Successful definitive radiation treatment of refractory canine oral papillomatosis.

This case report describes a three-year-old male intact border collie diagnosed with canine papillomavirus type 1 (CPV-1+) oral papillomas resistant to standard-of-care. With time, he developed lesions consistent with squamous cell carcinoma. Malignant tumors were incompletely excised and treated with definitive external beam radiation therapy (45 Gy, 3 Gy × 15 daily). The remaining oral cavity received 27 Gy (1.8 Gy x 15 daily) to treat the disseminated oral papillomatosis. A temporary treatment delay of 2 weeks was instituted due to grade 3 mucositis. The patient remained in complete remission after 10 months from radiotherapy. No tumor recurrences were noted by the owners after >1 year from treatment.

Cytokine production and the effects of oclacitinib in three canine mast cell tumour cell lines.

BACKGROUND: Cytokines are capable of manipulating the tumour microenvironment supporting tumour growth. Interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1, shown to be produced by various tumours, can negatively affect prognosis. The production of cytokines by canine mast cell tumours (MCT) has not been reported. HYPOTHESIS/OBJECTIVES: We hypothesise that MCT cell lines produce IL-8 and/or MCP-1 in addition to other cytokines, and that their production can be modulated by the Janus kinase (JAK) inhibitor oclacitinib. This pilot study aims to investigate the production of IL-8, MCP-1 and nine additional cytokines in three canine MCT cell lines, and determine the effects of oclacitinib on their production. METHODS AND MATERIALS: Reverse transcriptase-PCR was used to detect the expression of IL-8 and MCP-1 mRNA in three MCT cell lines (CoMS, CM-MC1 and VI-MC1). The supernatant of the cell lines was evaluated for the presence of 11 cytokines [IL-2, -6, -7, -8, -10, -15 and -18, and MCP-1, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)γ and tumour necrosis factor (TNF)α] by enzyme-linked immunosorbent assay (ELISA). The IC50 of oclacitinib was identified for each cell line. ELISA was performed again to compare changes in IL-8 and MCP-1 in treated cell lines versus untreated controls. RESULTS: Interleukin-8 and MCP-1 were produced by all MCT cell lines tested. Oclacitinib significantly decreased the release of IL-8 in the CoMS cell line and of MCP-1 in CoMS and VI-MC1 in clinically relevant concentrations. Furthermore, oclacitinib significantly decreased the proliferation of all three cell lines. CONCLUSIONS: Interleukin-8 and MCP-1 are produced by canine MCT cell lines. Modulation of their production is possible with oclacitinib.

Outcome of intensity-modulated radiation therapy-based stereotactic radiation therapy for treatment of canine nasal carcinomas.

Stereotactic radiation therapy (SRT) has emerged as a convenient definitive treatment modality in veterinary medicine, but few studies exist evaluating outcome with treatment for canine nasal tumors, and no studies report the treatment of one single tumor histotype. This retrospective, observational study evaluates toxicity, response, and survival in 17 dogs with nasal carcinomas treated with SRT. Dogs received a median of 3000 centigray in three fractions via 6-MV linear accelerator. Eighty-eight percent of patients (n = 15) demonstrated clinical benefit. Of dogs with repeated CT imaging (n = 10), 60% (n = 6) achieved a partial response and 10% (n = 1) achieved a complete response. Median progression-free survival (PFS) was 359 days. Median survival time (MST) was 563 days. Among dogs evaluable for acute toxicity, 50% (n = 10) developed low grade toxicity (grade 1, n = 4; grade 2, n = 1). No patients developed grade 3 toxicity. 16 dogs (87%) evaluable over the long term developed signs consistent with possible late toxicity. The majority of late toxicities were mild (alopecia, hyperpigmentation, and leukotrichia n = 10; ocular discharge and keratoconjunctivitis sicca n = 5). Thirty-seven percent of patients (n = 6) developed seven possible grade 3 late toxicities (blindness, n = 3; fistula, n = 1; seizures, n = 3), which were difficult to distinguish from progressive disease in most patients. Of the prognostic factors evaluated (demographics, tumor stage, dosimetric data, epistaxis, facial deformity, clinical response, image-based response, nonsteroidal anti-inflammatory drugs, and chemotherapy), only clinical response was a positive prognostic factor on MST (P < .00). No factors were found to be significantly associated with PFS.

