiple A. marginale strains suggests intracellular bacteria have more variable SNP retention rates than previously reported, and may have closed-core genomes in response to the host organism environment and/or reductive evolution.

Complete Urogenital and Anorectal Duplication in a Dog.

A 10-week-old sexually intact female golden retriever was evaluated for two functional anal openings and a bipartite vulva. Examination revealed haired skin between two separate anatomically smaller anal openings. On rectal palpation, a soft tissue septum (5 cm) with a mucosal surface between the two anal openings was palpated. In addition, circumferential rectal musculature was not appreciated on the ventral aspect. Urogenital evaluation revealed duplication of the vestibule and vagina with a complete centrally located septum extending dorsoventrally. Computed tomography (CT) of the abdomen and pelvis, vaginocystourethrogram, and colonogram were performed. Complete bifurcation of the urinary bladder with duplication of the urethra, cervix, and vaginal canal was noted. Approximately 2 cm from the rectum, there was a similar bifurcation that converged the colon into two rectal portions and separate anal openings. The owner was counseled on the severity of congenital malformations and a high likelihood of aging-related developmental complications in the future. The owner elected humane euthanasia and a necropsy was performed to confirm the malformations.