Diagnostic utility of ultrasound-guided fine-needle aspiration and needle-core biopsy sampling of canine splenic masses.

This prospective clinical study sought to determine the accuracy of cytopathologic examination and needle-core biopsy (NCB) against diagnoses obtained by excisional histopathology (EH) for canine splenic masses. Twenty-five masses were evaluated ex vivo by ultrasound-guided fine-needle aspiration (FNA) and NCB tissue sampling. Each spleen was placed in a container and artificial skin placed over its surface. Ultrasound-guided FNA using a 22-gauge needle and 2 NCB samples [14-gauge (NCB-14), 16-gauge (NCB-16)] were obtained and submitted for analysis. Results were compared to results obtained by splenic excisional histopathology (EH). There was no difference noted between FNA, NCB-14, or NCB-16 analyses. In addition, there was no difference in accuracy between FNA and NCB-14 or between FNA and NCB-14 versus NCB-16. Reported accuracy of FNA was 0.72, NCB-14 was 0.72, and NCB-16 was 0.64, respectively. Both FNA and NCB-14 displayed a sensitivity of 71% and NCB-16 a sensitivity of 53%. Both FNA and NCB-14 displayed a specificity of 75% and NCB-16 a specificity of 88%. The results demonstrated that NCB had no advantage clinically over FNA at diagnosing splenic pathology. This study further demonstrates that preoperative diagnostic evaluation of the spleen is not highly accurate and cannot be recommended prior to splenectomy.

Cette étude clinique prospective visait à déterminer la précision de l'examen cytopathologique et de la biopsie au trocart (NCB) par rapport aux diagnostics obtenus par histopathologie excisionnelle (EH) pour les masses spléniques canines. Vingt-cinq masses ont été évaluées ex vivo par aspiration à l'aiguille fine guidée par ultrasons (FNA) et prélèvement de tissu par NCB. Chaque rate a été placée dans un récipient et une peau artificielle placée sur sa surface. Une FNA guidée par ultrasons à l'aide d'une aiguille de calibre 22 et de 2 échantillons de NCB (calibre 14 (NCB-14), calibre 16 (NCB-16)) ont été obtenues et soumises pour analyse. Les résultats ont été comparés aux résultats obtenus par histopathologie excisionnelle splénique (EH). Aucune différence n'a été notée entre les analyses FNA, NCB-14 ou NCB-16. De plus, il n'y avait aucune différence de précision entre FNA et NCB-14 ou entre FNA et NCB-14 par rapport à NCB-16. La précision rapportée de FNA était de 0,72, celle de NCB-14 de 0,72 et de NCB-16 était de 0,64, respectivement. FNA et NCB-14 ont affiché une sensibilité de 71 % et NCB-16 une sensibilité de 53 %. FNA et NCB-14 ont affiché une spécificité de 75 % et NCB-16 une spécificité de 88 %. Les résultats ont démontré que la NCB n'avait aucun avantage clinique sur la FNA pour diagnostiquer la pathologie splénique. Cette étude démontre en outre que l'évaluation diagnostique préopératoire de la rate n'est pas très précise et ne peut être recommandée avant la splénectomie.(Traduit par Docteur Serge Messier).

Effect of perfusate volume on amikacin concentrations after saphenous intravenous regional limb perfusion in standing, sedated horses.

OBJECTIVE: To determine the influence of perfusate volume on synovial fluid amikacin concentrations in the joints of the hind limb after standing saphenous intravenous regional limb perfusion (IVRLP). STUDY DESIGN: Randomized crossover design. ANIMALS: Six adult horses. METHODS: Saphenous IVRLP was performed in 6 standing horses with 1 g of amikacin diluted with 0.9% NaCl to volumes of 10 ml, 60 ml, and 120 ml. Samples of synovial fluid from the tarsocrural, metatarsophalangeal, and hind limb distal interphalangeal joints were collected at 15 and 30 min after perfusate administration. Concentrations of 40 µg/ml and 160 µg/ml were considered therapeutic for susceptible and resistant pathogens, respectively. RESULTS: No difference in synovial fluid amikacin concentrations was detected between volumes in any joint (P = .4). All synovial fluid amikacin concentrations were higher at 30 min compared to 15 min (P = .003). All median synovial fluid amikacin concentrations at 30 min were > 40 µg/ml using the 60 ml and 120 ml volumes. Synovial fluid amikacin concentrations >40 µg/ml were only reached in the hind limb distal interphalangeal joint when the 10 ml volume was used. All median synovial fluid amikacin concentrations observed were < 160 µg/ml. CONCLUSIONS: Target concentrations for pathogens that were considered susceptible were consistently reached with perfusate volumes of 60 ml and 120 ml. However, median synovial fluid amikacin concentrations did not reach target levels for resistant pathogens. CLINICAL SIGNIFICANCE: Perfusate volumes of 60 ml or 120 ml are recommended to treat infections due to susceptible pathogens in the joints of the distal hind limb. These results justify investigation of saphenous IVRLP with different perfusate volumes using higher doses of amikacin.