Multimodality treatment including ONCEPT for canine oral melanoma: A retrospective analysis of 131 dogs.

Canine oral melanoma (OM) is an aggressive cancer with a high rate of metastasis. Surgery and/or radiotherapy (RT) are effective local treatments, yet many dogs succumb to distant metastasis. Immunotherapy represents an attractive strategy for this potentially immunogenic tumor. The objective of this multi-institutional retrospective study was to examine the clinical outcome of dogs with OM treated with ONCEPT melanoma vaccine. Most dogs also underwent surgery and/or RT (8 Gy × four weekly fractions). Dogs with distant metastasis at diagnosis and those receiving concurrent chemotherapy were excluded. One hundred thirty-one dogs treated with ONCEPT were included: 62 had adequate local tumor control defined as complete tumor excision or irradiation of residual microscopic disease; 15 were treated in the microscopic disease setting following an incomplete excision without adjuvant RT; and 54 had gross disease. Median time to progression, median progression-free survival, and median tumor-specific overall survival were 304, 260, and 510 days, respectively. In multivariable analysis, presence of gross disease correlated negatively with all measures of clinical outcome. Other negative prognostic indicators were primary tumor ≥2 cm, higher clinical stage (stages 2 and 3), presence of lymph node metastasis at diagnosis, and caudal location in the oral cavity. Radiotherapy had a protective effect against tumor progression. To date, this is the largest reported series of dogs with OM treated with ONCEPT. Several previously reported prognostic indicators were confirmed.

SAFETY AND TOXICITY OF AN ACCELERATED COARSELY FRACTIONATED RADIATION PROTOCOL FOR TREATMENT OF APPENDICULAR OSTEOSARCOMA IN 14 DOGS: 10 GY × 2 FRACTIONS.

Coarsely fractionated radiation is commonly used as a method for pain control in dogs with appendicular osteosarcoma, however there is little published information on optimal protocols. The aim of this retrospective, descriptive study was to report safety and toxicity findings in a sample of dogs with appendicular osteosarcoma that had been treated with a radiation scheme of 10 Gy delivered over two consecutive days for a total of 20 Gy. Dogs were included in the study if they had osteosarcoma that was treated with the aforementioned protocol. Dogs were excluded if treated with the same protocol for any other bone tumor besides osteosarcoma or inadequate follow-up. Thirteen of the 14 patients received adjuvant therapy with pamidronate and a nonsteroidal anti-inflammatory. Nine dogs received adjuvant chemotherapy with carboplatin after radiation was complete. Within a median of 14 days, 92.8% of dogs subjectively had improved pain control. Median duration of response (DOR) was 80 days (range 20-365). The majority of patients developed VRTOG grade one toxicity, primarily alopecia. Five dogs (35.7%) developed pathologic fracture postradiation treatment. Timing of fracture was variable ranging from 24 to 250 days. This radiation protocol was well tolerated, with minimal toxicity, subjectively improved survival time, and had the benefit of being completed in two consecutive days.

Comparison of Calculated Radiation Delivery Versus Actual Radiation Delivery in Military Macaws (Ara militaris).

