Safety and efficacy of AMG 820, an anti-colony-stimulating factor 1 receptor antibody, in combination with pembrolizumab in adults with advanced solid tumors.

Background: To determine the safety and efficacy of the anti-colony-stimulating factor 1 receptor (anti-CSF1R) monoclonal antibody AMG 820 in combination with pembrolizumab in patients with select solid tumors.

Patients And Methods: Patients had advanced, refractory mismatch repair-proficient colorectal cancer, pancreatic cancer, or non-small cell lung cancer (NSCLC) with low (<50%) programmed cell death-ligand 1 (PD-L1) expression and were naïve to anti-programmed cell death-1 (PD-1)/PD-L1 or had relapsed/refractory NSCLC after anti-PD-1/PD-L1 treatment with low or high (≥50%) PD-L1 expression; all were anti-CSF1/CSF1R naïve. Patients received 1100 mg or 1400 mg AMG 820 plus 200 mg pembrolizumab intravenously every 3 weeks.

Heterogeneity in the linear shiny white structures in melanomas seen with polarized light according to histopathological association: Cross-sectional observational study in 118 cutaneous melanomas.

Polarized dermoscopy enables visualization of linear shiny white structures in melanomas, thought to be due to the existence of fibrosis in the dermis. Our objective was to establish the existence of two types of linear shiny white structures and assess their association with different histological structures. We performed a cross-sectional study including all non-acral, non-facial melanomas from our hospital with linear shiny white structures.

Dual Targeting of Aurora Kinases with AMG 900 Exhibits Potent Preclinical Activity Against Acute Myeloid Leukemia with Distinct Post-Mitotic Outcomes.

Aurora kinase A and B have essential and non-overlapping roles in mitosis, with elevated expression in a subset of human cancers, including acute myeloid leukemia (AML). In this study, pan-aurora kinase inhibitor (AKI) AMG 900 distinguishes itself as an anti-leukemic agent that is more uniformly potent against a panel of AML cell lines than are isoform-selective AKIs and classic AML drugs. AMG 900 inhibited AML cell growth by inducing polyploidization and/or apoptosis.

Dose escalation results from a first-in-human, phase 1 study of glucocorticoid-induced TNF receptor-related protein agonist AMG 228 in patients with advanced solid tumors.

Background: This open-label, first-in-human, phase 1 study evaluated the safety, pharmacokinetics, pharmacodynamics, and maximum tolerated dose (MTD) of AMG 228, an agonistic human IgG1 monoclonal antibody targeting glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR), in patients with refractory advanced solid tumors.

Methods: AMG 228 was administered intravenously every 3 weeks (Q3W). Dose escalation was in two stages: single-patient cohorts (3, 9, 30, and 90 mg), followed by "rolling six" design (n = 2-6; 180, 360, 600, 900, and 1200 mg).

A phase 1, first-in-human study of AMG 900, an orally administered pan-Aurora kinase inhibitor, in adult patients with advanced solid tumors.

Background Aurora kinase overexpression or amplifications are associated with high proliferation, poor prognosis, and therapeutic resistance in human tumors. AMG 900 is an investigational, oral, selective pan-Aurora kinase inhibitor. Methods This first-in-human trial included dose-escalation and dose-expansion phases ( ClinicalTrials.

A phase 1 study of AMG 900, an orally administered pan-aurora kinase inhibitor, in adult patients with acute myeloid leukemia.

Aurora kinases are involved in the pathophysiology of several cancers including acute myeloid leukemia (AML). In this phase 1 study, we investigated the safety and efficacy of AMG 900, an orally administered, highly potent, selective, small-molecule inhibitor of both Aurora kinase A and B, in patients with AML . Patients with pathologically documented AML who either declined standard treatments or had relapsed from or were refractory to previous therapies were enrolled.

AMG 900, a potent inhibitor of aurora kinases causes pharmacodynamic changes in p-Histone H3 immunoreactivity in human tumor xenografts and proliferating mouse tissues.

