Iron, hepcidin, and microcytosis in canine hepatocellular carcinoma.

BACKGROUND: Erythrocyte microcytosis in some dogs with hepatocellular carcinoma (HCC) suggests a derangement in systemic iron. Hepcidin, the master regulator of iron, is secreted by the liver in response to interleukin 6 (IL-6) and/or bone morphogenetic protein 6 (BMP6) and can cause microcytosis. OBJECTIVES: Pilot study to compare the quantities of hepcidin, IL-6, and BMP6 RNA molecules in archival tumoral (HCC) and adjacent peritumoral (non-HCC) hepatic tissue to determine if they are different between tissue types or associated with microcytosis. METHODS: RNA was isolated from formalin-fixed, paraffin-embedded HCC and non-HCC tissue from seven microcytic dogs and four normocytic dogs. Digital RNA counts of hepcidin, IL-6, or BMP6, and six other iron-regulatory genes were determined using the Nanostring nCounter system. The area of blue on each section was digitally evaluated to measure the extent of Prussian blue staining objectively. Parameters were compared between HCC and non-HCC tissue and between microcytic and normocytic groups. RESULTS: Hepcidin was decreased, and transferrin receptor 1 (TfR1) was increased in HCC tissue compared with non-HCC tissue. Non-HCC hepcidin RNA counts correlated negatively with MCV and positively with the extent of iron staining. Hepcidin expression was higher in non-HCC tissue of microcytic cases than in normocytic cases. CONCLUSIONS: Canine HCC cases showed relatively increased iron staining in non-HCC tissue and decreased hepcidin RNA in HCC tissue. Microcytic cases had higher hepcidin RNA in non-HCC tissue than normocytic cases. Future studies may extend these findings to protein quantification, cellular localization of RNA changes, and determining if iron loading in canine liver is a predisposing factor for HCC.

Light chain myeloma and detection of free light chains in serum and urine of dogs and cats.

BACKGROUND: Detection of free light chains (fLC) in animals relies on protein electrophoresis or the Bence-Jones protein test on urine. OBJECTIVE: To describe the detection of both serum fLC (sfLC) and urine fLC (ufLC) in 8 dogs and 2 cats using a commercially available human immunofixation (IF) kit. ANIMALS: Archived serum or urine samples from 27 dogs and 2 cats submitted to the Colorado State University Veterinary Diagnostic Laboratory for routine diagnostics. METHODS: Retrospective study evaluating the presence of fLC in dogs and cats using agarose gel electrophoresis and routine and fLC IF performed on serum and urine. The performance of the fLC IF reagents was evaluated using samples characterized by routine IF, tandem mass spectrometry, and a combination of fLC IF and western blotting. Free light chains were documented by paired electrophoresis and fLC IF. RESULTS: The fLC only myeloma case developed end-stage renal failure 5 months post initial diagnosis. All electrophoresis-defined urinary Bence-Jones proteins were labeled by the anti-free λ light chain (anti-fλ) reagent; none were labeled by the anti-free κ light chain (anti-fκ); 2 of these were identified as fκ by mass spectrometry. An electrophoretically identical protein restriction that was labeled by the anti-fλ reagent was present in the paired serum from 5/8 of cases, documenting sfLC. CONCLUSIONS AND CLINICAL IMPORTANCE: Commercially available human IF reagents identified sfLC and ufLC in both dogs and cats. Free light chains may be nephrotoxic in dogs.

Identification of canine IgG and its subclasses, IgG1, IgG2, IgG3 and IgG4, by immunofixation and commercially available antisera.

