Scar revision for incompletely or narrowly excised soft tissue sarcomas in dogs.

Objective: Describe clinical features of dogs undergoing scar revision for incompletely or narrowly excised soft tissue sarcomas (STSs) in the absence of gross disease and to determine local recurrence rates following scar revision. Animals: Thirty-three dogs with 33 scars. Procedures: Medical records were reviewed to collect data on signalment, tumor details, pre-surgical diagnostic tests, surgical and pathologic findings for both the initial and revision surgeries, and clinical outcomes. Descriptive statistics were generated. Results: For the initial excision, cytology was performed before surgery in 45.5% (15/33) of dogs, and information on surgical margins was rarely reported [4.0% (1/25) of circumferential and 12.0% (3/25) of deep margins]. Microscopic evidence of residual STS was identified in 18.2% of scars. Recurrence occurred in 3.0% (1/33) of dogs [median follow-up of 1127 d (1 to 3192 d)]; this dog had had no evidence of residual tumor in the scar revision pathology. Conclusions: Despite the low identification rate of residual tumor, the local tumor recurrence rate was 3.0%, which is lower than what is historically reported for incompletely or narrowly excised STSs. Clinical relevance: Scar revision for incompletely or narrowly excised STSs resulted in durable tumor remission in the dogs of this study. Pre-surgical diagnostic tests were not often performed in this study; these may be considered before the first excision to plan surgical margins for potentially reducing the incidence of incomplete or narrow excision. Surgical reports should include details on circumferential and deep margins to guide pathologic interpretation and future scar revision, if required.

Révision des cicatrice pour les sarcomes des tissus mous incomplètement ou étroitement excisés chez le chien. Objectif: Décrire les caractéristiques cliniques des chiens subissant une révision de cicatrice pour des sarcomes des tissus mous (STSs) incomplètement ou étroitement excisés en l'absence de maladie macroscopique et pour déterminer les taux de récidive locale après la révision de cicatrice. Animaux: Trente-trois chiens avec 33 cicatrices. Procédures: Les dossiers médicaux ont été examinés pour recueillir des données sur le signalement, les détails de la tumeur, les tests de diagnostic pré-chirurgicaux, les résultats chirurgicaux et pathologiques pour les chirurgies initiales et de révision, et les résultats cliniques. Des statistiques descriptives ont été générées. Résultats: Pour l'excision initiale, une cytologie a été réalisée avant la chirurgie chez 45,5 % (15/33) des chiens, et les informations sur les marges chirurgicales ont été rarement rapportées [4,0 % (1/25) des marges circonférentielles et 12,0 % (3/25) des marges profondes]. Des preuves microscopiques de STS résiduel ont été identifiées dans 18,2 % des cicatrices. Une récidive est survenue chez 3,0 % (1/33) des chiens [suivi médian de 1127 jours (1 à 3192 jours)]; ce chien n'avait eu aucun signe de tumeur résiduelle dans la pathologie de révision de la cicatrice. Conclusions: Malgré le faible taux d'identification de tumeur résiduelle, le taux de récidive tumorale locale était de 3,0 %, ce qui est inférieur à ce qui est historiquement rapporté pour les STS incomplètement ou étroitement excisés. Pertinence clinique: La révision des cicatrices pour les STS incomplètement ou étroitement excisés a entraîné une rémission tumorale durable chez les chiens de cette étude. Les tests diagnostiques pré-chirurgicaux n'ont pas souvent été effectués dans cette étude; ceux-ci peuvent être envisagés avant la première excision pour planifier les marges chirurgicales afin de réduire potentiellement l'incidence de l'excision incomplète ou étroite. Les rapports chirurgicaux doivent inclure des détails sur les marges circonférentielles et profondes pour guider l'interprétation pathologique et la révision future de la cicatrice, si nécessaire.(Traduit par Dr Serge Messier).

Scar revision for incompletely or narrowly excised cutaneous mast cell tumors in dogs.

