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.

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

Comparison of Targeted Mass Spectrometry Techniques With an Immunoassay:A Case Study For HSP90α

PURPOSE: The objective of this study was to better understand factors governing the variability and sensitivity in SRM and PRM, compared to immunoassay. EXPERIMENTAL DESIGN: A 2D-LC-MS/MS-based SRM and PRM assay was developed for quantitative measurements of HSP90α in serum. Forty-three control sera were compared by SRM, PRM and ELISA following the manufacturer's instructions. Serum samples were trypsin-digested and fractionated by SCX chromatography prior to SRM and PRM measurements. Analytical parameters such as linearity, LOD, LOQ, repeatability and reproducibility of the SRM, PRM and ELISA were determined. RESULTS: PRM data obtained by high-resolution mass spectrometry correlated better with ELISA measurements than SRM data measured on a triple quadrupole mass spectrometer. While all three methods (SRM, PRM, ELISA) were able to quantify HSP90α in serum at the ng/mL level, the use of PRM on a high-resolution mass spectrometer reduced variation and showed comparable sensitivity to immunoassay. CONCLUSIONS AND CLINICAL RELEVANCE: Using fractionation, it is possible to measure ng/mL levels of HSP90α in a reproducible, selective and sensitive way using PRM in serum. This opens up the possibility to use PRM in a multiplexed way as an attractive alternative for immunoassays without the use of antibodies or comparable binders. This article is protected by copyright. All rights reserved

Susceptibility to COPD:Differential Proteomic Profiling after Acute Smoking

Cigarette smoking is the main risk factor for COPD (Chronic Obstructive Pulmonary Disease), yet only a subset of smokers develops COPD. Family members of patients with severe early-onset COPD have an increased risk to develop COPD and are therefore defined as "susceptible individuals". Here we perform unbiased analyses of proteomic profiles to assess how "susceptible individuals" differ from age-matched "non-susceptible individuals" in response to cigarette smoking. Epithelial lining fluid (ELF) was collected at baseline and 24 hours after smoking 3 cigarettes in young individuals susceptible or non-susceptible to develop COPD and older subjects with established COPD. Controls at baseline were older healthy smoking and non-smoking individuals. Five samples per group were pooled and analysed by stable isotope labelling (iTRAQ) in duplicate. Six proteins were selected and validated by ELISA or immunohistochemistry. After smoking, 23 proteins increased or decreased in young susceptible individuals, 7 in young non-susceptible individuals, and 13 in COPD in the first experiment; 23 proteins increased or decreased in young susceptible individuals, 32 in young non-susceptible individuals, and 11 in COPD in the second experiment. SerpinB3 and Uteroglobin decreased after acute smoke exposure in young non-susceptible individuals exclusively, whereas Peroxiredoxin I, S100A9, S100A8, ALDH3A1 (Aldehyde dehydrogenase 3A1) decreased both in young susceptible and non-susceptible individuals, changes being significantly different between groups for Uteroglobin with iTRAQ and for Serpin B3 with iTRAQ and ELISA measures. Peroxiredoxin I, SerpinB3 and ALDH3A1 increased in COPD patients after smoking. We conclude that smoking induces a differential protein response in ELF of susceptible and non-susceptible young individuals, which differs from patients with established COPD. This is the first study applying unbiased proteomic profiling to unravel the underlying mechanisms that induce COPD. Our data suggest that SerpinB3 and Uteroglobin could be interesting proteins in understanding the processes leading to COPD

Influence of clotting time on the protein composition of serum samples based on LC-MS data

Many large, disease-related biobanks of serum samples have been established prior to the widespread use of proteomics in biomarker research. These biobanks may contain relevant information about the disease process, response to therapy or patient classifications especially with respect to long-term follow-up that is otherwise very difficult to obtain based on newly initiated studies, particularly in the case of slowly developing diseases. An important parameter that may influence the composition of serum but that is often not exactly known is clotting time. We therefore investigated the influence of clotting time on the protein and peptide composition of serum by label-free and stable-isotope labeling techniques. The label-free analysis of trypsin-digested serum showed that the overall pattern of LC-MS data is not affected by clotting times varying from 2 to 8 h. However, univariate and multivariate statistical analyses revealed that proteins that are directly involved in blood clot formation, such as the clotting-derived fibrinopeptides, change significantly. This is most easily detected in the supernatant of acid-precipitated, immunodepleted serum. Stable-isotope labeling techniques show that truncated or phosphorylated forms of fibrinopeptides A and B increase or decrease depending on clotting time. These patterns can be easily recognized and should be taken into consideration when analyzing LC-MS data using serum sample collections of which the clotting time is not known. Next to the fibrinopeptides, leucine-rich alpha-2-glycoprotein (P02750) was shown to be consistently decreased in samples with clotting times of more than I h. For prospective studies, we recommend to let blood clot for at least 2 h at room temperature using glass tubes with a separation gel and micronized silica to accelerate blood clotting. (C) 2008 Elsevier B.V. All rights reserved

Multiple Testing Issues in Discriminating Compound-Related Peaks and Chromatograms from High Frequency Noise, Spikes and Solvent-Based Noise in LC – MS Data Sets

Liquid Chromatography - Mass Spectrometry (LC-MS) is a powerful method for sensitive detection and quantification of proteins and peptides in complex biological fluids like serum. LC-MS produces complex data sets, consisting of some hundreds of millions of data points per sample at a resolution of 0.1 amu in the m/z domain and 7000 data points in the time domain. However, the detection of the lower abundance proteins from this data is hampered by the presence of artefacts, such as high frequency noise and spikes. Moreover, not all of the tens of thousands of the chromatograms produced per sample are relevant for the pursuit of the biomarkers. Thus in analysing the LC-MS data, two critical pre-processing issues arise. Which of the thousands of the: 1. chromatograms per sample are relevant for the detection of the biomarkers?, and 2. signals per chromatogram are truly compound-related? Each of these issues involves assessing the significance (deviation from noise) of multiple observations and the issue of multiple comparisons arises. Current methods disregard the multiplicity and provide no concrete threshold for significance. However, with such procedures, the probability of one or more false-positives is high as the number of tests to be performed is large, and must be controlled. Realizing that the cut-offs for declaring a chromatogram (or a signal) to be compound-related can hugely influence which proteins are detected, it seems natural to define thresholds that are neither arbitrary nor subjective. We suggest the choice of thresholds guided by the critical aim of controlling the False Discovery Rate (FDR) in multiple hypotheses testing for significance over a large set of features produced per sample. This involves the use of the regression diagnostics to characterize the signals of a chromatogram (e.g. as outliers or influential) and to suggest suitable tests statistics for the multiple testing procedures (MTP) for discriminating noise and spikes from true signals. The role of the Generalized Linear Models (GLM) in this MTP is investigated. The method is applied to LC-MS datasets from trypsin-digested serum spiked with varying levels of horse heart cytochrome C (cytoc).