The skin and oral cavity are common sites of neoplasia in avian species. Radiation therapy has been described for the treatment of these tumors in birds; however, its observed effectiveness has been variable. One possible explanation for this variability when radiation is used to treat the head is the unique anatomy of the avian skull, which contains an elaborate set of sinuses not found in mammalian species. To compare a calculated dose of radiation intended to be administered and the actual amount of radiation delivered to the target area of the choana in 3 adult military macaws (Ara militaris), computed tomography scans were obtained and the monitor unit was calculated to deliver 100 cGy (1Gy) by using radiation planning software. The birds received 3-4 radiation treatments each from a megavoltage radiation therapy unit. A thermoluminescent dosimetry chip (TLD) placed in the choana of the birds was used to measure the amount of ionizing radiation delivered at each treatment. The TLDs were kept in place using Play-Doh as a tissue analog. The actual dose of radiation delivered was lower than the 100-cGy calculated dose, with the 95% confidence limits of predicted bias values between 2.35 and 5.39 (radiation dose from 94.61 to 97.65 cGy). A significant difference was identified between the actual amount of radiation delivered and the calculated radiation goal (P < .001). None of the TLDs received the intended dose of 100 cGy of radiation. The results revealed that the amount of radiation delivered did not reach intended levels. Because the combination of the significance of this discrepancy and the standard dose inhomogeneity could lead to greater than 10% dose inhomogeneity, future investigation is warranted for accurate dose calculation and efficacy of radiation therapy for neoplasia at the lumen of the choana in birds.

Reirradiation of canine nasal carcinomas treated with coarsely fractionated radiation protocols: 37 cases.

Data from 37 dogs with nasal carcinomas treated with two or more coarsely fractionated courses of radiation therapy (RT) were retrospectively reviewed. The median radiation dose for the first course of RT was 24 Gray (Gy). All dogs clinically responded, and 11 had complete resolution of signs for a median of 114 days. Dogs were retreated at relapse, with a median dose of 20 Gy, and 26 of 37 dogs (70%) had clinical responses. The second course of RT was initiated at a median of 150 days following completion of the first course. Side effects were mild: four dogs had chronic ocular conditions necessitating medication, one of which required enucleation. Median survival time (ST) from the first dose of RT was 453 days and 180 days from the first dose of the second course of RT. The following factors were examined but were not significant for survival: total RT dose, dose of the first course of RT, complete resolution of clinical signs, use of either chemotherapy or nonsteroidal anti-inflammatory drugs (NSAIDs), and stage (T1/T2 versus T3/T4). Dogs responded well to reirradiation with a subset experiencing chronic ocular side effects.

Characterization of expression and prognostic implications of transforming growth factor beta, programmed death-ligand 1, and T regulatory cells in canine histiocytic sarcoma.

Histiocytic sarcoma (HS) is an aggressive malignant neoplasm in dogs. Expression and prognostic significance of transforming growth factor beta (TGF-ß), programmed death-ligand 1 (PD-L1), and T regulatory cells (Tregs) in HS is unknown. The goal of this study was to investigate the expression and prognostic significance of TGF-ß, PD-L1, and FoxP3/CD25 in canine HS utilizing RNA in situ hybridization (RNAscope®). After validation was performed, RNAscope® on formalin-fixed paraffin-embedded (FFPE) patient HS tissue samples was performed for all targets and expression quantified with HALO® software image analysis. Cox proportional hazard model was conducted to investigate the association between survival time and each variable. Additionally, for categorical data, the Kaplan-Meier product-limit method was used to generate survival curves. TGF-ß and PD-L1 mRNA expression was confirmed in the DH82 cell line by reverse transcription polymerase chain reaction (RT-PCR) and CD25 + FoxP3 + cells were detected by flow cytometry in peripheral blood. Once the RNAscope® method was validated, TGF-ß H-score and dots/cell and FoxP3 dots/cell were assessed in HS samples and found to be significantly correlated with survival. Moderate positive correlations were found between FoxP3 and PD-L1 H-score, percent staining area, and dots/cell, and FoxP3 and TGF-ß dots/cell. In summary, RNAscope® is a valid technique to detect TGF-ß and PD-L1 expression and identify Tregs in canine HS FFPE tissues. Furthermore, canine HS expresses TGF-ß and PD-L1. Increased TGF-ß and FoxP3 correlated with worse prognosis. Prospective studies are warranted to further investigate TGF-ß, PD-L1, and Tregs effect on prognosis.