Objective: Describe clinical features and outcomes of dogs undergoing scar revision for incompletely or narrowly excised cutaneous mast cell tumors without gross disease. Animals: 52 dogs undergoing 54 scar revisions. Procedures: Retrospective record review with information collected on signalment, tumor type/location, pre-surgical diagnostics, surgical and pathologic findings for the first excision and scar revision surgeries, and follow-up. Descriptive statistics were generated. Results: Prior to initial excision, cytology was performed on 38.9% (21/54) of tumors and the initial surgery report rarely described surgical resection margins [14.6% (7/48) of surgeries]. Residual tumor was identified pathologically in 29.6% (16/54) of scars. Local recurrence following scar revision occurred in 3.7% (2/54) of all scars [median follow-up 881.5 d (range: 0 to 3317 d)]; both scars had either complete excision of residual mast cell tumor or no evidence of mast cell tumor on scar revision and neither dog received radiation therapy. Conclusions: Identification of residual disease was uncommon, and local recurrence was less common than previously reported for incomplete/narrow mast cell tumor excision. Clinical relevance: Scar revision of unplanned primary excisions appears to yield a high likelihood of durable tumor remission in patients. First opinion practitioners are encouraged to avoid unplanned excisions by informing the surgical plan with cytology of the primary tumor and detailing surgical margin excision which may facilitate revision, if necessary.

Révision des cicatrices pour des mastocytes cutanés incomplètement ou étroitement excisés chez le chien. Objectif: Décrire les caractéristiques cliniques et les résultats des chiens subissant une révision de cicatrice pour des tumeurs mastocytaires incomplètement ou étroitement excisées sans maladie grave. Animaux: Cinquante-deux chiens soumis à 54 révisions de cicatrice. Procédures: Examen rétrospectif des dossiers avec des informations recueillies sur le signalement, le type/l'emplacement de la tumeur, les diagnostics pré-chirurgicaux, les résultats chirurgicaux et pathologiques pour les premières chirurgies d'excision et de révision de cicatrice, et le suivi. Des statistiques descriptives ont été générées. Résultats: Avant l'excision initiale, une cytologie a été réalisée sur 38,9 % (21/54) des tumeurs et le rapport chirurgical initial décrivait rarement les marges de résection chirurgicale [14,6 % (7/48) des chirurgies]. Une tumeur résiduelle a été identifiée pathologiquement dans 29,6 % (16/54) des cicatrices. Une récidive locale après révision des cicatrices s'est produite dans 3,7 % (2/54) de toutes les cicatrices [suivi médian de 881,5 jours (intervalle : 0 à 3317 jours)] les deux cicatrices présentaient soit une excision complète du tumeur mastocytaire résiduel, soit aucun signe de tumeur mastocytaire lors de la révision de la cicatrice et aucun chien n'a reçu de radiothérapie. Conclusions: L'identification de la maladie résiduelle était rare et la récidive locale était moins fréquente que précédemment rapportée pour l'excision incomplète/étroite du tumeur mastocytaire. Pertinence clinique: La révision de la cicatrice des excisions primaires non planifiées semble donner une forte probabilité de rémission tumorale durable chez les patients. Les praticiens de première opinion sont encouragés à éviter les excisions non planifiées en informant le plan chirurgical avec la cytologie de la tumeur primaire et en détaillant les marges de l'excision chirurgicale ce qui peut faciliter la révision, si nécessaire.(Traduit par Dr Serge Messier).

Prognostic significance of CD25 expression in dogs with a noninvasive diagnosis of B-cell lymphoma treated with CHOP chemotherapy.

Prior studies have identified high CD25 expression in canine diffuse large B-cell lymphoma as a negative prognostic indicator. The objective of this retrospective study was to evaluate CD25 expression as a prognostic indicator in dogs with B-cell lymphoma (BCL) diagnosed with commonly used noninvasive diagnostics (cytology and flow cytometry [FC]) and treated with CHOP chemotherapy. Lymph node aspirates from 57 dogs with cytologic diagnosis of lymphoma composed of intermediate to large lymphocytes were analysed with FC. Percentage of neoplastic B-cells expressing CD25 and median fluorescence intensity (MFI) of CD25 were measured. Relationships of CD25 percent positivity and MFI to progression free survival (PFS) and survival time were evaluated. Median survival time (MST) of all dogs was 272 days (95% CI, 196-348 days) and median PFS was 196 days (95% CI, 172-220 days). Higher percentage of B-cells positive for CD25 was associated with decreased risk of death in multivariable analysis (p = .02). Dogs with higher CD25 positivity had longer MST and PFS than dogs with lower CD25 positivity (318 days versus 176 days and 212 days versus 148 days, respectively), but these differences were not significant. CD25 MFI was not significantly associated with outcome. Based on the results of this study, the association of CD25 expression and prognosis in dogs with BCL diagnosed using noninvasive methods should be interpreted with caution. Further evaluation, with studies that include histopathologic differentiation of lymphoma subtypes, is needed.

