Biomarker discovery for cervical cancer

Proteomics of human boy fluids is still in its early stage of development with major methodological challenges ahead. This implies that much attention is given to improving the methods and strategies. One major challenge is that many samples that have been acquired in the past may not fulfill the stringent requirements of storage and sample preparation to allow comparable proteomics analyses. It is therefore important to assess the factors that may affect the final proteomics result through systematic and reproducible analyses. Therefore accuracy and sensitivity of the analytical instrumentation is not the only critical factor in this research. Blood (plasma or serum) and urine can be easily sampled from patients or healthy volunteers and are therefore often the first choice when trying to discover novel biomarkers or biomarker patterns to diagnose cancer and other diseases. There are, however, drawbacks such as the masking of low-abundance by high abundance proteins and the possible effect of sampling and sample handling procedures (e.g. different times for blood clotting). A number of different approaches to deplete highly abundant proteins from human serum were studied throughout this thesis. Further, different analytical techniques were applied, such as a miniaturized, microfluidics-based LC-MS system (chip-LC-MS) to enhance overall sensitivity. It is shown that chip-LC-MS has at least twice the resolution of the previously used standard capillary LC-MS method. Since blood composition will change under the influence of external factors, the influence of clotting time on proteome of serum was studied. It was found that most proteins were not affected by clotting time except for those directly involved in this process, such as the fibrinopeptides. Next, we describe a more comprehensive approach for evaluating the influence of various pre-analytical parameters on the serum proteome. A factorial design strategy was applied to assess the importance of seven factors considered to be of relevance, including the level of hemolysis, the digestion conditions, and the storage conditions. Finally, we analyzed serum samples from cervical cancer patients at various stages of disease before and after treatment followed by data processing and statistical data analysis. While we did not discover major changes in the serum proteome using this method, subtle changes in the protein composition were observed in relation to treatment, the significance of which are being further investigated. It is thus demonstrated that the described methods are applicable to highly complex body fluids such as serum and that further studies into the relevance of the discovered changes of the serum proteome are warranted.

Analytical tools for the characterization of deamidation in monoclonal antibodies

This review focuses on the deamidation of asparagine in monoclonal antibodies, one of the well-known degradation pathways. The importance of close monitoring of deamidation from early developability assessment to in vivo studies is discussed consequentially. The application of chromatographic and electrophoretic instruments for the separation of deamidated variants is described. Current mass spectrometric approaches for characterization of deamidated antibody variants and localization of modification sites are discussed accordingly. The review also covers the challenges in bioanalysis of deamidated forms in complex biological matrices during in vivo studies.</p

Non-Antibody-Based Binders for the Enrichment of Proteins for Analysis by Mass Spectrometry

There is often a need to isolate proteins from body fluids, such as plasma or serum, prior to further analysis with (targeted) mass spectrometry. Although immunoglobulin or antibody-based binders have been successful in this regard, they possess certain disadvantages, which stimulated the development and validation of alternative, non-antibody-based binders. These binders are based on different protein scaffolds and are often selected and optimized using phage or other display technologies. This review focuses on several non-antibody-based binders in the context of enriching proteins for subsequent liquid chromatography-mass spectrometry (LC-MS) analysis and compares them to antibodies. In addition, we give a brief introduction to approaches for the immobilization of binders. The combination of non-antibody-based binders and targeted mass spectrometry is promising in areas, like regulated bioanalysis of therapeutic proteins or the quantification of biomarkers. However, the rather limited commercial availability of these binders presents a bottleneck that needs to be addressed

Meta-Inflammation and Metabolic Reprogramming of Macrophages in Diabetes and Obesity:The Importance of Metabolites