Characterization of expression and prognostic implications of GD2 and GD3 synthase in canine histiocytic sarcoma.

GD2 and GD3 are disialoganglioside oncofetal antigens important in oncogenesis. GD2 synthase (GD2S) and GD3 synthase (GD3S) are needed for GD2 and GD3 production. The objectives of this study are to validate the use of RNA in situ hybridization (RNAscope®) in the detection of GD2S and GD3S in canine histiocytic sarcoma (HS) in vitro and optimize this technique in canine formalin-fixed paraffin-embedded (FFPE) tissues. A secondary objective is to evaluate the prognostic significance of GD2S and GD3S on survival. Quantitative RT-PCR compared GD2S and GD3S mRNA expression between three HS cell lines followed by RNAscope® in fixed cell pellets from the DH82 cell line and FFPE tissues. Variables prognostic for survival were determined with Cox proportional hazard model. RNAscope® was validated for detection of GD2S and GD3S and optimized in FFPE tissues. GD2S and GD3S mRNA expression was variable between cell lines. GD2S and GD3S mRNA expression was detected and measured in all tumor tissues; there was no association with prognosis. GD2S and GD3S are expressed in canine HS and successfully detected using the high throughput technique of RNAscope® in FFPE samples. This study provides the foundation for future prospective research of GD2S and GD3S utilizing RNAscope®.

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.

Identification of the interleukin-8 (CXCL-8) pathway in feline oral squamous cell carcinoma - A pilot study.

The purpose of this pilot study was to detect the presence of interleukin-8 (IL-8) and the potential downstream effects of IL-8 receptor activation in 2 previously characterized feline oral squamous cell carcinoma cell lines (SCCF1 and SCCF2). Interleukin-8 messenger RNA (mRNA) was initially detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). A previously validated and commercially available enzyme-linked immunosorbent assay (ELISA) test was used to measure IL-8 production in the supernatant of the 2 cell lines. Western blot was used to detect phosphorylation of proteins (AKT, ERK1/2, JAK2, STAT3, and Src), known to be downstream of interleukin-8 receptor activation. The IL-8 receptor-specific antagonists, Reparixin and SCH527123, were used to identify effects on phosphorylation of these proteins. Interleukin-8 mRNA and protein were detected in both SCCF1 and SCCF2 by RT-PCR and ELISA, respectively. Phosphorylation of ERK1/2, STAT3, and Src was detected in both cell lines. Inhibition of the IL-8 receptor led to a decrease in phosphorylation of Src, but not ERK1/2 or STAT3. In conclusion, feline squamous cell carcinoma cell lines can produce IL-8. Phosphorylation of Src seems, at least in part, a consequence of IL-8 receptor activation. The phosphorylation of ERK1/2 and STAT3, although present, seems independent of IL-8 receptor activation. Due to its potential effects on the tumor microenvironment, in addition to its autocrine effects on Src phosphorylation, the inhibition of the IL-8 receptor may become a beneficial therapeutic tool. Evaluation of the presence of both IL-8 and Src in many cases should elucidate their importance.

