Antitumor activity of AZD0754, a dnTGFβRII-armored, STEAP2-targeted CAR-T cell therapy, in prostate cancer.

Prostate cancer is generally considered an immunologically "cold" tumor type that is insensitive to immunotherapy. Targeting surface antigens on tumors through cellular therapy can induce a potent antitumor immune response to "heat up" the tumor microenvironment. However, many antigens expressed on prostate tumor cells are also found on normal tissues, potentially causing on-target, off-tumor toxicities and a suboptimal therapeutic index.

Mass spectrometry quantifies target engagement for a KRASG12C inhibitor in FFPE tumor tissue.

Background: Quantification of drug-target binding is critical for confirming that drugs reach their intended protein targets, understanding the mechanism of action, and interpreting dose-response relationships. For covalent inhibitors, target engagement can be inferred by free target levels before and after treatment. Targeted mass spectrometry assays offer precise protein quantification in complex biological samples and have been routinely applied in pre-clinical studies to quantify target engagement in frozen tumor tissues for oncology drug development.

The addition of FAIMS increases targeted proteomics sensitivity from FFPE tumor biopsies.

Mass spectrometry-based targeted proteomics allows objective protein quantitation of clinical biomarkers from a single section of formalin-fixed, paraffin-embedded (FFPE) tumor tissue biopsies. We combined high-field asymmetric waveform ion mobility spectrometry (FAIMS) and parallel reaction monitoring (PRM) to increase assay sensitivity. The modular nature of the FAIMS source allowed direct comparison of the performance of FAIMS-PRM to PRM.

Global Extraction from Parallel Reaction Monitoring to Quantify Background Peptides for Improved Normalization and Quality Control in Targeted Proteomics.

Mass spectrometry-based targeted proteomics employs heavy isotope-labeled proteins or peptides as standards to improve accuracy and precision. The input sample amount is often determined by the total quantity of endogenous proteins or peptides, as defined by spectrophotometric assays, before the heavy-isotope standards are spiked into the samples. Errors in spectrophotometric measurements, which may be due to low sensitivity or chemical or biological interference, have a direct impact on the quantitative mass spectrometry results.

High throughput profiling of undifferentiated pleomorphic sarcomas identifies two main subgroups with distinct immune profile, clinical outcome and sensitivity to targeted therapies.

Background: Undifferentiated pleomorphic sarcoma (UPS) is the most frequent, aggressive and less-characterized sarcoma subtype. This study aims to assess UPS molecular characteristics and identify specific therapeutic targets.

Methods: High-throughput technologies encompassing immunohistochemistry, RNA-sequencing, whole exome-sequencing, mass spectrometry, as well as radiomics were used to characterize three independent cohorts of 110, 25 and 41 UPS selected after histological review performed by an expert pathologist.

Identification of a Blood-Based Protein Biomarker Panel for Lung Cancer Detection.

Lung cancer is the deadliest cancer worldwide, mainly due to its advanced stage at the time of diagnosis. A non-invasive method for its early detection remains mandatory to improve patients' survival. Plasma levels of 351 proteins were quantified by Liquid Chromatography-Parallel Reaction Monitoring (LC-PRM)-based mass spectrometry in 128 lung cancer patients and 93 healthy donors.

Data-Independent Acquisition Mass Spectrometry To Quantify Protein Levels in FFPE Tumor Biopsies for Molecular Diagnostics.

Mass spectrometry-based protein quantitation is currently used to measure therapeutically relevant protein biomarkers in CAP/CLIA setting to predict likely responses of known therapies. Selected reaction monitoring (SRM) is the method of choice due to its outstanding analytical performance. However, data-independent acquisition (DIA) is now emerging as a proteome-scale clinical assay.

A new ALK isoform transported by extracellular vesicles confers drug resistance to melanoma cells.