Diabetes mellitus type II and obesity are two important causes of death in modern society. They are characterized by low-grade chronic inflammation and metabolic dysfunction (meta-inflammation), which is observed in all tissues involved in energy homeostasis. A substantial body of evidence has established an important role for macrophages in these tissues during the development of diabetes mellitus type II and obesity. Macrophages can activate into specialized subsets by cues from their microenvironment to handle a variety of tasks. Many different subsets have been described and in diabetes/obesity literature two main classifications are widely used that are also defined by differential metabolic reprogramming taking place to fuel their main functions. Classically activated, pro-inflammatory macrophages (often referred to as M1) favor glycolysis, produce lactate instead of metabolizing pyruvate to acetyl-CoA, and have a tricarboxylic acid cycle that is interrupted at two points. Alternatively activated macrophages (often referred to as M2) mainly use beta-oxidation of fatty acids and oxidative phosphorylation to create energy-rich molecules such as ATP and are involved in tissue repair and downregulation of inflammation. Since diabetes type II and obesity are characterized by metabolic alterations at the organism level, these alterations may also induce changes in macrophage metabolism resulting in unique macrophage activation patterns in diabetes and obesity. This review describes the interactions between metabolic reprogramming of macrophages and conditions of metabolic dysfunction like diabetes and obesity. We also focus on different possibilities of measuring a range of metabolites intra-and extracellularly in a precise and comprehensive manner to better identify the subsets of polarized macrophages that are unique to diabetes and obesity. Advantages and disadvantages of the currently most widely used metabolite analysis approaches are highlighted. We further describe how their combined use may serve to provide a comprehensive overview of the metabolic changes that take place intracellularly during macrophage activation in conditions like diabetes and obesity

Effect of Trastuzumab-HER2 Complex Formation on Stress-Induced Modifications in the CDRs of Trastuzumab

Asparagine deamidation and aspartic acid isomerization in the complementarity determining regions (CDRs) of monoclonal antibodies may alter their affinity to the target antigen. Trastuzumab has two hot spots for deamidation and one position for isomerization in the CDRs. Little is known how complex formation with its target antigen HER2 affects these modifications. Modifications in the CDRs of trastuzumab were thus compared between the free antibody and the trastuzumab-HER2 complex when stressed under physiological conditions at 37°C. Complex formation and stability of the complex upon stressing were assessed by size-exclusion chromatography. Deamidation of light-chain Asn-30 (Lc-Asn-30) was extensive when trastuzumab was stressed free but reduced about 10-fold when the antibody was stressed in complex with HER2. Almost no deamidation of heavy-chain (Hc-Asn-55) was detected in the trastuzumab-HER2 complex, while deamidation was observed when the antibody was stressed alone. Hc-Asp-102 isomerization, a modification that critically affects biological activity, was observed to a moderate degree when the free antibody was stressed but was not detected at all in the trastuzumab-HER2 complex. This shows that complex formation has a major influence on critical modifications in the CDRs of trastuzumab.</p

Determination of Binding Sites on Trastuzumab and Pertuzumab to Selective Affimers Using Hydrogen-Deuterium Exchange Mass Spectrometry

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a method to probe the solvent accessibility and conformational dynamics of a protein or a protein-ligand complex with respect to exchangeable amide hydrogens. Here, we present the application of HDX-MS to determine the binding sites of Affimer reagents to the monoclonal antibodies trastuzumab and pertuzumab, respectively. Intact and subunit level HDX-MS analysis of antibody-affimer complexes showed significant protection from HDX in the antibody Fab region upon affimer binding. Bottom-up HDX-MS experiments including online pepsin digestion revealed that the binding sites of the affimer reagents were mainly located in the complementarity-determining region (CDR) 2 of the heavy chain of the respective antibodies. Three-dimensional models of the binding interaction between the affimer reagents and the antibodies were built by homology modeling and molecular docking based on the HDX data.</p