Bortezomib inhibits the proteasome, leading to cell death via apoptosis in feline injection site sarcoma cells in vitro.

OBJECTIVE: To determine the in vitro effects of the proteasome inhibitor bortezomib in feline injection site sarcoma (FISS) cell lines. SAMPLE: In vitro cultures of the FISS cell lines Ela-1, Hamilton, and Kaiser. PROCEDURES: Cells were treated with increasing doses of bortezomib or vehicle alone (dimethyl sulfoxide) and evaluated for cell viability via an adenosine triphosphate concentration assay, proteasome activity via a commercially available proteasome assay, accumulation of ubiquitinated proteins via Western blot, and apoptosis via flow cytometry. RESULTS: All 3 cell lines were sensitive to bortezomib with a 50% inhibitory concentration after 48 hours of treatment at 17.46 nM (95% CI, 15.47 to 19.72 nM) for Ela-1, 19.48 nM (95% CI, 16.52 to 23.00 nM) for Hamilton, and 21.38 nM (95% CI, 19.24 to 23.78 nM) for Kaiser. In the Ela-1 cell line, 20 nM bortezomib inhibited 20S proteasome activity by 90.9% compared with the vehicle-only control. In the Kaiser cell line, 20 nM bortezomib decreased 20S proteasome activity by 70%, compared with the untreated vehicle-only control. Last, treatment with bortezomib (25 and 40 nM) resulted in statistically significant decreases in viable cells accompanied by a statistically significant increase in apoptotic cells. CLINICAL RELEVANCE: Treatment options for FISS, especially nonresectable FISS, are currently very limited. These results support further investigation of bortezomib either alone or in combination with other treatments in such cases.

Fundamentals of integrated ferrohydrodynamic cell separation in circulating tumor cell isolation.

Methods to separate circulating tumor cells (CTCs) from blood samples were intensively researched in order to understand the metastatic process and develop corresponding clinical assays. However current methods faced challenges that stemmed from CTCs' heterogeneity in their biological markers and physical morphologies. To this end, we developed integrated ferrohydrodynamic cell separation (iFCS), a scheme that separated CTCs independent of their surface antigen expression and physical characteristics. iFCS integrated both diamagnetophoresis of CTCs and magnetophoresis of blood cells together via a magnetic liquid medium, ferrofluid, whose magnetization could be tuned by adjusting its magnetic volume concentration. In this paper, we presented the fundamental theory of iFCS and its specific application in CTC separation. Governing equations of iFCS were developed to guide its optimization process. Three critical parameters that affected iFCS's cell separation performance were determined and validated theoretically and experimentally. These parameters included the sample flow rate, the volumetric concentration of magnetic materials in the ferrofluid, and the gradient of the magnetic flux density. We determined these optimized parameters in an iFCS device that led to a high recovery CTC separation in both spiked and clinical samples.

Use of indirect computed tomography lymphangiography to determine metastatic status of sentinel lymph nodes in dogs with a pre-operative diagnosis of melanoma or mast cell tumour.