Background: The Aurora family of serine-threonine kinases are essential regulators of cell division in mammalian cells. Aurora-A and -B expression and kinase activity is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis. AMG 900 is a highly potent and selective pan-aurora kinase inhibitor that has entered clinical evaluation in adult patients with advanced cancers.

A method to quantitate the neutralizing capacity of anti-therapeutic protein antibodies in serum and their correlation to clinical impact.

A robust, quantitative method for assessing the neutralizing capacity of anti-therapeutic protein antibodies was developed and tested using 4 analytical assay platforms typically used for detection of anti-drug neutralizing antibodies. The method described here utilized titration of increasing concentrations of therapeutic protein into serum containing anti-therapeutic protein antibodies, either positive control antibodies or clinical samples. Neutralizing capacities were calculated by determining the EC50 from the titration curves.

Three-dimensional second-harmonic generation imaging of fibrillar collagen in biological tissues.

Multiphoton-induced second-harmonic generation (SHG) has developed into a very powerful approach for in depth visualization of some biological structures with high specificity. In this unit, we describe the basic principles of three-dimensional SHG microscopy. In addition, we illustrate how SHG imaging can be utilized to assess collagen fibrils in biological tissues.

Epitope characterization of pre-existing and developing antibodies to an aglycosylated monoclonal antibody therapeutic of G1m17,1 allotype.

Allotypes of IgG1 molecules can influence the immunogenicity of therapeutic monoclonal antibodies and may account for the presence of some pre-existing antibodies. An electrochemiluminescent (ECL) bridging immunoassay was used to characterize the binding epitopes of anti-therapeutic antibodies (ATAs) in a Phase 1 single ascending dose clinical trial of a therapeutic aglycosylated IgG1monoclonal antibody (mAb). There was no evidence for ATAs specific for a possible neo-epitope created due to the lack of glycosylation.

Leveraging image cytometry for the development of clinically feasible biomarkers: evaluation of activated caspase-3 in fine needle aspirate biopsies.

Quantitation of activated caspases in xenograft models by laser scanning cytometry has demonstrated mechanism-specific biological activity of Anti-Trail Receptor immunoglobulin therapies in situ. These preclinical data confirmed that caspase activation is an early event that precedes tumor regression. To apply this platform for clinical monitoring of caspase activation using fine needle aspirate (FNA) biopsies, additional assay feasibility and validation experiments need be addressed.

A first-in-human study of conatumumab in adult patients with advanced solid tumors.

Purpose: To determine the safety, tolerability, pharmacokinetics, and maximum tolerated dose (MTD) of conatumumab, an investigational, fully human monoclonal agonist antibody against human death receptor 5, in patients with advanced solid tumors.

Experimental Design: In the dose-escalation phase, patients received escalating intravenous doses of conatumumab (0.3, 1, 3, 10, or 20 mg/kg, 3-9 per cohort) every 2 weeks.

Measurement of conatumumab-induced apoptotic activity in tumors by fine needle aspirate sampling.

Conatumumab is a monoclonal antibody specific for death receptor 5 (DR5) that activates caspases leading to DNA fragmentation and tumor-cell death. Like other Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) receptor therapies, conatumumab is currently being evaluated in clinical trials across a variety of tumor types. However, molecular evidence of on-target drug activity in tumors is often an elusive goal for clinical investigation.

Conatumumab, a fully human agonist antibody to death receptor 5, induces apoptosis via caspase activation in multiple tumor types.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to death receptors 4 and 5 (DR4, DR5) to transduce apoptotic signals. Conatumumab (AMG 655) is an investigational, fully human monoclonal agonist antibody (IgG(1)) to human DR5, which induces apoptosis via caspase activation. In this study, we demonstrate that conatumumab binds to DR5, activating intracellular caspases in vitro in the presence of a cross-linker.

BRCA1 protein and nucleolin colocalize in breast carcinoma tissue and cancer cell lines.

