Stromal Expression Profiling Reveals Immune-Driven Adaption to Malignancy in Canine Melanoma Subtypes.

Canine mucosal melanoma (CMM) is the most common oral malignancy in dogs and is significantly more aggressive than its cutaneous counterpart (CCM), yet the reasons for this disparity remain unclear. Cancer-associated stroma (CAS) plays a crucial role in tumour progression, but a detailed understanding of CAS in canine melanoma is missing. To assess stromal reprogramming, we analysed CAS from 21 CMM, 14 CCM and normal stroma from 10 skin and 9 oral mucosa samples by laser-capture microdissection followed by RNA sequencing. Results were assessed in relation to subtypes, prognostic factors including mitotic count (MC), ulceration, necrosis, pigmentation and immune cell infiltration (CD3, CD20 and CD68), scored using immunohistochemistry and RNA in situ hybridisation. Stromal reprogramming was evident in both subtypes but significantly more pronounced in CMM. Immune-excluded tumours exhibited higher MC than desert/cold ones. MC strongly correlated with genes associated with B-cells, T-helper cells and CTLA4 in CCM, suggesting CAS reprogramming to depend on tumour malignancy. Finally, we identify an immune-suppressive stromal signature in a subset of CMM characterised by the downregulation of key immune checkpoints and pathways. Together, these findings provide a solid foundation for understanding the role of CAS in canine melanoma, specific to cutaneous and mucosal subtypes.

Bovine papillomavirus gene expression and inflammatory pathway activation vary between equine sarcoid tumour subtypes.

Equine sarcoids are common non-metastasising skin tumours in horses, associated with bovine papillomavirus (BPV) infection. Six subtypes are recognised (occult, verrucose, nodular, fibroblastic, mixed and malevolent lesions), with variable clinical behaviour. The pathophysiology underlying varying tumour phenotype is poorly understood, and previous data on associations with viral load have been conflicting. To better understand this clinical variation, we investigated associations between tumour subtype and viral load, viral early protein gene expression, and expression of 10 host genes by quantitative polymerase chain reaction in 27 sarcoids and 5 normal skin samples. Viral DNA copy number did not differ between subtypes but was significantly higher in animals with fewer tumours. Expression of BPV E2 and E6 was higher in occult lesions compared to fibroblastic or nodular lesions, while E5 expression was higher in previously-treated lesions. Of the host genes, only IL6 and IL1B differed between subtypes, with higher expression in fibroblastic lesions, while IL10 and CCL5 were elevated compared to skin in all lesion types, and elevations in TNF and TGFB1 were significant for occult lesions only. Expression of TLR9, ATR, VEGFA and PTGS2 in sarcoids was not significantly different from normal skin, suggesting differences between BPV and human papillomavirus tumorigenesis. Results for BPV viral load and gene expression differed from previous reports and are insufficient to explain the spectrum of tumour phenotypes. Activation of both pro-inflammatory and anti-inflammatory immune pathways in sarcoids could influence tumour growth and effective immune responses, and the contribution of specific infiltrating immune cells requires further investigation.

One-Year Outcome of an Ongoing Pre-Clinical Growing Animal Model for a Tissue-Engineered Valved Pulmonary Conduit.

Objectives: A self-constructed valved pulmonary conduit made out of a de-cellularized porcine small intestinal submucosal extracellular matrix biological scaffold was tested in a chronic growing lamb model. Methods: The conduit was implanted in pulmonary valve position in 19 lambs. We monitored clinical, laboratory, and echocardiographic findings until 12 months after surgery. In two animals, euthanasia was planned at nine and twelve months. Pre-mortem chest computed tomography and post-mortem pathologic work up were performed. Data are presented as frequency and percentage, median and range, or mean and standard deviation. Results: Twelve (63.2%) animals survived the perioperative period. Three unexpected deaths occurred during the follow-up period: one due to aspiration pneumonia at 23 days after surgery, and two due to early and late infective endocarditis of the conduit at 18 and 256 days. In the two animals with planned scarification, the pre-mortem CT scan revealed mild or no calcification within the conduit or valve leaflets. In the echocardiographic examination at 12 months, peak and mean systolic pressure gradients across the conduit valve were 6.5 (3-21) mmHg and 3 (2-12) mmHg, while valve regurgitation was none (n = 2), trivial (n = 5), moderate (n = 1), or severe (n = 1). No clinical or laboratory signs of hemolysis were seen. After 12 months of follow-up, the animals' body weights had increased from 33 (27-38) kg to 53 (38-66) kg (p = 0.010). Conclusions: Implantation of a valved pulmonary conduit in our growing lamb model was feasible. Infective endocarditis of the implanted valved conduit remained a significant complication.