Canine melanomas and mast cell tumours (MCTs) frequently metastasize to lymph nodes, worsening prognosis compared with dogs without metastasis. Sentinel lymph node (SLN) evaluation is more specific than evaluation of the lymph node closest to the tumour, which may not be the draining lymph node. Computed tomography lymphangiography (CTL) allows for SLN identification and one study of canine mammary tumours found that CTL was able to assist in determination of the metastatic status of inguinal SLNs prior to extirpation and histopathology. The objective of the present study was to evaluate CTL for use in determining metastasis to the SLN in dogs with a pre-operative diagnosis of melanoma or MCT in various locations by correlating CTL findings with histopathology. The hypothesis was that CTL would not be able to determine the metastatic status of lymph nodes, based on author experience. Dogs were prospectively enrolled and underwent CTL and subsequent SLN extirpation. Histopathology results for the primary tumour, SLN, and additional extirpated lymph nodes were recorded. Fifteen dogs were enrolled and 21 SLN were evaluated. The SLN enhancement pattern (heterogeneous, homogenous or peripheral) was not associated with metastasis, nor was the attenuation value at 1 minute, 5 minutes, or the change in attenuation value. No correlation was found between CTL findings and metastatic status of SLNs. Based on these results, CTL alone cannot be used to diagnose SLN metastasis. Extirpation of the SLN with histopathology is recommended to diagnose lymph node metastasis in dogs with melanoma and MCT.

Retrospective evaluation of a modified human lung cancer stage classification in dogs with surgically excised primary pulmonary carcinomas.

The stage classification for canine primary pulmonary carcinomas (PPC) was last updated in 1980. In people, the human lung cancer stage classification (HLCSC) (currently in its eighth edition) plays an integral role in diagnostic and therapeutic decision-making and is prognostic despite a heterogeneous population of tumours. The objective of this retrospective case study was to evaluate the prognostic significance of a canine lung carcinoma stage classification (CLCSC) adapted from the HLCSC by removal of substage for ease of application to canine PPC. A secondary objective was to evaluate the effect of adjuvant chemotherapy. Medical records of 71 dogs with histologically confirmed PPC were reviewed. All dogs underwent surgical excision of the primary lung tumour. Primary tumour features (referring to T1-T4 stages) and TNM stages (1-4) were assigned using the CLCSC. Canine lung carcinoma stage was I (n = 7), II (n = 32), III (n = 24) and IV (n = 8). Median survival time was 952, 658, 158 and 52 days for stages I-IV, respectively. Primary tumour features (T1-T4), incomplete surgical excision, presence of lymph node metastasis and tumour grade were independent prognostic indicators for overall survival. Twenty-six dogs received adjuvant chemotherapy; however, no statistically significant benefit was found. The CLCSC primary tumour features and stage classification were highly prognostic for survival in dogs with PPC. We propose further application and evaluation of this update to canine PPC stage classification. Given the poor prognosis of advanced stage canine PPC, novel treatments are needed.

Cutaneous and subcutaneous metastasis of appendicular osteosarcoma in dogs: 20 cases.

BACKGROUND: Metastasis of appendicular osteosarcoma is most common to the lungs and is generally considered a terminal event in dogs. Behavior and prognosis associated with cutaneous or subcutaneous metastases (CSM) is poorly defined. OBJECTIVE: Describe the population and gather prognostic information regarding appendicular osteosarcoma with CSM in dogs. ANIMALS: Twenty dogs with appendicular osteosarcoma and CSM. METHODS: Retrospective case series. Medical records were searched to identify dogs diagnosed with appendicular osteosarcoma that developed CSM. Demographic data, order of metastatic events, and CSM clinical features were evaluated. Kaplan-Meier survival curves were constructed and log-rank tests were used to compare survival between groups of dogs. RESULTS: In 19 dogs (95%), CSM was an incidental finding. Seventeen dogs (85%) developed pulmonary metastasis, and 1 dog (5%) developed bone metastasis. No other metastatic sites were detected before euthanasia. The median CSM-free interval and CSM survival time were 160 days (range: 0-542 days) and 55 days (range: 5-336 days), respectively. The median CSM survival time was significantly longer for dogs treated with surgery and chemotherapy (94 days) or chemotherapy only (64 days) than for dogs that did not receive these treatments (11 days) (P = .002 and P = .03, respectively). No other factors were associated with survival after diagnosis of CSM. CONCLUSION AND CLINICAL IMPORTANCE: The skin or subcutaneous tissue can be the first osteosarcoma metastatic site detected. After CSM diagnosis, the prognosis is grave with median survival <2 months. Although this finding could have been biased by case selection, treatment with surgery and chemotherapy may improve outcome.

Post-surgical outcome and prognostic factors in canine malignant melanomas of the haired skin: 87 cases (2003-2015).

