Caught in the act: Direct identification of Ligand-Receptor interactions
Many cellular responses are triggered by proteins, drugs or pathogens binding to cell-surface receptors, but it can be challenging to identify which receptors are bound by a given ligand. Here we describe TRICEPS, a chemoproteomic reagent with three moieties—one that binds ligands containing an amino group, a second that binds glycosylated receptors on living cells and a biotin tag for purifying the receptor peptides for identification by quantitative mass spectrometry. We validated this ligand-based, receptor-capture (LRC) technology using insulin, transferrin, apelin, epidermal growth factor, the therapeutic antibody trastuzumab and two DARPins targeting ErbB2. The LRC technology enables the identification of receptors for many types of ligands under under near-physiological conditions and without the need for genetic manipulations.
Stages in Biomarker Discovery and Translation to Clinical Practice
Thousands of candidate protein biomarkers have emerged from proteome studies in recent years, but so far none of these has been successfully translated into an FDA-cleared clinical test. Recently the techniques of multiple reaction monitoring (MRM) mass spectrometry and the use of stable isotope labeled standards (SIS) have been combined to provide precise relative quantitation of proteins of clinical interest. Such MS-based assays offer substantial advantages in comparison to the antigen capture immunoassays (e.g., ELISA) used to produce most quantitative protein measurement to date: MRM-MS provides almost absolute structural specificity (via multiple parent/product ion transitions), true internal standardization (via stable isotope labeled peptide standards) and facile multiplexing of disparate peptide targets.
Story of Successful Biomarker Development (YKL-40)
Ovarian cancer is the most lethal gynecologic malignancy and because symptoms of this disease are non-specific patients frequently present with advanced stage disease and an associated poor prognosis. Currently available screening techniques, pelvic ultrasound and serum CA125, have limited sensitivity and specificity and are therefore not recommended for routine ovarian cancer screening. We investigated more than twelve novel biomarkers for ovarian cancer and more recently distal fallopian tube carcinoma that included YKL-40, HE-4, Mesothelin and Osteopontin. In addition to measurements in serum, these biomarkers were also determined in peritoneal and uterine washings. All biomarkers were significantly elevated in peritoneal and uterine washings wherein YKL-40 was elevated 12-fold greater in cancer patients compared to controls. ROC curves constructed for each biomarker in serum and washings suggest that washings provide a new and improved approach to monitoring ovarian cancer than current serum measurements.
Transitioning Proteomic Biomarkers to a Clinical Diagnostic
During the last decade of proteomics research, significant progress has been made in the advances of proteomic technology and in our understanding of the human proteome. Many potential biomarkers have been discovered. However, little progress has been achieved in the translation of these biomarkers into clinical diagnostics. To be successful, we need to develop a roadmap and identify several key steps that are critical in this process - the 4Bs (bridge), 4Gs (gate) and 4Ps (partnership) for biomarker translation. Case studies will be presented for the successful transition of proteomic biomarkers to clinical diagnostics.
Metabolomics A Global Biochemical Approach for the Study of Human Disease and Drug Effects
Metabolomics the study of metabolism at a 'global' level provides powerful tools for defining perturbations in metabolic pathways and networks in human disease. The metabolome defines a metabolic state as regulated by net interactions between gene and environment influences and provides information that can possibly bridge the gap between genotype and phenotype. It provides tools for identification of biomarkers for disease, for disease progression and for response to therapy.
A Workflow for the Discovery, Validation, and Translation of Robust Expression Based Biomarker Signatures into Clinical Assays: Applications in Prostate and Breast Cancer
For most cancer patients, it is well accepted that a one-size-fits-all approach is not suitable for treatment purposes. Individualized patient therapy requires a comprehensive understanding of the disease and also an appreciation of the genotypic variation underlying the patient’s ability to respond to medication. Each tumor is unique and so is each patient. This combination of tumor subtypes and patient heterogeneity poses challenges for individualizing therapy for any one patient. Using examples from prostate and breast cancer gene expression studies, we describe a workflow for establishing multiplex profiles to serve as robust classifiers of disease subtyping. This workflow comprises distinct phases for microarray based biomarker discovery, signature validation & refinement using principle component analysis, and additional customized bioinformatics algorithms, and assay development. We subsequently incorporate these classifiers into custom designed molecular assays that can be validated and translated in the clinical laboratory for application to the management of cancer patients.
