Outcomes Based on Plasma Biomarkers for the Phase 3 CELESTIAL Trial of Cabozantinib versus Placebo in Advanced Hepatocellular Carcinoma.

INTRODUCTION: Cabozantinib, an inhibitor of MET, AXL, and VEGF receptors, significantly improved overall survival (OS) and progression-free survival (PFS) versus placebo in patients with previously treated advanced hepatocellular carcinoma (HCC). In this exploratory analysis, outcomes were evaluated according to plasma biomarker levels. METHODS: Baseline plasma levels were evaluated for MET, AXL, VEGFR2, HGF, GAS6, VEGF-A, PlGF, IL-8, EPO, ANG2, IGF-1, VEGF-C, and c-KIT for 674/707 randomized patients; and Week 4 levels were evaluated for MET, AXL, VEGFR2, HGF, GAS6, VEGF-A, PlGF, IL-8, and EPO for 614 patients. OS and PFS were analyzed by baseline levels as dichotomized or continuous variables and by on-treatment changes at Week 4 as continuous variables; biomarkers were considered potentially prognostic if p < 0.05 and predictive if p < 0.05 for the interaction between treatment and the biomarker. Multivariable analyses adjusting for clinical covariates were also performed. RESULTS: In the placebo group, high levels of MET, HGF, GAS6, IL-8, and ANG2 and low levels of IGF-1 were associated with shorter OS in univariate and multivariable analyses; these associations were also observed for MET, IL-8, and ANG2 in the cabozantinib group. Hazard ratios for OS and PFS favored cabozantinib over the placebo at low and high baseline levels for all biomarkers. No baseline biomarkers were predictive of a treatment benefit. Cabozantinib promoted pharmacodynamic changes in several biomarkers, including increases in VEGF-A, PlGF, AXL, and GAS6 levels and decreases in VEGFR2 and HGF levels; these changes were not associated with OS or PFS. CONCLUSION: Cabozantinib improved OS and PFS versus placebo at high and low baseline concentrations for all biomarkers analyzed. Low baseline levels of MET, HGF, GAS6, IL-8, and ANG2 and high levels of IGF-1 were identified as potential favorable prognostic biomarkers for survival in previously treated advanced HCC. Although cabozantinib promoted pharmacodynamic changes in several biomarkers, these changes were not associated with survival.

Biomarkers of Angiogenesis and Clinical Outcomes to Cabozantinib and Everolimus in Patients with Metastatic Renal Cell Carcinoma from the Phase III METEOR Trial.

PURPOSE: Antiangiogenic VEGF receptor (VEGFR) inhibitors are approved for metastatic clear cell renal cell carcinoma (mccRCC) and their efficacy is higher in high angiogenic tumors. As cabozantinib inhibits multiple tyrosine kinase receptors, including VEGFRs, we tested whether markers of angiogenesis, including microvascular density (MVD) and mast cell density (MCD), could predict benefit from cabozantinib versus everolimus, using RCC samples from the METEOR (NCT01865747) trial. EXPERIMENTAL DESIGN: MVD and MCD were studied in 430 patients (cabozantinib = 216, everolimus = 214) by double immunohistochemistry for CD31 (vascular marker) and tryptase (mast cell marker) coupled with automated image analysis. Results from evaluable cases (MVD = 360, MCD = 325) were correlated with progression-free survival (PFS), overall survival (OS), and objective response rate (ORR). RESULTS: MVD was positively correlated with MCD. In the whole cohort, high MVD and high MCD were associated with longer PFS; improved PFS was most evident in patients with high levels of both MCD and MVD. Cabozantinib was associated with improved PFS, OS, and ORR compared with everolimus, irrespective of MVD levels. Cabozantinib was also associated with improved ORR compared with everolimus, irrespective of MCD levels. For PFS and OS, the treatment effect for cabozantinib versus everolimus tended to be greater in tumors with low MCD. CONCLUSIONS: High MVD and high MCD are associated with improved outcome in mccRCC but do not predict efficacy to cabozantinib versus everolimus. The high efficacy of cabozantinib in low angiogenic tumors allows us to speculate that its antitumor activity is not exclusively mediated by VEGFR inhibition.