Le but de cette étude pilote était de détecter la présence d'interleukine-8 (IL-8) et les effets potentiels en aval de l'activation du récepteur IL-8 dans deux lignées cellulaires de carcinome épidermoïde oral félin (SCCF1 et SCCF2) précédemment caractérisées. L'ARN messager de l'interleukine-8 (ARNm) a été initialement détecté par amplification en chaîne par la polymérase à transcription inverse quantitative (qRT-PCR). Un test immuno-enzymatique ELISA précédemment validé et disponible dans le commerce a été utilisé pour mesurer la production d'IL-8 dans le surnageant des deux lignées cellulaires. L'immunobuvardage a été utilisé pour détecter la phosphorylation des protéines (AKT, ERK1/2, JAK2, STAT3 et Src), connues pour être en aval de l'activation du récepteur de l'interleukine-8. Les antagonistes spécifiques du récepteur IL-8, Reparixin et SCH527123, ont été utilisés pour identifier les effets sur la phosphorylation de ces protéines. L'ARNm et la protéine de l'interleukine-8 ont été détectés dans SCCF1 et SCCF2 par RT-PCR et ELISA, respectivement. La phosphorylation de ERK1/2, STAT3 et Src a été détectée dans les deux lignées cellulaires. L'inhibition du récepteur IL-8 a conduit à une diminution de la phosphorylation de Src, mais pas ERK1/2 ou STAT3. En conclusion, les lignées cellulaires de carcinome épidermoïde félin sont capables de produire de l'IL-8. La phosphorylation de Src semble, au moins en partie, une conséquence de l'activation du récepteur IL-8. La phosphorylation de ERK1/2 et STAT3, bien que présente, semble indépendante de l'activation du récepteur IL-8. En raison de ses effets potentiels sur le micro-environnement tumoral, en plus de ses effets autocrines sur la phosphorylation de Src, l'inhibition du récepteur IL-8 peut devenir un outil thérapeutique bénéfique. L'évaluation de la présence à la fois d'IL-8 et de Src dans un grand nombre de cas devrait élucider leur importance.(Traduit par Docteur Serge Messier).

A prospective, multi-centre, Veterinary Radiation Therapy Oncology Group study reveals potential efficacy of toceranib phosphate (Palladia) as a primary or adjuvant agent in the treatment of canine nasal carcinoma.

Radiation is the standard of care for dogs with nasal tumours. The addition of another therapy that could improve outcome without increasing toxicity is attractive. Medical therapy that could offer better outcome than maximally tolerated dose chemotherapy when radiation therapy (RT) is not possible or is declined is also attractive. This article reports the findings from a prospective, multi-centre, non-randomized, Veterinary Radiation Therapy Oncology Group clinical trial designed to evaluate whether toceranib phosphate (toceranib) has primary activity and if the addition of toceranib to RT could positively impact outcome. Owner's discretion determined enrolment in toceranib alone or toceranib + RT arm. Historical controls for radiation alone were selected from patients treated with identical RT and imaging protocols. Responses were evaluated with pre-treatment and week-16 CT scans. RT total dose of 42 Gy was completed in 10 fractions. Sixty-three dogs enrolled from 10 study sites. Overall response rates (CR + PR) were significantly improved in the toceranib + RT (79.4%) and RT alone (68.9%) arms over toceranib alone (22%) (p = .011). Clinical benefit rates (CR + PR + SD) were significantly improved in the toceranib + RT arm over the RT alone arm at 97.3% and 79.2% respectively (p = .036). Treatment with toceranib alone, toceranib + RT and RT alone resulted in median survival times of 298, 615 and 368 days respectively, but were not statistically significantly different (p = .0502). Adverse events associated with toceranib administration did not potentiate the RT side effect profile. Toceranib appears to have primary activity against nasal carcinoma.

C-kit, flt-3, PDGFR-ß, and VEGFR2 expression in canine adrenal tumors and correlation with outcome following adrenalectomy.

The objective of this retrospective study was to evaluate the expression of receptor tyrosine kinases (RTKs) in canine adrenal tumors and correlate this expression with features of tumor aggressiveness and survival in dogs undergoing adrenalectomy. Forty-three canine adrenal tumors were evaluated for expression of c-kit, fms-like tyrosine kinase 3 (flt-3), platelet-derived growth factor receptor-ß (PDGFR-ß), and vascular endothelial growth factor receptor 2 (VEGFR2) using immunohistochemistry. Tumor RTK staining characteristics were compared to normal adrenals. Medical records were reviewed for data regarding patient outcome and tumor characteristics. Expression of c-kit, flt-3, PDGFR-ß, and VEGFR2 was detected in 26.9%, 92.3%, 96.2%, and 61.5% of cortical tumors and 0%, 63.2%, 47.4%, and 15.8% of pheochromocytomas, respectively. Expression of RTKs was not significantly increased when compared to normal adrenals and did not correlate with survival after adrenalectomy. Receptor tyrosine kinases are not overexpressed in canine adrenal tumors compared to normal adrenal tissue. Therapeutic inhibition of these receptors may still represent an effective approach in cases where receptor activation is present.