Molecular markers for cervical cancer screening

This review gives an overview of current screening practices for cervical cancer. In the introduction, we will cover approaches of population screening focusing on high-risk Human Papilloma Virus (hrHPV) and the need for a better triage assay. We will further assess the impact of current vaccination programs on screening. Subsequently, the review will cover various technological aspects of nucleic acid- and protein-based biomarker assays. We will then detail different molecular markers in view of their use in triage assays, emphasizing epigenetic and protein markers. Finally, we will place this in the context of cost-effectiveness considerations in view of their implementation in high- as well as in low- to middle-income countries. Introduction: Cervical cancer remains a significant healthcare problem, notably in low- to middle-income countries. While a negative test for hrHPV has a predictive value of more than 99.5%, its positive predictive value is less than 10% for CIN2+ stages. This makes the use of a so-called triage test indispensable for population-based screening to avoid referring women, that are ultimately at low risk of developing cervical cancer, to a gynecologist. This review will give an overview of tests that are based on epigenetic marker panels and protein markers. Areas covered: There is a medical need for molecular markers with a better predictive value to discriminate hrHPV-positive women that are at risk of developing cervical cancer from those that are not. Areas covered are epigenetic and protein markers as well as health economic considerations in view of the fact that most cases of cervical cancer arise in low-to-middle-income countries. Expert opinion: While there are biomarker assays based on changes at the nucleic acid (DNA methylation patterns, miRNAs) and at the protein level, they are not widely used in population screening. Combining nucleic acid-based and protein-based tests could improve the overall specificity for discriminating CIN2+ lesions that carry a low risk of progressing to cervical cancer within the screening interval from those that carry an elevated risk. The challenge is to reduce unnecessary referrals without an undesired increase in false-negative diagnoses resulting in cases of cervical cancer that could have been prevented. A further challenge is to develop tests for low-and middle-income countries, which is critical to reduce the worldwide burden of cervical cancer

Change of charge variant composition of trastuzumab upon stressing at physiological conditions

Cation-exchange chromatography is a widely used approach to study charge heterogeneity of monoclonal antibodies. Heterogeneity may arise both in vitro and in vivo because of the susceptibility of monoclonal antibodies to undergo chemical modifications. Modifications may adversely affect the potency of the drug, induce immunogenicity or affect pharmacokinetics. In this study, we evaluated the application of optimized pH gradient systems for the separation of charge variants of trastuzumab after forced degradation study. pH gradient-based elution resulted in high-resolution separation of some 20 charge variants after 3 weeks at 37°C under physiological conditions. The charge variants were further characterized by LC-MS-based peptide mapping. There was no significant difference in the binding properties to HER2 or a range of Fcγ receptors between non-stressed and stressed trastuzumab.</p

Proteomic alterations in early stage cervical cancer

Laser capture microdissection (LCM) allows the capture of cell types or well-defined structures in tissue. We compared in a semi-quantitative way the proteomes from an equivalent of 8,000 tumor cells from patients with squamous cell cervical cancer (SCC, n = 22) with healthy epithelial and stromal cells obtained from normal cervical tissue (n = 13). Proteins were enzymatically digested into peptides which were measured by high-resolution mass spectrometry and analyzed by “all-or-nothing” analysis, Bonferroni, and Benjamini-Hochberg correction for multiple testing. By comparing LCM cell type preparations, 31 proteins were exclusively found in early stage cervical cancer (n = 11) when compared with healthy epithelium and stroma, based on criteria that address specificity in a restrictive “all-or-nothing” way. By Bonferroni correction for multiple testing, 30 proteins were significantly up-regulated between early stage cervical cancer and healthy control, including six members of the MCM protein family. MCM proteins are involved in DNA repair and expected to be participating in the early stage of cancer. After a less stringent Benjamini-Hochberg correction for multiple testing, we found that the abundances of 319 proteins were significantly different between early stage cervical cancer and healthy controls. Four proteins were confirmed in digests of whole tissue lysates by Parallel Reaction Monitoring (PRM). Ingenuity Pathway Analysis using correction for multiple testing by permutation resulted in two networks that were differentially regulated in early stage cervical cancer compared with healthy tissue. From these networks, we learned that specific tumor mechanisms become effective during the early stage of cervical cancer