Clinical, magnetic resonance imaging, and histological description of a choroid plexus papilloma with disseminated intraventricular and spinal cerebrospinal fluid drop metastases in a young adult dog: a case report.

A 2-year-old male entire Cane Corso was presented for investigations into a 1-week history of ambulatory paraparesis and pelvic limb ataxia gradually deteriorating. Magnetic resonance imaging (MRI) revealed intraventricular space-occupying lesions affecting the fourth ventricle and lateral apertures and intradural-extramedullary space-occupying lesions at the level of C7 vertebra, L4-L5, and L7-S1 intervertebral disk spaces. Due to poor quality of life, the patient was euthanized. A post-mortem examination revealed partially encapsulated, multifocally infiltrative, and moderately cellular neoplastic masses. The histological description was similar for all masses. The cells appeared cuboidal with round central nuclei and a moderate amount of eosinophilic cytoplasm and were arranged almost exclusively in single-layered papilliform patterns supported by a fibrovascular stroma. Mitoses were rarely observed (1/2.37 mm2). The primary neoplasm was morphologically most consistent with a choroid plexus papilloma despite drop metastases. This is the first report of a histologically confirmed primary ventricular choroid plexus papilloma causing disseminated MRI-apparent intraventricular and spinal drop metastases.

Deciphering Stromal Changes between Metastatic and Non-metastatic Canine Mammary Carcinomas.

Cancer-associated stroma (CAS) is widely recognized to influence development and progression of epithelial tumours including breast cancer. Canine mammary tumours (CMTs) such as simple canine mammary carcinomas represent valuable models for human breast cancer also with respect to stromal reprogramming. However, it remains unclear whether and how CAS changes in metastatic tumours compared to non-metastatic ones. To characterize stromal changes between metastatic and non-metastatic CMTs and identify potential drivers of tumour progression, we analysed CAS and matched normal stroma from 16 non-metastatic and 15 metastatic CMTs by RNA-sequencing of microdissected FFPE tissue. We identified 1438 differentially regulated genes between CAS and normal stroma, supporting previous results demonstrating stromal reprogramming in CMTs to be comparable with CAS in human breast cancer and validating deregulation of pathways and genes associated with CAS. Using primary human fibroblasts activated by treatment with TGFß, we demonstrate some of the strongest expression changes to be conserved in fibroblasts across species. Furthermore, we identify 132 differentially expressed genes between CAS from metastatic and non-metastatic tumours, with strong changes in pathways including chemotaxis, regulation of apoptosis, immune response and TGFß signalling and validate deregulation of several targets using RT-qPCR. Finally, we identify specific upregulation of COL6A5, F5, GALNT3, CIT and MMP11 in metastatic CAS, suggesting high stromal expression of these targets to be linked to malignancy and metastasis of CMTs. In summary, our data present a resource supporting further research into stromal changes of the mammary gland in relation to metastasis with implications for both canine and human mammary cancer.

Treatment with IgM-enriched intravenous immunoglobulins enhances clearance of stroke-associated bacterial lung infection.

Post-stroke infection is a common complication of stroke that is associated with poor outcome. We previously reported that stroke induces an ablation of multiple sub-populations of B cells and reduces levels of immunoglobulin M (IgM) antibody, which coincides with the development of spontaneous bacterial pneumonia. The loss of IgM after stroke could be an important determinant of infection susceptibility and highlights this pathway as a target for intervention. We treated mice with a replacement dose of IgM-enriched intravenous immunoglobulin (IgM-IVIg) prior to and 24 h after middle cerebral artery occlusion (MCAO) and allowed them to recover for 2- or 5-day post-surgery. Treatment with IgM-IVIg enhanced bacterial clearance from the lung after MCAO and improved lung pathology but did not impact brain infarct volume. IgM-IVIg treatment induced immunomodulatory effects systemically, including rescue of splenic plasma B cell numbers and endogenous mouse IgM and IgA circulating immunoglobulin concentrations that were reduced by MCAO. Treatment attenuated MCAO-induced elevation of selected pro-inflammatory cytokines in the lung. IgM-IVIg treatment did not increase the number of lung mononuclear phagocytes or directly modulate macrophage phagocytic capacity but enhanced phagocytosis of Staphylococcus aureus bioparticles in vitro. Low-dose IgM-IVIg contributes to increased clearance of spontaneous lung bacteria after MCAO likely via increasing availability of antibody in the lung to enhance opsonophagocytic activity. Immunomodulatory effects of IgM-IVIg treatment may also contribute to reduced levels of damage in the lung after MCAO. IgM-IVIg shows promise as an antibacterial and immunomodulatory agent to use in the treatment of post-stroke infection.