L'objectif de cette étude rétrospective était d'évaluer l'expression des récepteurs tyrosine kinases (RTKs) dans les tumeurs surrénales canines et de corréler cette expression avec des caractéristiques d'agressivité tumorale et de survie chez les chiens subissant une surrénalectomie.Quarante-trois tumeurs surrénales canines ont été évaluées pour l'expression de c-kit, de la tyrosine kinase 3 de type fms (flt-3), du récepteur du facteur de croissance dérivé des plaquettes-ß (PDGFR-ß) et du récepteur du facteur de croissance endothélial vasculaire 2 (VEGFR2) par immunohistochimie. Les caractéristiques de coloration de la tumeur RTK ont été comparées à celles des surrénales normales. Les dossiers médicaux ont été examinés pour les données concernant les résultats des patients et les caractéristiques de la tumeur. L'expression de c-kit, flt-3, PDGFR-ß et VEGFR2 a été détectée dans 26,9 %, 92,3 %, 96,2 % et 61,5 % des tumeurs corticales et 0 %, 63,2 %, 47,4 % et 15,8 % des phéochromocytomes, respectivement. L'expression des RTK n'était pas significativement augmentée par rapport aux surrénales normales et n'était pas corrélée avec la survie après surrénalectomie. Les récepteurs tyrosine kinases ne sont pas surexprimés dans les tumeurs surrénales canines par rapport au tissu surrénalien normal. L'inhibition thérapeutique de ces récepteurs peut encore représenter une approche efficace dans les cas où l'activation du récepteur est présente.(Traduit par Docteur Serge Messier).

Novel application of single-cell next-generation sequencing for determination of intratumoral heterogeneity of canine osteosarcoma cell lines.

Osteosarcoma (OSA) is a highly aggressive and metastatic neoplasm of both the canine and human patient and is the leading form of osseous neoplasia in both species worldwide. To gain deeper insight into the heterogeneous and genetically chaotic nature of OSA, we applied single-cell transcriptome (scRNA-seq) analysis to 4 canine OSA cell lines. This novel application of scRNA-seq technology to the canine genome required uploading the CanFam3.1 reference genome into an analysis pipeline (10X Genomics Cell Ranger); this methodology has not been reported previously in the canine species, to our knowledge. The scRNA-seq outputs were validated by comparing them to cDNA expression from reverse-transcription PCR (RT-PCR) and Sanger sequencing bulk analysis of 4 canine OSA cell lines (COS31, DOUG, POS, and HMPOS) for 11 genes implicated in the pathogenesis of canine OSA. The scRNA-seq outputs revealed the significant heterogeneity of gene transcription expression patterns within the cell lines investigated (COS31 and DOUG). The scRNA-seq data showed 10 distinct clusters of similarly shared transcriptomic expression patterns in COS31; 12 clusters were identified in DOUG. In addition, cRNA-seq analysis provided data for integration into the Qiagen Ingenuity Pathway Analysis software for canonical pathway analysis. Of the 81 distinct pathways identified within the clusters, 33 had been implicated in the pathogenesis of OSA, of which 18 had not been reported previously in canine OSA.

Clinical outcome in dogs with nasal tumors treated with intensity-modulated radiation therapy.