Background: Drug resistance remains an unsolved clinical issue in oncology. Despite promising initial responses obtained with BRAF and MEK kinase inhibitors, resistance to treatment develops within months in virtually all melanoma patients.

Methods: Microarray analyses were performed in BRAF inhibitor-sensitive and resistant cell lines to identify changes in the transcriptome that might play a role in resistance.

Identification of beta-arrestin-1 as a diagnostic biomarker in lung cancer.

Background: Distinguishing lung adenocarcinoma (ADC) from squamous cell carcinoma (SCC) has a tremendous therapeutic implication. Sometimes, the commonly used immunohistochemistry (IHC) markers fail to discriminate between them, urging for the identification of new diagnostic biomarkers.

Methods: We performed IHC on tissue microarrays from two cohorts of lung cancer patients to analyse the expression of beta-arrestin-1, beta-arrestin-2 and clinically used diagnostic markers in ADC and SCC samples.

Targeted data-independent acquisition for mass spectrometric detection of RAS mutations in formalin-fixed, paraffin-embedded tumor biopsies.

Genomic testing for KRAS and NRAS mutations in clinical biopsies of various cancers is routinely performed to predict futility of anti-epidermal growth factor receptor (anti-EGFR) therapies. We hypothesized that RAS mutations could be detected and quantified at the protein level for diagnostic purposes using data-independent acquisition (DIA)-based mass spectrometry in formalin-fixed, paraffin-embedded (FFPE) tumor samples. We developed a targeted DIA assay that surveys the specific mass range of all possible peptides harboring activating mutations in KRAS exon 2.

Cell-free DNA and next-generation sequencing in the service of personalized medicine for lung cancer.

Personalized medicine has emerged as the future of cancer care to ensure that patients receive individualized treatment specific to their needs. In order to provide such care, molecular techniques that enable oncologists to diagnose, treat, and monitor tumors are necessary. In the field of lung cancer, cell free DNA (cfDNA) shows great potential as a less invasive liquid biopsy technique, and next-generation sequencing (NGS) is a promising tool for analysis of tumor mutations.

Quantification of SAA1 and SAA2 in lung cancer plasma using the isotype-specific PRM assays.

The quantification of plasma proteins using the high resolution and accurate mass (HR/AM)-based parallel reaction monitoring (PRM) method provides an immediate benefit over the conventional SRM-based method in terms of selectivity. In this study, multiplexed PRM assays were developed to analyze isotypes of serum amyloid A (SAA) proteins in human plasma with a focus on SAA1 and SAA2. Elevated plasma levels of these proteins in patients diagnosed with lung cancer have been reported in previous studies.

Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts.

Exosomes derived from solid tumor cells are involved in immune suppression, angiogenesis, and metastasis, but the role of leukemia-derived exosomes has been less investigated. The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Here, we explore the role of CLL-derived exosomes in the cellular and molecular mechanisms by which malignant cells create this favorable surrounding.

Screening protein isoforms predictive for cancer using immunoaffinity capture and fast LC-MS in PRM mode.

Purpose: We report an immunocapture strategy to extract proteins known to harbor driver mutations for a defined cancer type before the simultaneous assessment of their mutational status by MS. Such a method bypasses the sensitivity and selectivity issues encountered during the analysis of unfractionated complex biological samples.

Experimental Design: Fast LC separations using short nanobore columns hyphenated with a high-resolution quadrupole-orbitrap mass spectrometer have been devised to take advantage of fast MS cycle times in conjunction with sharp chromatographic peak widths to accelerate the sample analysis throughput.

Verification of the biomarker candidates for non-small-cell lung cancer using a targeted proteomics approach.

Lung cancer, with its high metastatic potential and high mortality rate, is the worldwide leading cause of cancer-related deaths. High-throughput "omics"-based platforms have accelerated the discovery of biomarkers for lung cancer, and the resulting candidates are to be evaluated for their diagnostic potential as noninvasive biomarkers. The evaluation of the biomarker candidates involves the quantitative measurement of large numbers of proteins in bodily fluids using advanced mass spectrometric techniques.

