Current Molecular Medicine - Volume 10, Issue 2, 2010

As the “traditional” biomedical research efforts move us closer toward understanding why some people seem particularly vulnerable to one or another chronic disease, or why and how these diseases progress, the “translational” branch of the same field of science strives to develop molecular predictors for the course of the disease in given individual. These predictors are collectively known as biomarkers. More often than not, scientists inquiring about the mechanisms for particular human pathology serendipitously unearth a novel predictor, thus, venturing into the translational research and continue to moonlight in the biomarker area until the value of a novel biomarker for the bedside is proven or otherwise. Hence the root of the common belief holds the value of biomarker as somewhat proportional to its functional meaning within the pathogenetic realm of the disorder of interests. Contrary to this belief, many valuable biomarkers have no known function or their properties cannot be linked to underlying pathology without considerable speculative leap. In fact, even for the best-performing biomarkers, for example, carcinoembrionic antigen (CEA), PSA or hsCRP, the connection to underlying pathology assumes “missing link” similar to that of an ancient Chinese test for diabetes involving ants crawling to the patient's urine. In case of the described diabetes test, the “missing link” is obviously the glucose; however, it took an untold number of centuries to figure this out. The “missing link” does not interfere with correct diagnosis, as urine ant test was accidentally executed and corroborated by modern techniques even in relatively recent times [1, 2]. Since the discovery and the beginning of an industrial production of insulin, a great number of novel biomarkers for this disease have been introduced, but only few of these maintain direct connection to the pathogenesis (i.e. insulin and glucose levels in patient's blood). The best diabetic marker, HbA1c, had been chosen by American Diabetes Association (ADA) as the definitive test to diagnose diabetes, outperforming both the fasting plasma glucose test and the less commonly used oral glucose tolerance test [3]. Is there any particular role for HbA1c in diabetes pathogenesis? The answer is “No”. HbA1c is a simple sentinel that dutifully reports an extent of the damage done by unchecked glucose to the patients’ body. This marker is minimally invasive and cheap. Moreover, it delivers positive and negative predictive values superior to that of functionally important biomarkers. This wonderful biomarker was first discovered in the typical scientific “fishing expedition” of 1958, when it was first separated from other forms of hemoglobin using a chromatographic column [4]. Later, a serendipitous discovery of Rahbar revealed a substantial increase in its content in the samples of the blood taken from diabetic subjects [5, 6]. Since then, these two convoluted approaches, a “fishing expedition” and “serendipitous discovery”, provide for an unreliable road to success in biomarker discovery. An example of HbA1c illustrates that “biological plausibility” is not a prerequisite for any biomarker and certainly not an obstacle for successful future development. However desirable, truly biology-driven biomarker discovery would require a full understanding of a biological system including kinetics of interactions between its components, a “systems biology” vision we still are very far away from. Having that in mind, one should not be surprised to see that recent worldwide increases in funds allocated to the search for more biomarkers have not yet produced a breakthrough in the future of individualized medicine, but rather a promising trickle of the leads requiring an extensive validation. The biomarker discovery remains to be slow and often frustrating, even as the advent of genomics and related fields has dramatically accelerated this effort. In this issue we collected a number of important manuscripts either outlining recent efforts in the rationalization and the optimization of the biomarker discovery (Luchini et al., Sikaroodi et al. and Kohl, Current Molecular Medicine, this issue) or summarizing state-of-the-art in certain clinical areas desperately thirsty for the minimally invasive breakthroughs (Seli et al., Estep et al., Belousov et al., Claus et al. and Bonaterra et al., Current Molecular Medicine, this issue). Thus, this issue of “Current Molecular Medicine” reflects both medical need and therapeutic perspective for new biomarkers as well as the recent developments of the technology that facilitates these discoveries. With regard to the biomarker molecules, the articles encompass a variety of the biochemical entities, including RNA, micro RNA, proteins and small metabolites. While the use of single markers was commonplace in the past, it is now increasingly evident that complex diseases require marker panels to adequately reflect the status of patients. Along with technological progress in systems biology and with increasing coverage and data integration, a combination of different types of biomolecules into comprehensive panels may help to finally herald an era of personalized medicine.