Alzheimer-like pathology in the parietal cortex and hippocampus of aged donkeys.

Neurodegenerative disorders are gaining ever more importance in ageing populations of animals and people. Altered insulin signaling and type II diabetes have been linked to the development of Alzheimer's disease (AD) in humans and AD-like neurodegeneration in other long-lived animals. Donkeys are unusual amongst domestic species for their exceptional longevity and are additionally predisposed to abnormalities of insulin metabolism similar to those found in humans. In this study, the parietal lobe and hippocampus of 13 aged (>30 years) and 2 younger control donkeys were evaluated immunohistologically for the presence, distribution, and frequency of neurofibrillary tangles (NFT) and amyloid plaques (AP); the characteristic lesions of AD. AP were in parietal cortices of 9 donkeys, with a predilection for deep sulci, and NFT-like structures were observed in 7 donkeys, primarily within cortical areas. No changes were observed in the control donkeys. This represents the first identification of both AP and NFT in equids and is a stimulus for future work assessing their metabolic status in parallel.

New World camelids are sentinels for the presence of Borna disease virus.

Borna disease (BD), a frequently fatal neurologic disorder caused by Borna disease virus 1 (BoDV-1), has been observed for decades in horses, sheep, and other mammals in certain regions of Europe. The bicoloured white-toothed shrew (Crocidura leucodon) was identified as a persistently infected species involved in virus transmission. Recently, BoDV-1 attracted attention as a cause of fatal encephalitis in humans. Here, we report investigations on BoDV-1-infected llamas from a farm in a BD endemic area of Switzerland, and alpacas from holdings in a region of Germany where BD was last seen in the 1960s but not thereafter. All New World camelids showed apathy and abnormal behaviour, necessitating euthanasia. Histologically, severe non-suppurative meningoencephalitis with neuronal Joest-Degen inclusion bodies was observed. BoDV-1 was confirmed by immunohistology, RT-qPCR, and sequencing in selected animals. Analysis of the llama herd over 20 years showed that losses due to clinically suspected BD increased within the last decade. BoDV-1 whole-genome sequences from one Swiss llama and one German alpaca and-for comparison-from one Swiss horse and one German shrew were established. They represent the first published whole-genome sequences of BoDV-1 clusters 1B and 3, respectively. Our analysis suggests that New World camelids may have a role as a sentinel species for BoDV-1 infection, even when symptomatic cases are lacking in other animal species.

Pathology and Advanced Imaging-Characterization of a Congenital Cardiac Defect and Complex Hemodynamics in a Pig: A Case Report.

Domestic pigs are widely used in cardiovascular research as the porcine circulatory system bears a remarkable resemblance to that of humans. In order to reduce variability, only clinically healthy animals enter the study as their health status is assessed in entry examination. Like humans, pigs can also suffer from congenital heart disease, such as an atrial septal defect (ASD), which often remains undetected. Due to the malformation of the endocardial cushion during organ development, mitral valve defects (e.g., mitral clefts) are sometimes associated with ASDs, further contributing to hemodynamic instability. In this work, we report an incidental finding of a hemodynamically highly relevant ASD in the presence of incompetent mitral and tricuspid valves, in an asymptomatic, otherwise healthy juvenile pig. In-depth characterization of the cardiac blood flow by four-dimensional (4D) flow magnetic resonance imaging (MRI) revealed a prominent diastolic left-to-right and discrete systolic right-to-left shunt, resulting in a pulmonary-to-systemic flow ratio of 1.8. Severe mitral (15 mL/stroke) and tricuspid (22 mL/stroke) regurgitation further reduced cardiac output. Pathological examination confirmed the presence of an ostium primum ASD and found a serous cyst of lymphatic origin that was filled with clear fluid partially occluding the ASD. A large mitral cleft was identified as the most likely cause of severe regurgitation, and histology showed mild to moderate endocardiosis in the coaptation area of both atrio-ventricular valves. In summary, although not common, congenital heart defects could play a role as a cause of experimental variability or even intra-experimental mortality when working with apparently heathy, juvenile pigs.