Targeted proteomics strategy applied to biomarker evaluation.

The evaluation of biomarker candidates, involving quantitative measurement of a large number of proteins in bodily fluids, remains the main obstruction in the development of a biomarker validation pipeline. Although immunoassays are commonly used, high-throughput and multiplex-capable methods are required for expediting the evaluation process. MS-based approaches employing targeted proteomic strategies provide not only a sensitive, but in addition a precise quantification tool, which is versatile, systematic, and scalable.

Mass spectrometry-based detection and quantification of plasma glycoproteins using selective reaction monitoring.

Mass spectrometry-based targeted proteomics is a rapidly expanding method for quantifying proteins in complex clinical samples such as plasma. In conjunction with the stable isotope dilution method, selected reaction monitoring (SRM) assays provide unparalleled sensitivity and selectivity for detection and quantification. A crucial factor for robust SRM assays is the reduction of interference by lowering the background.

Structural mass spectrometry in protein therapeutics discovery.

The protein therapeutics market is one of the highest growing segments of the pharmaceutical industry with an estimated global market value of $77 billion by 2011 (Global Protein Therapeutics Market report by RNCOS: Delhi, India, 2009). This growth has been fueled by several advantages that protein drugs can offer such as higher specificity, reduced side effects, and faster development time compared to small molecule drugs. Major pharmaceutical companies are strategically shifting gears toward protein therapeutics and gradually increasing the biologics portion of their pipelines.

Reference map for liquid chromatography-mass spectrometry-based quantitative proteomics.

The accurate mass and time (AMT) tag strategy has been recognized as a powerful tool for high-throughput analysis in liquid chromatography-mass spectrometry (LC-MS)-based proteomics. Due to the complexity of the human proteome, this strategy requires highly accurate mass measurements for confident identifications. We have developed a method of building a reference map that allows relaxed criteria for mass errors yet delivers high confidence for peptide identifications.

Reproducibility assessment of relative quantitation strategies for LC-MS based proteomics.

The reproducibility of a given method for relative quantitation governs the reliability of liquid chromatography-mass spectrometry (LC-MS) based differential analysis in proteomic studies. Understanding the noise level introduced from biological, chemical, and instrumental sources not only helps to determine the experimental design but also aids in assessing the reliability of expression ratios used for quantitation. Here we present a reproducibility assessment method for relative quantitation based on the intensity ratio distribution of common features in LC-MS replicates.

Drug target identification and quantitative proteomics.

The emerging technologies in proteomic analysis provide great opportunity for the discovery of novel therapeutic drug targets for unmet medical needs through delivering of key information on protein expression, post-translational modifications and protein-protein interactions. This review presents a summary of current quantitative proteomic concepts and mass spectrometric technologies, which enable the acceleration of target discovery. Examples of the strategies and current technologies in the target identification/validation process are provided to illustrate the successful application of proteomics in target identification, in particular for monoclonal antibody therapies.

Identification of 12Cysbeta on tubulin as the binding site of tubulyzine.

We have undertaken quantitative binding site studies in order to identify the binding site of the known microtubule destabilizing agents, the tubulyzines, in the tubulin dimer. Two different approaches were employed that utilized the tubulyzines and their derivatives. The first approach was based on a chemical affinity labeling method using tubulyzine affinity derivatives, and the second approach employed the mass spectrometric measurement of the differential reactivity of cysteines using the tubulyzines and monobromobimane.

Structural characterization of the molten globule state of apomyoglobin by limited proteolysis and HPLC-mass spectrometry.

A method to characterize the structural conformation of an acidic molten globule apomyoglobin (apoMb) at pH 4.2 was developed using limited proteolysis and HPLC-mass spectrometry (HPLC-MS). Endoproteinase Glu-C, which has a double maximum activity at pH 4.