Malignant tumors induce humoral immune response in cancer patients, although the incidence of such autoantibody responses against individual tumor-associated antigens (TAA) is rather low. To increase predictive value of TAA-recognizing autoantibodies as potential cancer biomarkers, TAAs should be combined into protein arrays. Here we review recent advances in the application of such arrays and summarize data concerning most promising antigens. We also review the methods of cloning TAA-recognizing autoantibodies, generation of human hybridomas and screening of recombinant human immunoglobulin libraries.

DNA methylation plays a critical role in the regulation of gene expression, differentiation and in the development of cancer and other diseases. Hypermethylation of CpG islands located in the promoter regions of tumor suppressor genes is now firmly established as the most frequent mechanism for gene inactivation in cancers. Feasibility of using DNA methylation based biomarkers for early detection of cancer has been shown. Potential of using DNA methylation for prediction of therapeutic outcome and patient survival has also been shown. DNA originated from cancer cells has been routinely detected in clinical specimens (ex. Plasma/serum, sputum, urine etc.) from cancer patients. Presence of methylated DNA sequences in clinical specimens and potential of using them as biomarkers have been recognized. Novel methylation based biomarkers that can be used in clinical specimens, obtained non-invasively from cancer patients, offer significant practical advantages. More resources need to be committed to this area of biomarker research. Thus, we review recent findings on DNA methylation based cancer biomarkers with particular focus on these applicable to the clinical specimens obtained non-invasively from cancer patients.

Clinically relevant biomarkers exist in blood and body fluids in extremely low concentrations, are masked by high abundance high molecular weight proteins, and often undergo degradation during collection and transport due to endogenous and exogenous proteinases. Nanoparticles composed of a Nisopropylacrylamide hydrogel core shell functionalized with internal affinity baits are a new technology that can address all of these critical analytical challenges for disease biomarker discovery and measurement. Coreshell, bait containing, nanoparticles can perform four functions in one step, in solution, in complex biologic fluids (e.g. blood or urine): a) molecular size sieving, b) complete exclusion of high abundance unwanted proteins, c) target analyte affinity sequestration, and d) complete protection of captured analytes from degradation. Targeted classes of protein analytes sequestered by the particles can be concentrated in small volumes to effectively amplify (up to 100 fold or greater depending on the starting sample volume) the sensitivity of mass spectrometry, western blotting, and immunoassays. The materials utilized for the manufacture of the particles are economical, stable overtime, and remain fully soluble in body fluids to achieve virtually 100 percent capture of all solution phase target proteins within a few minutes.

Since the association of circulating DNA level changes with tumor growth was discovered many attempts have been made to develop the sensitive and robust blood-based tests for early tumor diagnostics. Both genomic as well as mitochondrial DNA quantification in the circulation have been extensively evaluated as a diagnostic and prognostic tool to monitor cancer therapy. Cell-free DNA bearing the same genetic and epigenetic changes as the tumor tissues were shown to be detectable in plasma / serum of cancer patients indicating the principal possibility to create the minimally invasive diagnostic tests based on tumor-specific DNA markers. Apart from circulating DNA, tumor-derived RNA in plasma / serum was found to be a promising approach for the development of cancer markers. Results of the last two years establish the quantification of the tumor-derived microRNAs in plasma / serum as an extremely promising approach for cancer diagnostics. The aim of this publication was to review the recently reported studies on the circulating DNA and RNA in cancer patients and to estimate their impact on making the ongoing research closer to clinical application.

Non-alcoholic fatty liver disease (NAFLD) is a clinico-pathologic spectrum of conditions ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). Although simple or bland steatosis follows a relatively benign clinical course, NASH can potentially progress to cirrhosis (approximately 10 to 15 percent) and hepatocellular carcinoma. NAFLD occurs in an estimated 25 to 30 percent of the US general population, while NASH is reported in 2 to 3 percent of the population. Even though common explanation for the increased prevalence of NAFLD is the increased rate of obesity, the risk of developing NAFLD and NASH is not limited to overweight and obese individuals. Currently, the only way to diagnose NASH or to assess the stage of fibrosis is by obtaining a liver biopsy. Liver biopsy is invasive, expensive, and associated with potential risks, including post biopsy pain, bleeding, organ perforation, and even death; serious complications can occur in 0.3 percent of liver biopsies with 0.01 percent being fatal. This review examines the current strategies for development of the non-invasive techniques that will one day replace liver biopsy and serve as a non-invasive gold standard for the diagnosis and staging of NASH.