Immunofixation is a diagnostic and research tool used for characterizing the electrophoretic location of immunoglobulin fractions in serum and urine. Commercially available polyclonal antisera which discriminate two IgG subclasses (IgG1 and IgG2) are available and commonly used. More recently, four IgG subclasses have been defined in the dog based on cDNA data. Archived serum from 16 dogs with naturally occurring monoclonal or biclonal gammopathies were characterized using routine serum protein electrophoresis, routine immunofixation and LCMS/MS as 3 IgA, 3 IgM, 2 IgG2, 7 IgG3 and 2 IgG4 heavy chain predominant cases. Immunofixation reactivity of a panel of commercially available antisera to these cases was characterized. The anti-human IgG antisera was the only tested antisera which bound all canine IgG restricted bands without also labelling IgA or IgM heavy chains or light chains. The tested polyclonal antisera labeled as reacting with canine IgG2 bound canine IgG2, IgG3, IgA and IgM and may label IgG1. The tested polyclonal antisera labeled as reacting with canine IgG1 bound the canine IgG4 bands but not those identified as IgA, IgM, IgG2 or IgG3 and likely did not bind IgG1. This data suggests that commercially available polyclonal IgG1 antisera (Bethyl A40 - 120A and Bio-Rad AHP947) can be used to positively but possibly not selectively identify canine IgG4 by immunofixation.

Cobimetinib and trametinib inhibit platelet MEK but do not cause platelet dysfunction.

The MEK inhibitors cobimetinib and trametinib are used in combination with BRAF inhibitors to treat metastatic melanoma but increase rates of hemorrhage relative to BRAF inhibitors alone. Platelets express several members of the MAPK signalling cascade including MEK1 and MEK2 and ERK1 and ERK2 but their role in platelet function and haemostasis is ambiguous as previous reports have been contradictory. It is therefore unclear if MEK inhibitors might be causing platelet dysfunction and contributing to increased hemorrhage. In the present study we performed pharmacological characterisation of cobimetinib and trametinib in vitro to investigate potential for MEK inhibitors to cause platelet dysfunction. We report that whilst both cobimetinib and trametinib are potent inhibitors of platelet MEK activity, treatment with trametinib did not alter platelet function. Treatment with cobimetinib results in inhibition of platelet aggregation, integrin activation, alpha-granule secretion and adhesion but only at suprapharmacological concentrations. We identified that the inhibitory effects of high concentrations of cobimetinib are associated with off-target inhibition on Akt and PKC. Neither inhibitor caused any alteration in thrombus formation on collagen under flow conditions in vitro. Our findings demonstrate that platelets are able to function normally when MEK activity is fully inhibited, indicating MEK activity is dispensable for normal platelet function. We conclude that the MEK inhibitors cobimetinib and trametinib do not induce platelet dysfunction and are therefore unlikely to contribute to increased incidence of bleeding reported during MEK inhibitor therapy.

Carbon Monoxide-Releasing Molecule Enhances Coagulation and Decreases Fibrinolysis in Normal Canine Plasma.

The dog is an important companion animal and also purpose-bred for research studies. Coagulopathies in dogs are common, although the availability of blood products for therapy is inconsistent throughout the profession. A pro-coagulant therapeutic that is readily available and easily stored would be useful for the treatment of coagulopathies. Tricarbonyldichlororuthenium (II) dimer [Carbon monoxide-releasing molecule-2 (CORM-2)] acts as a prothrombotic agent in plasma by increasing the velocity of clot formation and clot strength, and by decreasing the clot's vulnerability to fibrinolysis. We sought to test CORM-2's effect on coagulation and fibrinolysis in vitro in canine plasma using thromboelastography. Measures of the rate of clot formation and clot strength in plasma without CORM-2 were highly correlated with fibrinogen concentration. We found that CORM-2 significantly enhanced the rate of clot formation and clot strength and significantly reduced the rate of fibrinolysis and the clot lysis time. The per cent change in rate of clot formation and clot strength was not significantly correlated with fibrinogen concentration, indicating that CORM-2's pro-coagulant effect is not dependent on fibrinogen concentration. This study corroborates studies in other species that show that CORM-2 is pro-coagulant in plasma, and lays the groundwork for developing CORM-2 as a therapeutic agent for canine coagulopathies. Future studies will evaluate the effect of CORM-2 on whole blood both in vitro and in vivo.