Outcomes based on plasma biomarkers in METEOR, a randomized phase 3 trial of cabozantinib vs everolimus in advanced renal cell carcinoma.

BACKGROUND: In the phase 3 METEOR trial, cabozantinib improved progression-free survival (PFS) and overall survival (OS) versus everolimus in patients with advanced RCC after prior antiangiogenic therapy. METHODS: In this exploratory analysis, plasma biomarkers from baseline and week 4 from 621 of 658 randomized patients were analyzed for CA9, HGF, MET, GAS6, AXL, VEGF, VEGFR2, and IL-8. PFS and OS were analyzed by baseline biomarker levels as both dichotomized and continuous variables using univariate and multivariable methods. For on-treatment changes, PFS and OS were analyzed using fold change in biomarker levels at week 4. Biomarkers were considered prognostic if p < 0.05 and predictive if pinteraction < 0.05 for the interaction between treatment and biomarker. RESULTS: Hazard ratios for PFS and OS favored cabozantinib versus everolimus for both low and high baseline levels of all biomarkers (hazard ratios ≤0.78). In univariate analyses, low baseline HGF, AXL, and VEGF were prognostic for improvements in both PFS and OS with cabozantinib, and low HGF was prognostic for improvements in both PFS and OS with everolimus. Low AXL was predictive of relative improvement in PFS for cabozantinib versus everolimus. Results were generally consistent when baseline biomarkers were expressed as continuous variables, although none were predictive of benefit with treatment. In multivariable analysis, low baseline HGF was independently prognostic for improved PFS for both cabozantinib and everolimus; low HGF, GAS6, and VEGF were independently prognostic for improved OS with cabozantinib. No biomarkers were independently prognostic for OS with everolimus. On-treatment increases in some biomarkers appeared prognostic for PFS or OS with cabozantinib in univariate analyses; however, none were independently prognostic in multivariable analysis. CONCLUSIONS: PFS and OS were improved with cabozantinib versus everolimus at high and low baseline levels of all biomarkers. Low baseline HGF was consistently identified as a prognostic biomarker for improved PFS or OS with cabozantinib or everolimus, supporting further prospective evaluation of the prognostic significance of HGF in advanced RCC. TRIAL REGISTRATION: ClinicalTrials.gov NCT01865747 (registered on 05/31/2013).

Immune-related cutaneous adverse events due to checkpoint inhibitors.

OBJECTIVE: To familiarize the reader with the most common cutaneous adverse events with immune checkpoint inhibitors (CPIs) and their grading and treatment. DATA SOURCES: Recent research articles, relevant review articles, and case series/reports in English from the PubMed database mostly, from 2010 onward. STUDY SELECTIONS: Most data are from retrospective studies and case series. Older studies regarding the mechanism were included if they were of particular importance. RESULTS: An understanding of this review should enable the reader to identify specific skin disorders in patients receiving immune CPIs, grade the adverse event, and be able to treat or refer the patient as needed. CONCLUSION: Allergists/immunologists need to be familiar with these immune-related cutaneous adverse events because their incidence will increase with the ever-expanding use of CPIs and, in particular, because patients will certainly continue to be referred suspecting drug allergies.

Transport-Based Modeling of Bubble Nucleation on Gas Evolving Electrodes.

Bubble nucleation is ubiquitous in gas evolving reactions that are instrumental for a variety of electrochemical systems. Fundamental understanding of the nucleation process, which is critical to system optimization, remains limited as prior works generally focused on the thermodynamics and have not considered the coupling between surface geometries and different forms of transport in the electrolytes. Here, we establish a comprehensive transport-based model framework to identify the underlying mechanism for bubble nucleation on gas evolving electrodes. We account for the complex effects on the electrical field, ion migration, ion diffusion, and gas diffusion arising from surface heterogeneities and gas pockets initiated from surface crevices. As a result, we show that neglecting these effects leads to significant underprediction of the energy needed for nucleation. Our model provides a non-monotonic relationship between the surface cavity size and the overpotential required for nucleation, which is physically more consistent than the monotonic relationship suggested by a traditional thermodynamics-based model. We also identify the significance of the gas diffuse layer thickness, a parameter controlled by external flow fields and overall electrode geometries, which has been largely overlooked in previous models. Our model framework offers guidelines for practical electrochemical systems whereby, without changing the surface chemistry, nucleation on electrodes can be tuned by engineering the cavity size and the gas diffuse layer thickness.