The medical records of 87 dogs treated with surgery for cutaneous malignant melanoma (CMM) of the haired skin were retrospectively reviewed for overall survival time (OST), progression-free survival time (PFS), and prognostic factors. The post-surgery median PFS and median OST were 1282 days and 1363 days, respectively. The post-surgery metastatic rate was 21.8% with a local recurrence rate of 8%. Increasing mitotic index (MI) was predictive of a significantly decreased OST and PFS on multivariable analysis [hazard ratio (HR): 1.05, 95% confidence interval (CI): 1.02 to 1.07 and HR: 1.04, 95% CI: 1.02 to 1.06, respectively]. Increasing age was likewise predictive of a significantly decreased OST and PFS on multivariable analysis (HR: 1.39, 95% CI: 1.17 to 1.65 and HR: 1.33, 95% CI: 1.14 to 1.54, respectively). These results confirm clinical impressions that long survival times are likely in dogs diagnosed with malignant melanoma of the haired skin when treated with surgery alone.

Résultat post-chirurgical et facteurs de pronostic pour les mélanomes malins canins de la peau poilue : 87 cas (2003­2015). Les dossiers médicaux de 87 chiens traités à l'aide d'une chirurgie pour le mélanome malin cutané (MMC) de la peau poilue ont été évalués rétrospectivement pour le temps de survie global (TSG), le temps de survie sans progression (TSSP) et les facteurs de pronostic. Le TSSP médian après la chirurgie et le TSG médian étaient de 1282 jours et de 1363 jours, respectivement. Le taux métastasique après la chirurgie était de 21,8 % avec un taux de récurrence local de 8 %. L'augmentation de l'indice mitotique (IM) était prédictive d'un TSG et d'un TSSP réduits à l'analyse multivariable (ratio de risque [RR] : 1,05, intervalle de confiance [IC] de 95 % : 1,02 à 1,07 et RR : 1,04, IC de 95 % : 1,02 à 1,06, respectivement). La progression de l'âge était aussi prédictive d'une réduction importante du TSG et du TSSP à l'analyse multivariable (RR : 1,39, IC de 95 % : 1,17 à 1,65 et RR : 1,33, IC de 95 % : 1,14 à 1,54, respectivement). Ces résultats confirment les impressions cliniques que des longs délais de survie sont probables chez les chiens diagnostiqués avec le mélanome malin de la peau poilue lorsqu'ils sont uniquement traités à l'aide d'une chirurgie.(Traduit par Isabelle Vallières).

An NF-κB p65-cIAP2 link is necessary for mediating resistance to TNF-α induced cell death in gliomas.

Malignant gliomas are diffusively infiltrative and remain among the deadliest of all cancers. NF-κB is a transcription factor that mediates cell growth, migration and invasion, angiogenesis and resistance to apoptosis. Normally, the activity of NF-κB is tightly regulated by numerous mechanisms. However, in many cancers, NF-κB is constitutively activated and may function as a tumor promoter. Herein, we show that in gliomas, NF-κB is constitutively activated and the levels of cIAP2, Bcl-2, Bcl-xL and Survivin are elevated. These genes are regulated by NF-κB and can inhibit apoptosis. To understand the potential role of NF-κB p65 in suppressing apoptosis, we generated human glioma cell lines that inducibly express shRNA molecules specific for p65. We demonstrate that in the absence of p65, TNF-α induced cIAP2 expression is significantly reduced while the levels of Bcl-2, Bcl-xL and Survivin are not affected. These data suggest that of these genes, only cIAP2 is a direct target of p65, which was confirmed using RT-PCR and chromatin immunoprecipitation (ChIP) assays. By reducing the levels of p65 and/or cIAP2 levels, we demonstrate that the levels of RIP poly-ubiquitination are reduced, and that p65-deficient glioma cells are more sensitive to the cytotoxic effects of TNF-α. Specifically, in the presence of TNF-α glioma cells lacking p65 and/or cIAP2 showed cellular proliferation defects and underwent cell death. These data suggest that NF-κB and/or cIAP2 may be therapeutically relevant targets for the treatment of malignant gliomas.