Intensity-modulated radiation therapy (IMRT) is a valuable tool in human radiation oncology, but information on its use in veterinary medicine is lacking. In this study, 12 dogs with nasal tumors were treated with IMRT at a median radiation dose of 54 Gy. Patient survival times and frequency and severity of side effects on ocular structures, oral mucosa, and skin were recorded. Eight dogs (67%) had resolution of clinical signs during radiation therapy. Median overall survival time was 446 d with a 50% 1-year and a 25% 2-year survival rate. Minimal grade 2 or 3 acute skin toxicity, no grade 2 or 3 late skin toxicity, and no grade 2 or 3 toxicity to oral mucosa or the eye opposite the tumor were identified in the dogs treated with IMRT in this study. The ipsilateral eye could not be routinely spared due to its proximity to the tumor.

Molecular diagnosis using RNAscope in-situ hybridization in canine malignancies.

Immunohistochemistry has been used extensively to evaluate protein expression in clinical and research settings. However, immunohistochemistry is not always successful in veterinary medicine due to the lack of reliable antibody options, poor tissue preservation, labor-intensive staining, and antigen-retrieval optimization processes. RNAscope in-situ hybridization (ISH) is a powerful technology that uses a specific sequence probe to identify targeted mRNA. In this study, we demonstrate RNAscope ISH in 4 common canine malignancies, which are traditionally diagnosed by histopathology and immunohistochemistry. Probes were designed for commonly targeted mRNA markers of neoplastic tumors; these included c-kit in mast cell tumor, microphthalmia-associated transcription factor in malignant melanoma, ionized calcium-binding adapter molecule-1 in histiocytic sarcoma, and alkaline phosphatase in osteosarcoma. A strong staining signal was obtained by these 4 targets in each canine malignancy. These results support the use of RNAscope ISH for definitive diagnosis in canine malignancies.

L'immunohistochimie a grandement été utilisée pour évaluer l'expression de protéines dans des environnements clinique et de recherche. Toutefois, l'immunohistochimie n'est pas toujours un gage de réussite en médecine vétérinaire étant donné le manque d'options pour des anticorps fiables, la mauvaise conservation des tissus, la demande intensive de travail pour la coloration et les processus d'optimisation de recouvrement des antigènes. L'utilisation de l'hybridation in situ (ISH) et du RNAscope est une technologie puissante qui utilise une sonde avec une séquence spécifique afin d'identifier l'ARNm ciblé. Dans la présente étude, nous démontrons l'utilisation de l'ISH RNAscope pour quatre tumeurs canines fréquentes, qui sont traditionnellement diagnostiquées par histopathologie et immunohistochimie. Les sondes furent élaborées pour des marqueurs ARNm fréquemment ciblés de tumeurs néoplasiques; ceux-ci incluaient c-kit pour les tumeurs à cellules mastocytaires, le facteur associé à la transcription de la microphtalmie lors de mélanome malin, la molécule-1 adaptative à l'attachement du calcium ionisé lors de sarcome histiocytaire et la phosphatase alcaline lors d'ostéosarcome. Un signal fort de coloration fut obtenu par ces quatre cibles dans chacun des types de tumeurs. Ces résultats supportent l'utilisation d'ISH RNAscope pour le diagnostic définitif lors de tumeurs canines.(Traduit par Docteur Serge Messier).

In vitro effects of the chemotherapy agent water-soluble micellar paclitaxel (Paccal Vet) on canine hemangiosarcoma cell lines.

Paccal Vet (Osamia Pharmaceuticals) is a water-soluble nanoparticle micellar formulation of the drug paclitaxel that is well tolerated in dogs. This study evaluated the in vitro effect of Paccal Vet on two canine hemangiosarcoma (HSA) cell lines and their expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Paccal Vet caused HSA cell death in a dose- and time-dependent manner. The 50% inhibitory concentration (IC50 ) for the two HSA cell lines were 7 to 610 ng/mL, which are clinically achievable. Cell cycle analysis through flow cytometry showed cell cycle arrest at G2/M phase. Annexin-V and caspase 3/7 activity assays showed significant increases in apoptosis in correlation with the IC50 in each cell line. Reverse transcriptase-PCR was performed on the cell lines to validate the gene expression of VEGF and bFGF. Results obtained from this study support future studies involving the use of paclitaxel (micellar) for treatment of canine HSA.