Structural and Functional Analyses of an Allosteric EYA2 Phosphatase Inhibitor That Has On-Target Effects in Human Lung Cancer Cells.

EYA proteins (EYA1-4) are critical developmental transcriptional cofactors that contain an EYA domain (ED) harboring Tyr phosphatase activity. EYA proteins are largely downregulated after embryogenesis but are reexpressed in cancers, and their Tyr phosphatase activity plays an important role in the DNA damage response and tumor progression. We previously identified a class of small-molecule allosteric inhibitors that specifically inhibit the Tyr phosphatase activity of EYA2. Herein, we determined the crystal structure of the EYA2 ED in complex with NCGC00249987 (a representative compound in this class), revealing that it binds to an induced pocket distant from the active site. NCGC00249987 binding leads to a conformational change of the active site that is unfavorable for Mg2+ binding, thereby inhibiting EYA2's Tyr phosphatase activity. We demonstrate, using genetic mutations, that migration, invadopodia formation, and invasion of lung adenocarcinoma cells are dependent on EYA2 Tyr phosphatase activity, whereas growth and survival are not. Further, we demonstrate that NCGC00249987 specifically targets migration, invadopodia formation, and invasion of lung cancer cells, but that it does not inhibit cell growth or survival. The compound has no effect on lung cancer cells carrying an EYA2 F290Y mutant that abolishes compound binding, indicating that NCGC00249987 is on target in lung cancer cells. These data suggest that the NCGC00249987 allosteric inhibitor can be used as a chemical probe to study the function of the EYA2 Tyr phosphatase activity in cells and may have the potential to be developed into an antimetastatic agent for cancers reliant on EYA2's Tyr phosphatase activity.

Unsupervised discovery of phenotype-specific multi-omics networks.

MOTIVATION: Complex diseases often involve a wide spectrum of phenotypic traits. Better understanding of the biological mechanisms relevant to each trait promotes understanding of the etiology of the disease and the potential for targeted and effective treatment plans. There have been many efforts towards omics data integration and network reconstruction, but limited work has examined the incorporation of relevant (quantitative) phenotypic traits. RESULTS: We propose a novel technique, sparse multiple canonical correlation network analysis (SmCCNet), for integrating multiple omics data types along with a quantitative phenotype of interest, and for constructing multi-omics networks that are specific to the phenotype. As a case study, we focus on miRNA-mRNA networks. Through simulations, we demonstrate that SmCCNet has better overall prediction performance compared to popular gene expression network construction and integration approaches under realistic settings. Applying SmCCNet to studies on chronic obstructive pulmonary disease (COPD) and breast cancer, we found enrichment of known relevant pathways (e.g. the Cadherin pathway for COPD and the interferon-gamma signaling pathway for breast cancer) as well as less known omics features that may be important to the diseases. Although those applications focus on miRNA-mRNA co-expression networks, SmCCNet is applicable to a variety of omics and other data types. It can also be easily generalized to incorporate multiple quantitative phenotype simultaneously. The versatility of SmCCNet suggests great potential of the approach in many areas. AVAILABILITY AND IMPLEMENTATION: The SmCCNet algorithm is written in R, and is freely available on the web at https://cran.r-project.org/web/packages/SmCCNet/index.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

SIX2 Mediates Late-Stage Metastasis via Direct Regulation of SOX2 and Induction of a Cancer Stem Cell Program.

The capacity for tumor cells to metastasize efficiently is directly linked to their ability to colonize secondary sites. Here we identify Six2, a developmental transcription factor, as a critical regulator of a breast cancer stem cell program that enables metastatic colonization. In several triple-negative breast cancer (TNBC) models, Six2 enhanced the expression of genes associated with embryonic stem cell programs. Six2 directly bound the Sox2 Srr2 enhancer, promoting Sox2 expression and downstream expression of Nanog, which are both key pluripotency factors. Regulation of Sox2 by Six2 enhanced cancer stem cell properties and increased metastatic colonization. Six2 and Sox2 expression correlated highly in breast cancers including TNBC, where a Six2 expression signature was predictive of metastatic burden and poor clinical outcome. Our findings demonstrate that a SIX2/SOX2 axis is required for efficient metastatic colonization, underscoring a key role for stemness factors in outgrowth at secondary sites. SIGNIFICANCE: These findings provide novel mechanistic insight into stemness and the metastatic outgrowth of triple-negative breast cancer cells.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/4/720/F1.large.jpg.