Identification of disease-promoting stromal components by comparative proteomic and transcriptomic profiling of canine mammary tumors using laser-capture microdissected FFPE tissue.

Cancer-associated stroma (CAS) profoundly influences progression of tumors including mammary carcinoma (mCA). Canine simple mCA represent relevant models of human mCA, notably also with respect to CAS. While transcriptomic changes in CAS of mCA are well described, it remains unclear to what extent these translate to the protein level. Therefore, we sought to gain insight into the proteomic changes in CAS and compare them with transcriptomic changes in the same tissue. To this end, we analyzed CAS and matched normal stroma using laser-capture microdissection (LCM) and LC-MS/MS in a cohort of 14 formalin-fixed paraffin embedded (FFPE) canine mCAs that we had previously characterized using LCM-RNAseq. Our results reveal clear differences in protein abundance between CAS and normal stroma, which are characterized by changes in the extracellular matrix, the cytoskeleton, and cytokines such as TNF. The proteomics- and RNAseq-based analyses of LCM-FFPE show a substantial degree of correlation, especially for the most deregulated targets and a comparable activation of pathways. Finally, we validate transcriptomic upregulation of LTBP2, IGFBP2, COL6A5, POSTN, FN1, COL4A1, COL12A1, PLOD2, COL4A2, and IGFBP7 in CAS on the protein level and demonstrate their adverse prognostic value for human breast cancer. Given the relevance of canine mCA as a model for the human disease, our analysis substantiates these targets as disease-promoting stromal components with implications for breast cancer in both species.

Contrast-enhanced ultrasound for sentinel lymph node mapping in the routine staging of canine mast cell tumours: A feasibility study.

Canine mast cell tumours (MCTs) typically spread to lymph nodes (LNs) before reaching distant sites, and LN assessment is an important part of MCT staging. Sentinel LN (SLN) mapping techniques to identify draining LNs are being developed and could improve the accuracy of MCT staging. The primary objective of this feasibility study was to determine the safety and effectiveness of contrast-enhanced ultrasound (CEUS) to identify SLNs. Secondary objectives were to determine if the SLNs identified by CEUS coincided with the regional LN predicted by the anatomical lymphosomes, if previous MCT excision altered CEUS SLN findings, and if CEUS could identify MCT nodal metastases. Between June 2017 and March 2019, 59 dogs with 62 MCTs were enrolled. No adverse events related to CEUS were reported. CEUS detected at least 1 SLN in 59/62 MCTs (95.2%, 95% CI: 86.5-99.0%). In only 32/59 (54.2%) MCTs, clinicians would have correctly predicted the SLN(s) identified by CEUS. Among the 35 MCTs that had histological examination of the SLN(s), the prevalence of metastasis was 60% (95% CI: 42.1-76.1%). Additional staging procedures did not reveal any metastases in dogs with histologically non-metastatic SLNs. Integration of CEUS SLN mapping into the routine staging of MCTs is promising, but future studies are required to refine this procedure and to investigate if it would translate into a clinical benefit.

Colony Stimulating Factors in Early Feline Infectious Peritonitis Virus Infection of Monocytes and in End Stage Feline Infectious Peritonitis; A Combined In Vivo And In Vitro Approach.