The ING4 tumor suppressor attenuates NF-kappaB activity at the promoters of target genes.

The NF-kappaB family mediates immune and inflammatory responses. In many cancers, NF-kappaB is constitutively activated and induces the expression of genes that facilitate tumorigenesis. ING4 is a tumor suppressor that is absent or mutated in several cancers. Herein, we demonstrate that in human gliomas, NF-kappaB is constitutively activated, ING4 expression is negligible, and NF-kappaB-regulated gene expression is elevated. We demonstrate that an ING4 and NF-kappaB interaction exists but does not prevent NF-kappaB activation, nuclear translocation, or DNA binding. Instead, ING4 and NF-kappaB bind simultaneously at NF-kappaB-regulated promoters, and this binding correlates with reductions in p65 phosphorylation, p300, and the levels of acetylated histones and H3-Me3K4, while enhancing the levels of HDAC-1 at these promoters. Using a knockdown approach, we correlate reductions in ING4 protein levels with increased basal and inducible NF-kappaB target gene expression. Collectively, these data suggest that ING4 may specifically regulate the activity of NF-kappaB molecules that are bound to target gene promoters.

IFN-beta-mediated inhibition of IL-8 expression requires the ISGF3 components Stat1, Stat2, and IRF-9.

Interleukin-8 (IL-8) is a key component of the innate immune response because of its ability to recruit inflammatory cells to sites of inflammation. Although the effects of IL-8 are largely beneficial, aberrant expression of IL-8 is known to contribute to a number of pathologic states. Interferon-beta (IFN-beta), an antiviral cytokine, is known to inhibit the expression of IL-8, although the exact mechanism by which this occurs has yet to be elucidated. In this study, we dissect the role of each member of the IFN-stimulated gene factor 3 (ISGF3) signaling complex in contributing to IFN-beta inhibition of IL-8 gene expression. To date, no IFN-stimulated response element (ISRE) (the DNA binding target for ISGF3) has been identified within the promoter region of the IL-8 gene. We conclude, through use of cell lines deficient for ISGF3 components, that all three members of this complex, Stat1, Stat2, and IFN regulatory factor-9 (IRF-9), are required for IFN-beta-mediated inhibition of IL-8 expression. In contrast to positive signaling by ISGF3 to activate gene expression, we find that the transactivation domains of Stat1 and Stat2 are not essential to IFN-beta inhibition of IL-8. Taken together, these data define the role of the ISGF3 members in IFN-beta inhibitory signaling.

Mechanism of IFN-beta-mediated inhibition of IL-8 gene expression in astroglioma cells.

IL-8 is a chemokine that recruits migrating neutrophils and leukocytes to areas of inflammation. In noninflamed tissue, IL-8 expression is low but can be rapidly induced by proinflammatory cytokines. Typically, inflammation and transient IL-8 expression are beneficial. However, some diseases are characterized by excessive inflammation and high levels of IL-8. Previous studies have shown that IFN-beta can inhibit the expression of IL-8, although the mechanism is unknown. Using chromatin immunoprecipitation assays, we define the IL-8 transcriptional program in the absence or presence of inducing stimuli and/or inhibition by IFN-beta. In the absence of stimuli, the IL-8 promoter is acetylated but negatively regulated by corepressor proteins. Upon PMA stimulation, the levels of these corepressors are reduced and the promoter is rapidly bound and activated by transcription factors, including NF-kappaB p65, C/EBPbeta, and c-Fos. In addition, RNA polymerase II is recruited to the IL-8 promoter to initiate transcription. However, in the presence of both PMA and IFN-beta, there are diminished levels of histone acetylation, reduced levels of transcription factors such as NF-kappaB p65 and RNA polymerase II, and an increased presence of corepressor proteins such as histone deacetylases 1 and 3 and silencing mediator of retinoic acid and thyroid hormone receptors. IFN-gamma-inducible protein-10 and MCP-1 genes, also regulated by NF-kappaB, are unaffected by IFN-beta, and IFN-beta does not prevent the activation, nuclear migration, or binding of NF-kappaB p65 to the kappaB element of the IFN-gamma-inducible protein-10 promoter. As such, these data show that the inhibitory effects of IFN-beta are specific to the IL-8 promoter.