Author Correction: Eya3 partners with PP2A to induce c-Myc stabilization and tumor progression.

In the original version of this Article, the title of the legend to Fig. 7 incorrectly read 'Knockdown of B55α increases breast cancer metastasis' instead of 'Knockdown of B55α decreases breast cancer metastasis'. This has now been corrected in both the PDF and HTML versions of the Article.

Eya3 promotes breast tumor-associated immune suppression via threonine phosphatase-mediated PD-L1 upregulation.

Eya proteins are critical developmental regulators that are highly expressed in embryogenesis but downregulated after development. Amplification and/or re-expression of Eyas occurs in many tumor types. In breast cancer, Eyas regulate tumor progression by acting as transcriptional cofactors and tyrosine phosphatases. Intriguingly, Eyas harbor a separate threonine (Thr) phosphatase activity, which was previously implicated in innate immunity. Here we describe what we believe to be a novel role for Eya3 in mediating triple-negative breast cancer-associated immune suppression. Eya3 loss decreases tumor growth in immune-competent mice and is associated with increased numbers of infiltrated CD8+ T cells, which, when depleted, reverse the effects of Eya3 knockdown. Mechanistically, Eya3 utilizes its Thr phosphatase activity to dephosphorylate Myc at pT58, resulting in a stabilized form. We show that Myc is required for Eya3-mediated increases in PD-L1, and that rescue of PD-L1 in Eya3-knockdown cells restores tumor progression. Finally, we demonstrate that Eya3 significantly correlates with PD-L1 in human breast tumors, and that tumors expressing high levels of Eya3 have a decreased CD8+ T cell signature. Our data uncover a role for Eya3 in mediating tumor-associated immune suppression, and suggest that its inhibition may enhance checkpoint therapies.

Eya3 partners with PP2A to induce c-Myc stabilization and tumor progression.

Eya genes encode a unique family of multifunctional proteins that serve as transcriptional co-activators and as haloacid dehalogenase-family Tyr phosphatases. Intriguingly, the N-terminal domain of Eyas, which does not share sequence similarity to any known phosphatases, contains a separable Ser/Thr phosphatase activity. Here, we demonstrate that the Ser/Thr phosphatase activity of Eya is not intrinsic, but arises from its direct interaction with the protein phosphatase 2A (PP2A)-B55α holoenzyme. Importantly, Eya3 alters the regulation of c-Myc by PP2A, increasing c-Myc stability by enabling PP2A-B55α to dephosphorylate pT58, in direct contrast to the previously described PP2A-B56α-mediated dephosphorylation of pS62 and c-Myc destabilization. Furthermore, Eya3 and PP2A-B55α promote metastasis in a xenograft model of breast cancer, opposing the canonical tumor suppressive function of PP2A-B56α. Our study identifies Eya3 as a regulator of PP2A, a major cellular Ser/Thr phosphatase, and uncovers a mechanism of controlling the stability of a critical oncogene, c-Myc.

Impaired B cell function during viral infections due to PTEN-mediated inhibition of the PI3K pathway.

Transient suppression of B cell function often accompanies acute viral infection. However, the molecular signaling circuitry that enforces this hyporesponsiveness is undefined. In this study, experiments identify up-regulation of the inositol phosphatase PTEN (phosphatase and tensin homolog) as primarily responsible for defects in B lymphocyte migration and antibody responses that accompany acute viral infection. B cells from mice acutely infected with gammaherpesvirus 68 are defective in BCR- and CXCR4-mediated activation of the PI3K pathway, and this, we show, is associated with increased PTEN expression. This viral infection-induced PTEN overexpression appears responsible for the suppression of antibody responses observed in infected mice because PTEN deficiency or expression of a constitutively active PI3K rescued function of B cells in infected mice. Conversely, induced overexpression of PTEN in B cells in uninfected mice led to suppression of antibody responses. Finally, we demonstrate that PTEN up-regulation is a common mechanism by which infection induces suppression of antibody responses. Collectively, these findings identify a novel role for PTEN during infection and identify regulation of the PI3K pathway, a mechanism previously shown to silence autoreactive B cells, as a key physiological target to control antibody responses.