Cabozantinib in Combination with Immunotherapy for Advanced Renal Cell Carcinoma and Urothelial Carcinoma: Rationale and Clinical Evidence.

The treatment landscape for metastatic renal cell carcinoma (mRCC) and urothelial carcinoma (mUC) has evolved rapidly in recent years with the approval of several checkpoint inhibitors. Despite these advances, survival rates for metastatic disease remain poor, and additional strategies will be needed to improve the efficacy of checkpoint inhibitors. Combining anti-VEGF/VEGFR agents with checkpoint inhibitors has emerged as a potential strategy to advance the immunotherapy paradigm, because VEGF inhibitors have immunomodulatory potential. Cabozantinib is a tyrosine kinase inhibitor (TKI) whose targets include MET, AXL, and VEGFR2. Cabozantinib has a unique immunomodulatory profile and has demonstrated clinical efficacy as a monotherapy in mRCC and mUC, making it a potentially suitable partner for checkpoint inhibitor therapy. In this review, we summarize the current status of immunotherapy for mRCC and mUC and discuss the development of immunotherapy-TKI combinations, with a focus on cabozantinib. We discuss the rationale for such combinations based on our growing understanding of the tumor microenvironment, and we review in detail the preclinical and clinical studies supporting their use.

PD-L1 Expression and Clinical Outcomes to Cabozantinib, Everolimus, and Sunitinib in Patients with Metastatic Renal Cell Carcinoma: Analysis of the Randomized Clinical Trials METEOR and CABOSUN.

PURPOSE: Programmed death-ligand 1 (PD-L1) status by IHC is prognostic in metastatic renal cell carcinoma (mRCC), and its role as a potential predictive biomarker is under investigation. Using tumor tissue from the METEOR (NCT01865747) and CABOSUN (NCT01835158) clinical trials, we explored whether PD-L1 expression and the extent of the immune cell infiltrate can serve as prognostic and/or predictive biomarkers for cabozantinib and other targeted agents. EXPERIMENTAL DESIGN: IHC double staining for PD-L1 and CD45/CD163 (immune cell markers) was performed on tumor tissue from METEOR (n = 306) and CABOSUN (n = 110) clinical trials. Immune cell density and MET expression levels were also analyzed. Our primary aim was to correlate progression-free survival (PFS) by independent central review with PD-L1 status in patients treated with cabozantinib, everolimus (METEOR), or sunitinib (CABOSUN). Overall survival (OS) was also interrogated. RESULTS: Tumor cell (TC) PD-L1 expression (≥1% cutoff) was detected in 29% and 23% of tumors from patients in the METEOR and CABOSUN trials, respectively. On univariate analysis, patients with PD-L1-positive TC had poorer PFS and OS than patients with PD-L1-negative TC on both trials, independent of therapy. On multivariable analysis and when combining the two trials, the association between TC PD-L1 expression and OS was statistically significant for all patients (P = 0.034) and for patients treated with cabozantinib only (P = 0.038). Cabozantinib was associated with improved PFS (HR < 0.70) and OS (HR < 0.85) compared with everolimus and sunitinib irrespective of PD-L1 expression. CONCLUSIONS: Higher PD-L1 expression results in worse clinical outcomes in mRCC treated with targeted therapy. Furthermore, PD-L1 expression is not predictive of response to cabozantinib therapy.

Multiple Reaction Monitoring for Direct Quantitation of Intact Proteins Using a Triple Quadrupole Mass Spectrometer.