The breast and ovarian cancer susceptibility gene BRCA1 encodes a tumor suppressor. BRCA1 protein, which is involved in DNA damage response, has been thought to be found primarily in cell nuclei. In the present investigation, immunohistological studies of BRCA1 protein in frozen breast cancer tissue and MCF7 and HeLa cell lines revealed BRCA1 expression in both nucleoli and nucleoplasmic foci.

Antagonists of CD117 (cKit) signaling inhibit mast cell accumulation in healing skin wounds.

Mast cells (MCs) have important functional roles in leukocyte recruitment, pain, and wound healing, and increased tissue resident MC function has been associated with several fibrotic diseases. Consequently, the study of MCs in situ can be a direct approach to studying the pharmacodynamic impact of MC-directed therapeutics in tissues. Here we describe an automated laser scanning cytometry assay that was used to characterize the kinetics of MC accumulation in healing skin wounds and to study the effect of inhibiting CD117 (cKit) signaling.

Differential staining of DNA and RNA.

Cell cycle analysis by means of differential staining of RNA and DNA permits determination of RNA content, which in turn allows one to discriminate G(0) versus G(1) cells and to detect cell differentiation. This unit presents two protocols for differential staining, one using the metachromatic dye acridine orange (AO) and the other a combination of pyronin Y (PY) and Hoechst 33342. Each method has its advantages and limitations, and the Hoechst-PY method is not applicable to single-laser instruments.

Detection of mitotic cells.

In preparation for cell division, nuclear chromatin undergoes a vital rearrangement required for the organization of chromosomes and their allocation to daughter cells. This process is initiated during G(2) phase with the most remarkable morphological manifestation being chromatin condensation. This unit provides protocols for identification and quantification of mitotic cells based on immunocytochemical detection of histone H3 phosphorylated on Ser 10 (H3-P), the critical event occurring during the G(2) to M transition (essential for chromatin condensation), using anti-H3-P, a commercially available antibody to which apoptotic cells are not reactive, concurrently with differential staining of cellular DNA.

Comprehensive analysis of tumoral DNA content reveals clonal ploidy heterogeneity as a marker with prognostic significance in locoregional neuroblastoma.

The clinical management of locoregional neuroblastoma (LR NB) is controversial. In a previous study we showed that diploidy was a strong prognostic predictor of outcome and detected the existence of clonal ploidy heterogeneity in a select group of cases. The aims of this study were (1) to assess the frequency of ploidy heterogeneity in LR NB, (2) to ascertain the best method to detect heteterogeneity, and (3) to correlate ploidy populations with clinical outcome.

Rb inactivation promotes genomic instability by uncoupling cell cycle progression from mitotic control.

Advanced human cancers are invariably aneuploid, in that they harbour cells with abnormal chromosome numbers. However, the molecular defects underlying this trait, and whether they are a cause or a consequence of the malignant phenotype, are not clear. Mutations that disable the retinoblastoma (Rb) pathway are also common in human cancers.

Cytometry of cell cycle regulatory proteins.

Immunocytochemical detection of cyclins, inhibitors of cyclin-dependent kinases and other cell cycle regulatory proteins, combined with analysis of cell cycle position (DNA content) by multiparameter cytometry offers unique analytical possibilities. This review focuses on applications of the methodology that yield information on the abundance, modification or interactions of these proteins in relation to cell cycle progression that cannot be obtained by other methods. Particular attention is given to the use of multiparameter cytometry in analysis of the mechanism by which antitumor drugs affect cell cycle progression and induce cell cycle phase-specific apoptosis.

Separation of live cells in different phases of the cell cycle for gene expression analysis.

Background: Homogeneity of cell populations is a basic requirement for gene expression analyses of the cell cycle, such as those based on microarrays. The most common approach to obtain specific populations is the use of synchronization methods that increase the number of cells representing a certain cell cycle stage. On the one hand, conventional synchronization usually causes undesirable effects.