Feline coronavirus (FCoV) infection initiates monocyte-associated viremia and viral persistence. Virus-infected, -activated monocytes also trigger feline infectious peritonitis (FIP), a fatal systemic disease of felids typified by granulomatous (peri)phlebitis. Currently, the exact mechanisms inducing monocyte activation and FIP are unknown. This study attempted to identify the potential immediate effect of virulent FCoV on colony-stimulating factor (CSF) (granulocyte (G)-CSF, monocyte (M)-CSF and granulocyte-monocyte (GM)-CSF levels through in vitro assessment, alongside prototypical pro- and anti-inflammatory mediators (interleukin (IL)-1, IL-6, IL-12p40, tumor necrosis factor (TNF)-α, and IL-10); this was assessed alongside the in vivo situation in the hemolymphatic tissues of cats euthanized with natural end-stage FIP. For the in vitro work, isolated monocytes from SPF cats were cultured short-term and infected with the FIP virus (FIPV) strain DF2. Mediator transcription was assessed by quantitative reverse transcriptase PCR (RT-qPCR) at 3, 6 and 9 h post infection (hpi), and in the post-mortem samples of bone marrow, spleen, and mesenteric lymph nodes (MLN) of cats with FIP. We observed limited and transient changes in cytokine transcription in monocytes after infection, i.e., a significant increase of IL-6 at 3 hpi and of GM-CSF over the 3 and 6 hpi period, whereas M-CSF was significantly decreased at 9 hpi, with a limited effect of age. The findings indicate that the infection induces expansion of the monocyte/macrophage population, which would ensure the sufficient supply of cells for consistent viral replication. In natural disease, the only upregulation was of G-CSF in the MLN, suggesting either immune exhaustion or an active downregulation by the host as part of its viral response.

The Effect of Natural Feline Coronavirus Infection on the Host Immune Response: A Whole-Transcriptome Analysis of the Mesenteric Lymph Nodes in Cats with and without Feline Infectious Peritonitis.

Feline infectious peritonitis (FIP) is a coronavirus-induced disease of cats, in which the immune system is known to play a crucial, but complex, role in the pathogenesis. This role is still incompletely understood, with involvement of both host and viral factors. To evaluate differential gene expression and pathway involvement in feline coronavirus (FCoV) infection and FIP, we applied next-generation RNA-sequencing of the mesenteric lymph nodes from cats with naturally-acquired FIP, as well as those with systemic FCoV infection without FIP, and those with neither. Viral infection was associated with upregulation of viral defenses regardless of the disease state, but to a greater degree in FIP. FIP was associated with higher pro-inflammatory pathway enrichment, whilst non-FIP FCoV-positive cats showed lower enrichment of humoral immunity pathways, below that of uninfected cats in the case of immunoglobulin production pathways. This host response is presumed to be protective. In FIP, downregulation of T cell-related processes was observed, which did not occur in non-FIP FCoV-positive cats. These results emphasize the importance of the host's immune balance in determining the outcome of the FCoV infection.

Establishing a pre-clinical growing animal model to test a tissue engineered valved pulmonary conduit.

BACKGROUND: Many valvular pathologies of the heart may be only sufficiently treated by replacement of the valve if a reconstruction is not feasible. However, structural deterioration, thrombosis with thromboembolic events and infective endocarditis are commonly encountered complications over time and often demand a re-operation. In congenital heart disease the lack of small diameter valves with the potential to grow poses additional challenges and limits treatment options to homo- or xenograft implants. METHODS: In this study, a chronic sheep model (24 months follow-up), a self-constructed valved conduit was created out of a tissue engineered (TE) patch (CorMatrix® Cardiovascular, Inc, USA) and implanted in orthotopic right ventricular (RV)-pulmonary artery (PA) position. Thereafter, the sheep were regularly monitored by clinical, laboratory and echocardiographic examinations to evaluate cardiac function and the implanted RV-PA-conduit. DISCUSSION: Here, we summarize the study protocol and our experiences during the perioperative phase and the follow up period and explain how we constructed a valved conduit out of a commercially available TE patch. TRIAL REGISTRATION: License number: ZH 284/14.

Differential stromal reprogramming in benign and malignant naturally occurring canine mammary tumours identifies disease-modulating stromal components.

While cancer-associated stroma (CAS) in malignant tumours is well described, stromal changes in benign forms of naturally occurring tumours remain poorly characterized. Spontaneous canine mammary carcinomas (mCA) are viewed as excellent models of human mCA. We have recently reported highly conserved stromal reprogramming between canine and human mCA based on transcriptome analysis of laser-capture-microdissected FFPE specimen. To identify stromal changes between benign and malignant mammary tumours, we have analysed matched normal and adenoma-associated stroma (AAS) from 13 canine mammary adenomas and compared them to previous data from 15 canine mCA. Our analyses reveal distinct stromal reprogramming even in small benign tumours. While similarities between AAS and CAS exist, the stromal signature clearly distinguished adenomas from mCA. The distinction between AAS and CAS is further substantiated by differential enrichment in several hallmark signalling pathways as well as differential abundance in cellular composition. Finally, we identify COL11A1, VIT, CD74, HLA-DRA, STRA6, IGFBP4, PIGR, and TNIP1 as strongly discriminatory stromal genes between adenoma and mCA, and demonstrate their prognostic value for human breast cancer. Given the relevance of canine CAS as a model for the human disease, our approach identifies disease-modulating stromal components with implications for both human and canine breast cancer.