Methods that can efficiently and effectively quantify proteins are needed to support increasing demand in many bioanalytical fields. Triple quadrupole mass spectrometry (QQQ-MS) is sensitive and specific, and it is routinely used to quantify small molecules. However, low resolution fragmentation-dependent MS detection can pose inherent difficulties for intact proteins. In this research, we investigated variables that affect protein and fragment ion signals to enable protein quantitation using QQQ-MS. Collision induced dissociation gas pressure and collision energy were found to be the most crucial variables for optimization. Multiple reaction monitoring (MRM) transitions for seven standard proteins, including lysozyme, ubiquitin, cytochrome c from both equine and bovine, lactalbumin, myoglobin, and prostate-specific antigen (PSA) were determined. Assuming the eventual goal of applying such methodology is to analyze protein in biological fluids, a liquid chromatography method was developed. Calibration curves of six standard proteins (excluding PSA) were obtained to show the feasibility of intact protein quantification using QQQ-MS. Linearity (2-3 orders), limits of detection (0.5-50 µg/mL), accuracy (<5% error), and precision (1%-12% CV) were determined for each model protein. Sensitivities for different proteins varied considerably. Biological fluids, including human urine, equine plasma, and bovine plasma were used to demonstrate the specificity of the approach. The purpose of this model study was to identify, study, and demonstrate the advantages and challenges for QQQ-MS-based intact protein quantitation, a largely underutilized approach to date.

PARP1 expression, activity and ex vivo sensitivity to the PARP inhibitor, talazoparib (BMN 673), in chronic lymphocytic leukaemia.

In chronic lymphocytic leukemia (CLL), mutation and loss of p53 and ATM abrogate DNA damage signalling and predict poorer response and shorter survival. We hypothesised that poly (ADP-ribose) polymerase (PARP) activity, which is crucial for repair of DNA breaks induced by oxidative stress or chemotherapy, may be an additional predictive biomarker and a target for therapy with PARP inhibitors.We measured PARP activity in 109 patient-derived CLL samples, which varied widely (192 - 190052 pmol PAR/106 cells) compared to that seen in healthy volunteer lymphocytes (2451 - 7519 pmol PAR/106 cells). PARP activity was associated with PARP1 protein expression and endogenous PAR levels. PARP activity was not associated with p53 or ATM loss, Binet stage, IGHV mutational status or survival, but correlated with Bcl-2 and Rel A (an NF-kB subunit). Levels of 8-hydroxy-2'-deoxyguanosine in DNA (a marker of oxidative damage) were not associated with PAR levels or PARP activity. The potent PARP inhibitor, talazoparib (BMN 673), inhibited CD40L-stimulated proliferation of CLL cells at nM concentrations, independently of Binet stage or p53/ATM function.PARP activity is highly variable in CLL and correlates with stress-induced proteins. Proliferating CLL cells (including those with p53 or ATM loss) are highly sensitive to the PARP inhibitor talazoparib.

Affinity mesh screen materials for selective extraction and analysis of antibiotics using transmission mode desorption electrospray ionization mass spectrometry.

The extraction of active compounds from natural sources has shown to be an effective approach to drug discovery. However, the isolation and identification of natural products from complex extracts can be an arduous task. A novel approach to drug discovery is presented through the use of polymer screens functionalized with an l-lysine-d-alanine-d-alanine (Kaa) peptide to create new affinity capture mesh screen materials. The Kaa sequence is a well-characterized specific binding site for antibiotics that inhibit cell wall synthesis in Gram-positive bacteria. The detailed synthesis and characterization of these novel screen materials are presented in this work. Polypropylene mesh screens were first coated with a poly(acrylic acid) film by pulsed plasma polymerization. The synthesized Kaa peptide was then covalently attached to carboxylic acid groups through a condensation reaction. An analysis of captured compounds was performed in a rapid fashion with transmission-mode desorption electrospray ionization (TM-DESI) mass spectrometry. A proof of principle was demonstrated to show the ability of the novel affinity capture materials to select for a macrocyclic antibiotic, vancomycin, over a negative control compound, spectinomycin. With further development, this method may provide a rapid screening technique for new antibacterial compounds, for example, those extracted from natural product sources having a limited supply. Here, we show that the screen can capture vancomycin preferentially over spectinomycin in a spiked extract of tea leaves.

Immersion condensation on oil-infused heterogeneous surfaces for enhanced heat transfer.

Enhancing condensation heat transfer is important for broad applications from power generation to water harvesting systems. Significant efforts have focused on easy removal of the condensate, yet the other desired properties of low contact angles and high nucleation densities for high heat transfer performance have been typically neglected. In this work, we demonstrate immersion condensation on oil-infused micro and nanostructured surfaces with heterogeneous coatings, where water droplets nucleate immersed within the oil. The combination of surface energy heterogeneity, reduced oil-water interfacial energy, and surface structuring enabled drastically increased nucleation densities while maintaining easy condensate removal and low contact angles. Accordingly, on oil-infused heterogeneous nanostructured copper oxide surfaces, we demonstrated approximately 100% increase in heat transfer coefficient compared to state-of-the-art dropwise condensation surfaces in the presence of non-condensable gases. This work offers a distinct approach utilizing surface chemistry and structuring together with liquid-infusion for enhanced condensation heat transfer.