Immunomics: Discovering New Targets for Vaccines and Therapeutics
Immunoinformatics techniques enable to us look at the world through the lens of the immune system, identifying proteins and constituent peptides that are likely to induce an immune response. I will present such an analysis that we have been doing of the human genome, a variety of bacterial and viral genomes, and the human microbiome, in order to discover leads that are suitable for vaccine development. I will also discuss our analogous characterization of therapeutic proteins and identification of possible immunogenic contaminants in the production of therapeutic proteins
Effects of Increased Oversight on the Translation of Biomarkers to Clinical Settings
New diagnostic tests must offer improved clinical utility to succeed in the marketplace. In initial stages, researchers may focus on sensitivity, AUROC, or positive/negative predictive value. During translation, those who validate the new test must consider pre—analytical issues, manufacturability, sources of clinical interference, ease of use and ease of interpretation. The current US diagnostic environment encompasses multiple options for new test introduction. There may be minimal control over raw materials, minimal clinical validation, and a risk of significant changes in performance over time. The responsibility for assuring consistent, informative results is shared by both the test developers and oversight groups that perform reviews. Different considerations may apply for the introduction of new analytes, as compared to improved methods for established analytes.
Effects of Regulation on Transition of new Biomarkers to Clinical Diagnostics
In vitro diagnostic developers face a number of hurdles in bringing innovative biomarker tests to the clinic. Furthermore, the emergence of high-throughput technologies and the use of biomarkers in guiding the selection of therapy pose additional challenges. I will focus on the regulatory issues that must be considered when developing biomarker tests, including how to evaluate the risk associated with the use of a biomarker test, what must be considered when performing an investigation of a novel biomarker test, and what types of evidence are required to demonstrate the safety and effectiveness of a test. Special attention will be paid to the special challenges involved in the regulation of omics-based tests, and to the use of biomarker tests as companion diagnostics.
Funding Issues for Biomarker Discovery, Validation, and Translation
Each stage of biomarker development faces technical, clinical, scientific, and financial challenges. The NIH and specifically, the National Cancer Institute (NCI) have developed a number of funding opportunities to enable researchers to overcome these challenges from biomarker discovery to clinical translation. Additionally, the NCI has a number of resources designed to aid affinity-based assay development. These reagent-based resources can facilitate the transition from target discovery to validation. Dr. Kinsinger will provide an overview of these programs including scientific objectives and findings, resources available, and current funding opportunities in cancer biomarker development.
Copy Number Alterations in DNA Repair/Repair-Related Genes Identify Disease-Specific Biomarkers with Potential Clinical Relevance to Hematologic Malignancies
Microarray copy number alterations (CNAs) were analyzed for 155 DNA repair-related genes in 425 cases representing diverse hematologic malignancies. Expected disease associations in RB1, TP53 and ATM were seen. Newly defined relationships included deletion of ERCC5 seen in B-ALL, with co-deletion of ERCC5 and LIG4 confined to NHL. CNAs specific to NHL (GTF2H4, POLQ, REV1L, FANCL, BRCA2, and NUDT1), AML [UBE2V2, APTX, APTX, MGMT and RPA2, with the latter found only in cases with translocations (PML/RARA or MLL)] myeloma (ALKBH3) and MDS (ALKBH5, TOP3A) were seen. Other CNAs occurred in multiple disorders (NEIL1, PMS2, ENDOV, CHEK2, and HELQ). The results reveal disease specificity for CNAs in DNA repair related genes. Further study is warranted to validate these alterations as disease specific diagnostic/prognostic biomarkers.