Feline Infectious Peritonitis as a Systemic Inflammatory Disease: Contribution of Liver and Heart to the Pathogenesis.

Feline infectious peritonitis (FIP) is a fatal immune-mediated disease of cats, induced by feline coronavirus (FCoV). A combination of as yet poorly understood host and viral factors combine to cause a minority of FCoV-infected cats to develop FIP. Clinicopathological features include fever, vasculitis, and serositis, with or without effusions; all of which indicate a pro-inflammatory state with cytokine release. As a result, primary immune organs, as well as circulating leukocytes, have thus far been of most interest in previous studies to determine the likely sources of these cytokines. Results have suggested that these tissues alone may not be sufficient to induce the observed inflammation. The current study therefore focussed on the liver and heart, organs with a demonstrated ability to produce cytokines and therefore with huge potential to exacerbate inflammatory processes. The IL-12:IL-10 ratio, a marker of the immune system's inflammatory balance, was skewed towards the pro-inflammatory IL-12 in the liver of cats with FIP. Both organs were found to upregulate mRNA expression of the inflammatory triad of cytokines IL-1ß, IL-6, and TNF-α in FIP. This amplifying step may be one of the missing links in the pathogenesis of this enigmatic disease.

Next-generation RNA sequencing of FFPE subsections reveals highly conserved stromal reprogramming between canine and human mammary carcinoma.

Spontaneous canine simple mammary carcinomas (mCA) are often viewed as models of human mCA. Cancer-associated stroma (CAS) is central for initiation and progression of human cancer, and is likely to play a key role in canine tumours as well. However, canine CAS lacks characterisation and it remains unclear how canine and human CAS compare. Formalin-fixed paraffin embedded (FFPE) tissue constitutes a valuable resource of patient material, but chemical crosslinking has largely precluded its analysis by next-generation RNA sequencing (RNAseq). We have recently established a protocol to isolate CAS and normal stroma from archival FFPE tumours using laser-capture microdissection followed by RNAseq. Using this approach, we have analysed stroma from 15 canine mCA. Our data reveal strong reprogramming of canine CAS. We demonstrate a high-grade molecular homology between canine and human CAS, and show that enrichment of upregulated canine CAS genes strongly correlates with the enrichment of an independently derived human stromal signature in the TCGA breast tumour dataset. Relationships between different gene signatures observed in human breast cancer are largely maintained in the canine model, suggesting a close interspecies similarity in the network of cancer signalling circuitries. Finally, we establish the prognostic potential of the canine CAS signature in human samples, emphasising the relevance of studying canine CAS as a model of the human disease. In conclusion, we provide a proof-of-principle to analyse specific subsections of FFPE tissue by RNAseq, and compare stromal gene expression between human and canine mCA to reveal molecular drivers in CAS supporting tumour growth and malignancy.

κ Light-chain monoclonal gammopathy and cast nephropathy in a horse with multiple myeloma.

CASE DESCRIPTION A 27-year-old Dutch Warmblood mare was evaluated because of a history of lethargy, reluctance to move, weight loss, persistent hyperproteinemia, and recurrent episodes of mild lameness. CLINICAL FINDINGS Hematologic evaluation revealed anemia (RBC concentration, 3.84 × 106 cells/µL), thrombocytopenia (47 × 103 thrombocytes/µL), and hyperproteinemia (total protein concentration, 11.2 g/dL) with hyperglobulinemia and hypoalbuminemia. Results of protein electrophoresis of serum and urine samples indicated a monoclonal gammopathy; the paraprotein was identified as a κ light chain. On abdominal ultrasonographic examination, hypoechoic nodules were visualized in the spleen. Results of cytologic examination of a splenic fine-needle aspirate and histologic examination of a bone marrow biopsy sample were consistent with plasma cell myeloma. TREATMENT AND OUTCOME Treatment was declined owing to the age of the horse and poor prognosis. The horse was discharged from the hospital, and the owner was given palliative care instructions. The horse was euthanized 2 weeks later because of recurrent episodes of lethargy, anorexia, and signs of colic. Necropsy confirmed the diagnosis of multiple (plasma cell) myeloma. Plasma cell aggregates in the liver, spleen, bone marrow, and kidney and the presence of cast nephropathy were identified on histologic examination. CLINICAL RELEVANCE Multiple myeloma is rarely reported in horses. A monoclonal peak on serum protein electrophoresis should raise the suspicion of neoplasia, specifically multiple myeloma. The findings for this patient confirmed the importance of considering neoplasia in horses with nonspecific clinical signs.

