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Biomarkers in molecular medicine: cancer detection and diagnosis
 
Padma Maruvada, Wendy Wang, Paul D. Wagner, Sudhir Srivastava
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It is uncertain whether protein profiling will prove to be as valuable a diagnostic tool as the initial papers suggest. Factors, such as interfering or high-abundant proteins in the sample, variations in diet, or inflammation, which can influence the protein patterns and peak intensity, need to be taken into consideration. Other factors, such as sample quality, variability between instruments, inter-lab variations, and differences in chip composition from separate batches and companies, can all influence the reproducibility and resolution of the patterns. There is also need for robust algorithms that can effectively detect molecular patterns reproducibly and consistently. Currently many labs, including NCI's Early Detection Research Network (EDRN)-sponsored labs, are conducting validation studies to address these issues.

Another proteomic approach gaining attention for molecular screening is protein arrays composed of either recombinant proteins or antibodies. These protein arrays are similar to DNA microarrays in their layout and utilize the principles of immunoassays for detection of tumor antigens. Robust platforms with high analytical sensitivity enable simultaneous detection of several tumor-specific antigens. Specific antigen-antibody interactions make this approach sufficiently precise to identify cancer-specific antigens and enable accurate diagnosis of cancer. Its portability and throughput make it attractive as a screening tool.

Metabolomic Technologies

Metabolomics refers to the study of metabolites present in cells, tissues, and bodily fluids. The potential usefulness of metabolomics for detecting and monitoring cancer is that the identities, concentrations, and fluxes of these molecules are the final products of interactions between gene expression, protein expression, and the cellular environment. The limited number of metabolites and metabolic products makes them suitable for analysis by high-throughput methods. Carcinogenic transformation often involves changes in cellular metabolites, and metabolites of environmental toxins that play an important role in carcinogenic transformation are detectable in bodily fluids. Metabolomic approaches use analytical techniques such as nuclear magnetic resonance spectroscopy (NMR), high-performance liquid chromatography (HPLC), gas-liquid chromatography (GLC), and MS to measure populations of low-molecular-weight metabolites. Advanced statistical and bioinformatic tools are then employed to maximize the recovery of information and to interpret the large data sets. An advantage of the metabolomic approach is that it is possible to interrogate more than one type of molecule (lipids, carbohydrates, nucleotides) at a time, giving a better description of cellular events. For example, oxidative damage may change cellular antioxidant status, resulting in differences in a variety of cellular pro-oxidant and antioxidant molecules. It is, therefore, always advantageous to analyze all the molecules and pathways. Metabolomic patterns may be influenced by the same factors as proteomic analyses. Recently, metabolomic approaches have been utilized to study the metabolic changes of hypoxia inducible factor-1 deficient tumors (16). The potential of metabolomics for cancer detection is just beginning to be explored.

Multiplex Approaches

A limitation of many, if not all, currently used cancer biomarkers, is that they do not detect all individuals with cancer (false negatives). This results from both the progressive nature of cancer and its heterogeneity. Cancer is not a single disease but rather an accumulation of several events, genetic and epigenetic, arising in a single cell over a period of time. The problem of biomarkers indicating the presence of cancer when none is present (false positives) results because these biomarkers are not uniquely present in tumors. With no single biomarker providing 100% sensitivity and specificity, there is a need for an ensemble of biomarkers to enable molecular screening to reduce false positive and false negative cases. Consequently, methods that allow for the simultaneous measurement of several biomarkers are needed.

Sophisticated molecular technologies that can simultaneously identify a variety of promising markers are employed in the discovery phase. The same technologies may be used for clinical application. However, often as a biomarker progresses from discovery to validation and to subsequent clinical application, its measurement needs to be adapted to various platforms. Not all technologies are robust enough to pass through all the phases of biomarker development. For large-scale screenings, technologies need to be robust, flexible, and cost-efficient. Multiplex platforms allow for simultaneous analysis of several different biomarkers and are, therefore, attractive platforms for screening. For example, quantitative multiplex-methylation-specific PCR (QM-MSP) is a highly sensitive method that can determine the hypermethylation status of multiple genes, such as RASSF1A, TWIST, Cyclin D2, and HIN1, in a single tube (17). Using Invader® assays (Third Wave Technologies, Madison, WI, USA), it is possible to simultaneously quantitate both RNA and DNA (6). LabChip® technology in combination with the 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) enables the analysis of DNA, RNA, protein, and cellular substances from a single sample (18). These approaches decrease the laborious sample preparation times. Another revolution in molecular detection is microfluidics technology that allows detection of molecules in suspension using lasers and fluorescent dyes (18). It is now possible to simultaneously perform 100 bioassays in a single reaction with high sensitivity. Such approaches also require analytical tools to analyze the multidimensional, high-throughput data (19,20,21). While the optimization of the technologies can be very complex, these multiplex technologies offer great promise for molecular screening.

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