Since the different, so-called omics disciplines generate huge amount of data, the application of appropriate, sophisticated statistical methods for developing and validating predictive molecular signatures for drug development, for prevention, screening, diagnosis, monitoring of treatment or aftertreatment of diseases as well as for stratification of individuals is fundamental. The development and validation require several steps and it is quite a long journey from the detection of a molecular predictive signature to the routine use in clinical practice. In our review we focus on data obtained from cDNA expression microarrays. We describe the necessary development and validation steps including recent results of the second phase of the MAQC project (MAQC-II) and emphasise on potential pitfalls.

Motivated by the challenge of risk assessment in a heterogeneous population and guided by advances in our knowledge of the pathobiology of cardiovascular diseases (CVD), basic and clinical scientists have maintained substantial interest in the development and application of novel biomarkers for risk stratification of CVD. In particular, strategies to identify and combine multiple biomarkers, which may reflect diverse pathobiological contributors to the onset and complications of CVD, have been arising as an approach to improve more effectively the risk assessment and target therapy. Moreover, comparative evaluations of novel markers are necessary to estimate these candidates for integration into present and future strategies. In this review we consider the recent in-depth knowledge and advances with the use of systemic biomarkers in the area of CVD with special attention on inflammatory markers and those that can predict an individual arteriosclerotic disease stage.

A key step in assisted reproduction is the assessment of embryo viability in order to identify the embryo(s) most likely to result in pregnancy. Currently used embryo assessment systems are largely based on morphology and cleavage rate. While these systems have been pivotal in improving implantation and pregnancy rates and reducing multiple gestations, their precision is still insufficient. The limitations of strategies based on morphology have led to the investigation of adjunctive technologies for non-invasive assessment of embryo viability in assisted reproduction. These include the measurement of glucose, pyruvate, or amino acid levels in the embryo culture media, assessment of oxygen consumption by the embryo, genomic and proteomic profiling, and most recently, analytical examination of the embryonic metabolome. As the number of ART cycles increases worldwide, improvements in the ability to quickly and non-invasively identify the best embryos for transfer become an increasingly more important goal for reproductive medicine.

The gap of the post-genomic era is increasingly being filled by the metabolomics approach, comprising a technology for analyzing small molecule endogenous metabolites (≤1500 Dalton) in complex biological samples. This new analytical science has progressed within the last years particularly with regard to improvements in mass spectrometry based detection, now allowing highly robust, reproducible, selective and sensitive qualitative or quantitative analysis of endogenous metabolites. The precise and accurate quantitation of these metabolites via targeted metabolomics, now critically contributes to the quantitative analysis of endogenous compounds in biomarker discovery and validation thus to future personalized therapy. The analytical methods of choice in (MS-based) targeted metabolomics primarily are HPLC-API-MS/MS, FIA-APIMS/ MS and GC-MS. In the parent paper, we provide an introduction and brief survey on the technological basis of targeted metabolomics in biomarker research, discuss various relevant analytical aspects in mass spectrometry including comparison to non-targeted approaches, effects of sample preparation, impact of sample stability, carryover- and matrix effects, need for standardization and for proficiency tests, standardization of analytical methods as well as the requirement for method validation.