Hydrogen sulfide is an endogenous potentiator of T cell activation.

H(2)S is an endogenous signaling molecule that may act via protein sulfhydrylation to regulate various physiological functions. H(2)S is also a byproduct of dietary sulfate metabolism by gut bacteria. Inflammatory bowel diseases such as ulcerative colitis are associated with an increase in the colonization of the intestine by sulfate reducing bacteria along with an increase in H(2)S production. Consistent with its increased production, H(2)S is implicated as a mediator of ulcerative colitis both in its genesis or maintenance. As T cells are well established mediators of inflammatory bowel disease, we investigated the effect of H(2)S exposure on T cell activation. Using primary mouse T lymphocytes (CD3+), OT-II CD4+ T cells, and the human Jurkat T cell line, we show that physiological levels of H(2)S potentiate TCR-induced activation. Nanomolar levels of H(2)S (50-500 nM) enhance T cell activation assessed by CD69 expression, interleukin-2 expression, and CD25 levels. Exposure of T cells to H(2)S dose-dependently enhances TCR-stimulated proliferation with a maximum at 300 nM (30% increase, p < 0.01). Furthermore, activation increases the capacity of T cells to make H(2)S via increased expression of cystathionine γ-lyase and cystathionine ß-synthase. Disrupting this response by silencing these H(2)S producing enzymes impairs T cell activation, and proliferation and can be rescued by the addition of 300 nM H(2)S. Thus, H(2)S represents a novel autocrine immunomodulatory molecule in T cells.

Role for 53BP1 Tudor domain recognition of p53 dimethylated at lysine 382 in DNA damage signaling.

Modification of histone proteins by lysine methylation is a principal chromatin regulatory mechanism (Shi, Y., and Whetstine, J. R. (2007) Mol. Cell 25, 1-14). Recently, lysine methylation has been shown also to play a role in regulating non-histone proteins, including the tumor suppressor protein p53 (Huang, J., and Berger, S. L. (2008) Curr. Opin. Genet. Dev. 18, 152-158). Here, we identify a novel p53 species that is dimethylated at lysine 382 (p53K382me2) and show that the tandem Tudor domain of the DNA damage response mediator 53BP1 acts as an "effector" for this mark. We demonstrate that the 53BP1 tandem Tudor domain recognizes p53K382me2 with a selectivity relative to several other protein lysine methylation sites and saturation states. p53K382me2 levels increase with DNA damage, and recognition of this modification by 53BP1 facilitates an interaction between p53 and 53BP1. The generation of p53K382me2 promotes the accumulation of p53 protein that occurs upon DNA damage, and this increase in p53 levels requires 53BP1. Taken together, our study identifies a novel p53 modification, demonstrates a new effector function for the 53BP1 tandem Tudor domain, and provides insight into how DNA damage signals are transduced to stabilize p53.

Modulation of p53 function by SET8-mediated methylation at lysine 382.

Reversible covalent methylation of lysine residues on histone proteins constitutes a principal molecular mechanism that links chromatin states to diverse biological outcomes. Recently, lysine methylation has been observed on nonhistone proteins, suggesting broad cellular roles for the enzymes generating and removing methyl moieties. Here we report that the lysine methyltransferase enzyme SET8/PR-Set7 regulates the tumor suppressor protein p53. We find that SET8 specifically monomethylates p53 at lysine 382 (p53K382me1). This methylation event robustly suppresses p53-mediated transcription activation of highly responsive target genes but has little influence on weak targets. Further, depletion of SET8 augments the proapoptotic and checkpoint activation functions of p53, and accordingly, SET8 expression is downregulated upon DNA damage. Together, our study identifies SET8 as a p53-modifying enzyme, identifies p53K382me1 as a regulatory posttranslational modification of p53, and begins to dissect how methylation may contribute to a dynamic posttranslational code that modulates distinct p53 functions.

