Translation and evaluation of a pre-clinical 5-protein response prediction signature in a breast cancer phase Ib clinical trial.

Human protein biomarker discovery relies heavily on pre-clinical models, in particular established cell lines and patient-derived xenografts, but confirmation studies in primary tissue are essential to demonstrate clinical relevance. We describe in this study the process that was followed to clinically translate a 5-protein response signature predictive for the activity of an anti-HER3 monoclonal antibody (lumretuzumab) originally measured in fresh frozen xenograft tissue. We detail the development, qualification, and validation of the multiplexed targeted mass spectrometry assay used to assess the signature performance in formalin-fixed, paraffin-embedded human clinical samples collected in a phase Ib trial designed to evaluate lumretuzumab in patients with metastatic breast cancer.

Retrospective Assessment of a Serum Proteomic Test in a Phase III Study Comparing Erlotinib plus Placebo with Erlotinib plus Tivantinib (MARQUEE) in Previously Treated Patients with Advanced Non-Small Cell Lung Cancer.

Background: The VeriStrat test provides accurate predictions of outcomes in all lines of therapy for patients with non-small cell lung cancer (NSCLC). We investigated the predictive and prognostic role of VeriStrat in patients enrolled on the MARQUEE phase III trial of tivantinib plus erlotinib (T+E) versus placebo plus erlotinib (P+E) in previously treated patients with advanced NSCLC.

Methods: Pretreatment plasma samples were available for 996 patients and were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry to generate VeriStrat labels (good, VS-G, or poor, VS-P).

Therapeutically Induced Changes in HER2, HER3, and EGFR Protein Expression for Treatment Guidance.

Protein-targeted therapies are expected to selectively kill tumor cells that express the targeted protein biomarker. Although a tumor mass may initially respond to targeted therapies based on expression of the targeted protein, all cells within a tumor may not express the targeted protein above a critical threshold level; therefore, those cells that do not express, or that downregulate expression of, the targeted protein may not be responsive to therapy. The ability to monitor the dynamic expression of these protein biomarkers throughout the course of therapy may allow for treatment to be personalized in real-time in response to the evolving nature of the tumor.

High-Throughput Microdissection for Next-Generation Sequencing.

Precision medicine promises to enhance patient treatment through the use of emerging molecular technologies, including genomics, transcriptomics, and proteomics. However, current tools in surgical pathology lack the capability to efficiently isolate specific cell populations in complex tissues/tumors, which can confound molecular results. Expression microdissection (xMD) is an immuno-based cell/subcellular isolation tool that procures targets of interest from a cytological or histological specimen.

Mass-spectrometry-based quantitation of Her2 in gastroesophageal tumor tissue: comparison to IHC and FISH.

Background: Trastuzumab has shown a survival benefit in cases of Her2-positive gastroesophageal cancer (GEC). Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) currently determine eligibility for trastuzumab-based therapy. However, these low-throughput assays often produce discordant or equivocal results.

The serotonin 5-HT(2A) receptor agonist TCB-2: a behavioral and neurophysiological analysis.

Rationale: There are few reports on the high-affinity 5-HT(2A) agonist (4-Bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (TCB-2).

Objectives: Here we provide the first behavioral and neurophysiological profile of TCB-2 in C57BL/6J mice, with direct comparisons to the 5-HT(2A/2C) agonist (+/-)-2,5-dimethoxy-4-iodophenyl-2-aminopropane (DOI), in addition to determinations of 5-HT(2A) mediation via pretreatment with the selective 5-HT(2A) antagonist MDL 11,939.

Results: In a dose-dependent manner, TCB-2 induced head twitches, decreased food consumption in food-deprived mice, induced hypothermia, and increased corticosterone levels, with no effects on locomotor activity or anxiety-like behaviors in the open field.

Chromatographic benefits of elevated temperature for the proteomic analysis of membrane proteins.

Integral membrane proteins (IMPs) perform crucial cellular functions and are the primary targets for most pharmaceutical agents. However, the hydrophobic nature of their membrane-embedded domains and their intimate association with lipids make them difficult to handle. Numerous proteomic platforms that include LC separations have been reported for the high-throughput profiling of complex protein samples.

A shotgun proteomic method for the identification of membrane-embedded proteins and peptides.

Integral membrane proteins perform crucial cellular functions and are the targets for the majority of pharmaceutical agents. However, the hydrophobic nature of their membrane-embedded domains makes them difficult to work with. Here, we describe a shotgun proteomic method for the high-throughput analysis of the membrane-embedded transmembrane domains of integral membrane proteins which extends the depth of coverage of the membrane proteome.

Proteomic characterization of membrane protein topology.

Integral membrane proteins are represented by 20-30% of the eukaryotic genome and crucial for cellular functions including cell signaling, nutrient influx, toxin efflux, and maintenance of osmotic balance. Importantly, over 70% of all drugs are targeted at membrane proteins. Because of their hydrophobicity, however, methods used to characterize the structure of soluble proteins, such as NMR and X-ray crystallography, are generally not suitable to the study of membrane proteins (1).

Label-free comparative analysis of proteomics mixtures using chromatographic alignment of high-resolution muLC-MS data.

Label-free relative quantitative proteomics is a powerful tool for the survey of protein level changes between two biological samples. We have developed and applied an algorithm using chromatographic alignment of microLC-MS runs to improve the detection of differences between complex protein mixtures. We demonstrate the performance of our software by finding differences in E.

Shotgun analysis of integral membrane proteins facilitated by elevated temperature.

The beneficial effects on peak selectivity and resolution of conducting liquid chromatography (LC) at elevated temperature (e.g., 30-80 degrees C) are generally well-known; however, its importance for peptide recovery is not nearly as well recognized.

Quantitative comparison of proteomic data quality between a 2D and 3D quadrupole ion trap.

A 2D ion trap has a greater ion trapping efficiency, greater ion capacity before observing space-charging effects, and a faster ion ejection rate than a traditional 3D ion trap mass spectrometer. These hardware improvements should result in a significant increase in protein identifications from complex mixtures analyzed using shotgun proteomics. In this study, we compare the quality and quantity of peptide identifications using data-dependent acquisition of tandem mass spectra of peptides between two commercially available ion trap mass spectrometers (an LTQ and an LCQ XP Max).