The ‘systemic inflammatory response syndrome (SIRS)’ reflects a non-specific inflammatory reaction to various insults. In sepsis, defined as SIRS triggered by infection, a complex and overwhelming network of mediators contributes to the clinical syndrome. The host response in sepsis is characterized by unspecific physiologic criteria, which are unable to identify patients adequately who might benefit from either conventional anti-infective therapies or from novel therapies targeting specific mediators of sepsis. The early diagnosis of sepsis, the identification of the origin, adequate therapeutical management and the monitoring of the disease may help to overcome sepsis-associated mortality, which is unacceptably high and the third leading cause of death in Western Countries. Molecular techniques for identification of pathogens, their associated molecular patterns (PAMPs) and the ensuing host response may help to stratify patients with the urgent need for antibiotic therapy and those where it is safe to withhold or to de-escalate therapy. Beyond analysis of danger associated molecular patterns (DAMPs) at a single molecular level, the advent of genome-wide screening allows for an assessment of a wide variety of effectors and mediators in response to PAMPs. Also their purposeful targeting in animal models of sepsis revolutionized our understanding of pathophysiology in the critically ill. Molecular tools are about to challenge “state-of-the-art” diagnostic tests such as blood culture as they not only increase sensitivity but also dramatically reduce time requirements to identify pathogens and their resistance patterns. Mounting evidence suggests that our pathophysiological understanding might in the near future help to identify “patients at risk”, i.e. those with a high likelihood to develop organ dysfunction and/or to guide therapeutic interventions in particular regarding resource-consuming and expensive therapies (“theragnostics”). The clinical utility for most of the discussed markers for monitoring systemic inflammation and sepsis has still to be evaluated in prospective trails. In conclusion, there is an unmet medical need for identification and validation of reliable biomarkers of sepsis; the clinical information obtained from the use of novel biomarkers might contribute to transform sepsis from a physiologic syndrome to a group of distinct biochemical disorders, to improve diagnosis and therapeutic decision making for high-risk patients, to monitor the response to therapy and to ensure the enrollment of seriously characterized patients in clinical studies.

Purpose: This review investigates and highlights the activity of Willebrand factor (VWF) and its cleaving protease as biomarkers of the development of multiple organ dysfunction in infectious and noninfectious systemic inflammatory response syndrome. State of the Art: Ultra-large VWF (ULVWF) multimers activate platelets resulting in a prothrombotic situation. Systemic inflammation is associated with increased ULVWF plasma level and a decreased ADAMTS13 activity. The potential role of ADAMTS13 as a diagnostic and prognostic marker of disseminated intravascular coagulopathy is largely underestimated. Summary: VWF is an acute phase protein and its plasma level increases in systemic inflammation. When released from endothelial cells and platelets, the native multimeric glycoprotein is mostly present in the ultralarge form (ULVWF), which may have a major clinical significance under proinflammatory conditions. ULVWFmultimers may activate endothelial cells and platelets simultaneously. The multimers undergo limited proteolysis by a specific plasma metalloprotease known as ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motif), thus, in healthy individuals only marginal amounts of circulating ULVWF are detectable. Severe hereditary or acquired ADAMTS13 deficiency causes thrombotic thrombocytopenic purpura (TTP), which contributes to prothrombotic coagulation abnormalities preceding organ dysfunction systemic inflammatory response syndrome (SIRS). In proinflammatory conditions, ADAMTS13 activity decreases due to various mechanisms, (i) down regulation on a transcriptional level, (ii) proteolytic degradation, and (iii) consumption due to the high substrate level. Marked dysbalance as found in patients with severe sepsis or septic shock results in substantial amounts of plasma ULVWF. This level of dysbalance is negatively correlated with platelet count and positively correlated with the severity of inflammation and the degree of organ failure.

Tumor markers are the molecules that indicate the presence or prognosis of malignancy. Most often, tumor markers are produced by the cancer tissue itself. Many of them could be secreted into the body fluids in small quantities. Thus, tumor markers could be useful for early diagnostics of primary tumors and relapsed disease, as well as for determining tumor prognosis and predicting likely response of the tumor to therapy. Tumor markers are part of the clinical routine. Nevertheless, lack of sensitivity and specificity precludes routine usage of single tumor markers in population-based screening. Shortcomings of single tumor markers could be solved by parallel evaluation of multiple tumor markers that can perform with required certainty. Genome and proteome-wide approaches currently lead to identification and initial characterization of hundreds new tumor marker candidates. Most prominent of such methods are serological analyses of recombinant cDNA expression libraries (SEREX), 2-dimensional polyacrylamide gel electrophoresis, mass spectrometry, as well as protein and DNA microarrays. Last but not the least is a computational approach allowing high-throughput detection of tumor marker candidate genes in publicly available datasets. Listed approaches are critically discussed in this review as well as the most crucial tumor-related findings. Finally, a perspective on the future of tumor markers in the tailored medicine is given.