An optimised protocol for isolation of RNA from small sections of laser-capture microdissected FFPE tissue amenable for next-generation sequencing.

BACKGROUND: Formalin-fixed paraffin embedded (FFPE) tissue constitutes a vast treasury of samples for biomedical research. Thus far however, extraction of RNA from FFPE tissue has proved challenging due to chemical RNA-protein crosslinking and RNA fragmentation, both of which heavily impact on RNA quantity and quality for downstream analysis. With very small sample sizes, e.g. when performing Laser-capture microdissection (LCM) to isolate specific subpopulations of cells, recovery of sufficient RNA for analysis with reverse-transcription quantitative PCR (RT-qPCR) or next-generation sequencing (NGS) becomes very cumbersome and difficult. METHODS: We excised matched cancer-associated stroma (CAS) and normal stroma from clinical specimen of FFPE canine mammary tumours using LCM, and compared the commonly used protease-based RNA isolation procedure with an adapted novel technique that additionally incorporates a focused ultrasonication step. RESULTS: We successfully adapted a protocol that uses focused ultrasonication to isolate RNA from small amounts of deparaffinised, stained, clinical LCM samples. Using this approach, we found that total RNA yields could be increased by 8- to 12-fold compared to a commonly used protease-based extraction technique. Surprisingly, RNA extracted using this new approach was qualitatively at least equal if not superior compared to the old approach, as Cq values in RT-qPCR were on average 2.3-fold lower using the new method. Finally, we demonstrate that RNA extracted using the new method performs comparably in NGS as well. CONCLUSIONS: We present a successful isolation protocol for extraction of RNA from difficult and limiting FFPE tissue samples that enables successful analysis of small sections of clinically relevant specimen. The possibility to study gene expression signatures in specific small sections of archival FFPE tissue, which often entail large amounts of highly relevant clinical follow-up data, unlocks a new dimension of hitherto difficult-to-analyse samples which now become amenable for investigation.

Analysis of Gene Expression Signatures in Cancer-Associated Stroma from Canine Mammary Tumours Reveals Molecular Homology to Human Breast Carcinomas.

Cancer-associated stroma (CAS) plays a key role in cancer initiation and progression. Spontaneously occurring canine mammary carcinomas are viewed as excellent models of human breast carcinomas. Considering the importance of CAS for human cancer, it likely plays a central role in canine tumours as well. So far, however, canine CAS lacks characterisation, and it remains unclear whether the biology between CAS from canine and human tumours is comparable. In this proof-of-principle study, using laser-capture microdissection, we isolated CAS and normal stroma from 13 formalin-fixed paraffin embedded canine simple mammary carcinomas and analysed the expression of seven known human CAS markers by RT-qPCR (Reverse Transcription quantitative PCR) and validated some targets by immunohistochemistry. We found that Col1a1 (Collagen1α1), αSMA (alpha Smooth Muscle Actin), FAP (Fibroblast activation protein), PDGFRß (Platelet-derived growth factor receptor beta), and Caveolin-1 were significantly upregulated in canine CAS, and the expression of CXCL12 (Stromal cell derived factor 1) significantly decreased, whereas MMP2 (Matrix Metalloproteinase 1) and IL6 (Interleukin 6) did not change. Our results suggest strong similarities in CAS biology in canine and human mammary carcinomas but also reveal some differences. To the best of our knowledge, this is the first report to provide a comprehensive expression analysis of the most important CAS markers in canine simple mammary carcinomas and further supports the validity of the dog as model for human cancer.