Immunogenicity and Efficacy of a Novel Multi-Antigenic Peptide Vaccine Based on Cross-Reactivity between Feline and Human Immunodeficiency Viruses.

For the development of an effective HIV-1 vaccine, evolutionarily conserved epitopes between feline and human immunodeficiency viruses (FIV and HIV-1) were determined by analyzing overlapping peptides from retroviral genomes that induced both anti-FIV/HIV T cell-immunity in the peripheral blood mononuclear cells from the FIV-vaccinated cats and the HIV-infected humans. The conserved T-cell epitopes on p24 and reverse transcriptase were selected based on their robust FIV/HIV-specific CD8⁺ cytotoxic T lymphocyte (CTL), CD4⁺ CTL, and polyfunctional T-cell activities. Four such evolutionarily conserved epitopes were formulated into four multiple antigen peptides (MAPs), mixed with an adjuvant, to be tested as FIV vaccine in cats. The immunogenicity and protective efficacy were evaluated against a pathogenic FIV. More MAP/peptide-specific CD4⁺ than CD8⁺ T-cell responses were initially observed. By post-third vaccination, half of the MAP/peptide-specific CD8⁺ T-cell responses were higher or equivalent to those of CD4⁺ T-cell responses. Upon challenge, 15/19 (78.9%) vaccinated cats were protected, whereas 6/16 (37.5%) control cats remained uninfected, resulting in a protection rate of 66.3% preventable fraction (p = 0.0180). Thus, the selection method used to identify the protective FIV peptides should be useful in identifying protective HIV-1 peptides needed for a highly protective HIV-1 vaccine in humans.

Intratumoral bleomycin and IL-12 electrochemogenetherapy for treating head and neck tumors in dogs.

Bleomycin and Interleukin 12 have been used clinically to treat tumors; however, the co-administration of Bleomycin and Interleukin 12 followed by electroporation has not been tested clinically. In this study, dogs with spontaneous head and neck tumors were treated with one co-administration of Bleomycin and Interleukin 12 plasmid DNA followed by electroporation. The regression of the recurrent papillary tumor and the adjacent metastatic bone tumor was analyzed by multiple CT scans. The papillary tumor was completely eradicated in less than 2 weeks, and the bone tumor was not visible 23 weeks after the administration.

Evaluation of a ferret-specific formula for determining body surface area to improve chemotherapeutic dosing.

OBJECTIVE: To use CT-derived measurements to create a ferret-specific formula for body surface area (BSA) to improve chemotherapeutic dosing. ANIMALS: 25 adult ferrets (19 live and 6 cadavers). PROCEDURES: Live subjects were weighed, and body measurements were obtained by each of 3 observers while ferrets were awake and anesthetized. Computed tomography was performed, and a 3-D surface model was constructed with open-source imaging software, from which BSA was estimated. The CT-derived values were compared with BSA calculated on the basis of the traditional tape method for 6 cadavers. To further validate CT analysis software, 11 geometric shapes were scanned and their CT-derived values compared with those calculated directly via geometric formulas. Agreement between methods of surface area estimation was assessed with linear regression. Ferret-specific formulas for BSA were determined with nonlinear regression models. RESULTS: Repeatability among the 3 observers was good for all measurements, but some measurements differed significantly between awake and anesthetized ferrets. Excellent agreement was found between measured versus CT-derived surface area of shapes, traditional tape- versus CT-derived BSA of ferret cadavers, and CT-derived BSA of cadavers with and without monitoring equipment. All surface area formulas performed relatively similarly. CONCLUSIONS AND CLINICAL RELEVANCE: CT-derived BSA measurements of ferrets obtained via open-source imaging software were reliable. On the basis of study results, the recommended formula for BSA in ferrets would be 9.94 × (body weight)(2/3); however, this represented a relatively minor difference from the feline-derived formula currently used by most practitioners and would result in little practical change in drug doses.