Detection of “oncometabolite” 2-hydroxyglutarate by Magnetic Resonance Analysis as a Biomarker of IDH1/2 Mutations in Glioma
Somatic mutations in isocitrate dehydrogenase (IDH)1 and 2 have been detected in a subset of gliomas, rendering these tumors with elevated levels of “oncometabolite”, D-2-Hydroxyglutarate (2HG). We demonstrate the detection of 2HG as a biomarker of this subset of gliomas ex vivo using a magnetic resonance (MR) approach. This study thus highlights the feasibility of using MR detection of 2HG for the diagnosis and classification of IDH1/2 mutation-positive brain tumors and opens up the possibility of performing analogous non-invasive MR-based imaging and spectroscopy studies directly in humans in the neuro-oncology clinic.
Mass Spectrometry Used To Define Plasma Protein-Based Classifiers That Discriminate Patients With Colon Polyps or Adenomas As Compared to Colonoscopy
Plasma samples were collected from patients undergoing colonoscopy and their plasma protein profiles were quantified via LCMS. 100 matched samples were used in this analysis. Approximately 150,000 molecular features were observed in at least 50% of the samples in at least one group. Ten rounds of 10-fold cross validation using Elastic Net feature selection, top 100 feature ranking, and SVM classifier assembly were performed to determine potential classifier performance. The average cross-validated AUC was 0.92 ± 0.12, indicating a high degree of predictive performance. These results demonstrate the feasibility of blood-based protein tests to help manage colonoscopy screen compliance.
ApoE typing from small amounts of blood samples
In clinical testing, ApoE levels in blood are determined by immunoassay. Combining ApoE immunoassay reagents with mass spectrometric analysis, we attempted to resequence ApoE protein from small amounts of sera for typing. Most ApoE mutant isoforms, such as ApoE2 and ApoE4, involve substitutions of lysine and arginine residues, and on trypsin digestion will result in fragments with a larger mass difference than a single amino acid substitution of other amino acids. ApoE types from sera, including heterozygous combinations (ApoE2/E3, E2/E4, E3/E4) corresponded exactly with the APOE genotyping results in each of the subjects. ApoE types from sera may be a novel clinical test that is performed using blood samples remaining from those collected for routine clinical tests and may enable retrospective studies using preserved body fluids.
Immuno-MALDI Assay for Plasma Renin Activity: Proof of Principle for a Translatable Proteomic Assay without Chromatography
The renin-angiotensin-aldosterone system (RAAS) plays an essential role in maintaining arterial blood pressure. Determination of plasma renin activity (PRA) is essential for screening and diagnosis of primary aldosteronism and other derangements of the RAAS pathway. Clinical laboratories have traditionally used radioimmunoassays (RIAs) for angiotensin-I in the determination of PRA. Recently however, there has been pressure to discontinue radioisotope-dependent assays in favour of stable isotope techniques. We have developed a MS approach for the determination of plasma renin activity utilizing immuno-MALDI (iMALDI) MS and have compared with both RIA and LC-MS/MS. The iMALDI method has the benefit of requiring no chromatography.
Differential solublization method to extract low-molecular-weight proteins/peptides for successful serum SRM analyses
Selected reaction monitoring mass spectrometry (SRM-MS) has been used to measure low abundance proteins/peptides in serum/plasma, owing to its high sensitivity and selectivity. However, the presence of highly abundant proteins in serum/plasma and the huge dynamic range of serum proteins/peptides make SRM analysis challenging. We previously developed differential solubilization (DS) method to extract low-molecular-weight proteins/peptides in serum with good reproducibility. Here we present data indicating that the DS method is useful as pretreatment for serum SRM analyses for biomarker validation.
Validation of Arsenic Speciation Method for Human Urine Using HPLC-ICP-MS
Arsenic is carcinogenic depending on its chemical nature and oxidation states. Quantitative determination of inorganic and organic Arsenic species in clinical samples could be used as potential biomarker for its toxic exposure. Arsenic can be ingested in form of one or more species. Arsenic species can be metabolized into several different inorganic and organic forms, including As-III and As-V, and the methylated species such as Monomethyl arsonic acid (MMA), and Dimethyl arsinic acid (DMA) or Arsenobetaine (AsB) and Arsenocholine (AsC) which most likely stay unaffected in the body. We have developed an analytical method for quantitative determination of six Arsenic species in clinical samples using isocratic LC coupled with ICP-MS. The results showed that each of the six Arsenic species was well separated and LOD of each of the species was approximately 1.0 ppb.