Proteomic analysis of reporter genes for molecular imaging of transplanted embryonic stem cells.

Study of stem cells may reveal promising treatment for diseases. The fate and function of transplanted stem cells remain poorly defined. Recent studies demonstrate that reporter genes can monitor real-time survival of transplanted stem cells in living subjects. We examined the effects of a novel and versatile triple fusion (TF) reporter gene construction on embryonic stem (ES) cell function by proteomic analysis. Murine ES cells were stably transduced with a self-inactivating lentiviral vector containing fluorescence (firefly luciferase; Fluc), bioluminescence (monomeric red fluorescence protein; mRFP), and positron emission tomography (herpes simplex virus type 1 truncated thymidine kinase; tTK) reporter genes. Fluorescence-activated cell sorting (FACS) analysis isolated stably transduced populations. TF reporter gene effects on cellular function were evaluated by quantitative proteomic profiling of control ES cells versus ES cells stably expressing the TF construct (ES-TF). Overall, no significant changes in protein quantity were observed. TF reporter gene expression had no effect on ES cell viability, proliferation, and differentiation capability. Molecular imaging studies tracked ES-TF cell survival and proliferation in living animals. In summary, this is the first proteomic study, demonstrating the unique potential of reporter gene imaging for tracking ES cell transplantation non-invasively, repetitively, and quantitatively.

Monomeric IgE enhances human mast cell chemokine production: IL-4 augments and dexamethasone suppresses the response.

BACKGROUND: Mouse monoclonal IgE antibodies can promote the survival of mouse bone marrow-derived cultured mast cells and induce the cells to secrete mediators in the absence of known specific antigen. OBJECTIVE: To determine whether human IgE, in the absence of known specific antigen, had effects on the mediator secretion or survival of human mast cells. METHODS: We tested whether human IgE induced human cord blood-derived mast cells to secrete mediators or enhanced their survival on withdrawal of stem cell factor. RESULTS: Exposure to IgE, but not IgG, at concentrations as low as 2.5 microg/mL significantly enhanced the release of IL-8 and monocyte chemoattractant protein 1, but not histamine or cysteinyl leukotrienes. However, under the conditions tested, chemokine production in response to IgE alone was significantly less than that induced when aliquots of the same IgE-sensitized populations of human mast cells were stimulated with anti-IgE. The production of IL-8 and monocyte chemoattractant protein 1 in response to either IgE alone or IgE and anti-IgE was enhanced by preincubation of the cells in IL-4 and was inhibited by preincubation of the cells with dexamethasone. By contrast, we did not detect any ability of IgE to enhance mast cell survival on withdrawal of stem cell factor. CONCLUSION: Exposure to human IgE in vitro in the absence of known specific antigen can enhance chemokine production by human mast cells, and this secretory response can be enhanced by preincubation of the mast cells with IL-4 and can be suppressed by dexamethasone.

Functional proteomics of the active cysteine protease content in Drosophila S2 cells.

The fruit fly genome is characterized by an evolutionary expansion of proteases and immunity-related genes. In order to characterize the proteases that are active in a phagocytic Drosophila model cell line (S2 cells), we have applied a functional proteomics approach that allows simultaneous detection and identification of multiple protease species. DCG-04, a biotinylated, mechanism-based probe that covalently targets mammalian cysteine proteases of the papain family was found to detect Drosophila polypeptides in an activity-dependent manner. Chemical tagging combined with tandem mass spectrometry permitted retrieval and identification of these polypeptides. Among them was thiol-ester motif-containing protein (TEP) 4 which is involved in insect innate immunity and shares structural and functional similarities with the mammalian complement system factor C3 and the pan-protease inhibitor alpha2-macroglobulin. We also found four cysteine proteases with homologies to lysosomal cathepsin (CTS) L, K, B, and F, which have been implicated in mammalian adaptive immunity. The Drosophila CTS equivalents were most active at a pH of 4.5. This suggests that Drosophila CTS are, similar to their mammalian counterparts, predominantly active in lysosomal compartments. In support of this concept, we found CTS activity in phagosomes of Drosophila S2 cells. These results underscore the utility of activity profiling to address the functional role of insect proteases in immunity.