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European Biopharmaceutical Review

Cause and Effect

Translational and clinical research fundamentally depends on specifi c and accurate biomarkers. Accordingly, discovery and development of innovative biomarkers is crucial for the cost-effective and successful development and validation of improved diagnostics and novel therapeutics.

Successful innovations in diagnosis and therapy will depend on novel approaches rather than adhering to the conventional repertoire of usual suspects, in terms of potentially active compounds as well as with respect to applied tools for development. Recent technological advances, especially in the fi eld of molecular investigation techniques, pave the way for novel innovative approaches to answer old questions and address new objectives. Nucleic acid biomarker discovery, development and applications based on advanced technologies – such as next generation sequencing, sensitive fl exible-content microarrays and improved quantitative real-time polymerase chain reaction methods – provide promising new opportunities for preclinical and clinical research and trials. Nucleic acid biomarkers, such as microRNAs, single nucleotide polymorphisms (SNPs) and DNA methylation patterns, are amenable to highly sensitive analysis from limited amounts of easily accessible clinical sample material, such as whole-blood, serum, plasma, urine or other bodily fl uids. The signifi cance of biomarkers for the monitoring and judgment of study progress, and as diagnostic and predictive tools with clinical utility, probably cannot be overestimated. Co-development and extended use of biomarkers throughout the entire drug development process, referred to as ‘companion diagnostics’, are considered to provide the desired surveillance to yield signifi cant increases in study effectiveness.

Biomarker applications can be roughly divided into four categories (1,2):

  • Diagnosis/prognosis – these biomarkers allow for the assessment of a disease state. This group of biomarkers discriminates between healthy and diseased individuals and/ or differentiates between (related) diseases. In addition, it might be exploited to stage a disease and predict the outcome with and without treatment
  • Effi cacy – effi cacy biomarkers monitor biological changes in the diseased patient in response to treatment
  • Pharmacodynamics – surveilling the effect of a drug or treatment on a patient’s disease state and organism, pharmacodynamic biomarkers allow judgment and adjustment of drug doses and administration schedules
  • Patient selection/stratifi cation – these biomarkers are intended to identify patients which should receive a certain treatment based on their inherent genetic and/or phenotypic characteristics and to choose the most appropriate drug with the highest potential to yield the desired outcome. This group of biomarkers is of special interest during the design and successful conduct of clinical trials

This article highlights the broad range utilities and high performance of genomic biomarkers, in particular microRNA signatures, from easily accessible bodily fl uids for virtually all biomarker applications. It also briefl y elaborates on the importance of standardised workfl ows for pre-analytic and analytic processing and data analyses to yield robust reliable results.

Pre-analytic Requirements

Successful molecular biomarker development and application essentially depend on the consideration and defi nition of pre-analytic procedures to minimise undesirable variations. To avoid confounding the results obtained from blood or other bodily fl uid samples (for example plasma) sampling equipment – beginning with the type of collection tubes (EDTA versus Citrate versus heparin as an anticoagulant) down to the gauge of the needle used for phlebotomy – have to be standardised. Fasting versus non-fasting state at blood collection, time elapsed between whole blood collection and processing to plasma or serum as well as the duration of storage all impact on the results and require defi nition (3). However, all of these requirements can be easily met if standardised procedures are available prior to sample collection or selection. Additional sources of variation originate in different methods of extraction. Serum, plasma or urine have minute contents of nucleic acids, and therefore require modifi ed, highly effi cient, and reproducible extraction protocols accompanied by reliable methods for accurate quantifi cation and quality control of the low amounts of nucleic acids recovered from these starting materials. Synthetic, non-human spike-in controls allow for the assessment and normalisation of preparative technical variation between samples. In essence, expert, standardised pre-analytic handling, storage, shipping and preparation protocols are essential to yield robust, reproducible results.

Non-invasive Methods and Minute Sample Requirements

Blood-derived cellular and cell-free nucleic acid biomarkers are easily accessible by non-invasive clinical standard methods. This provides the major advantage of inexpensive repetitive real-time monitoring of molecular events and dynamic changes – for example, in response to treatment. Therefore, minimally invasive blood analyses of nucleic acids are expected to gain clinical utility (4). In addition, depending on optimised standard sample preparations and highly sensitive detection methods, like next-generation sequencing, microarrays or qRT-PCR, only minute amounts of sample are required for the development and application of this biomarker class. This provides the excellent opportunity to analyse early stage model organisms, like rats or mice, with the same standardised methods like the patient cohorts. The resulting highly comparable data sets can then be easily integrated to a comprehensive holistic picture.

Broad Range Utilities of (Circulating) Genomic Biomarkers

Genomic biomarkers cover the whole range of DNA and RNA molecules and their biological variations and modifications. DNA biomarkers, which range from single-nucleotide polymorphisms (SNP) over copy number variations (CNV) and larger mutational re-arrangement to epigenetic modifications, have not only been successfully associated with different disorders and diseases and are exploited as diagnostic biomarkers, but also have been efficiently applied for patient stratification and selection to define patient cohorts expected to show the most beneficial response to a treatment, as judged by their inherent genetic and/ or phenotypic make-up. RNA biomarkers – either mRNA expression profiles or more recently microRNA profiles – provide more dynamic read-outs especially required for additional temporal resolution of altered cellular processes, for example in response to treatment or onset and progression of disease. Elevated levels of different circulating nucleic acids (DNA, mRNA and microRNA) in the blood have been reported to reflect diverse pathological processes, including malignant and benign lesions, inflammatory diseases, stroke and sepsis (4). The contributions to the content of circulating nucleic acids from different cellular sources, whether passively by apoptotic and necrotic cells and/or by active secretion and shedding of exosomes/microvesicles, is subject to active research and to lively debate.

Several studies report that circulating DNA from cancer patients carries tumourrelated genetic and epigenetic alterations directly linked to cancer development, progression and resistance to therapy. The variety of detectable alterations ranges from mutations in tumour suppressor and oncogenes to the integrity of noncoding repeat sequences and epigenetic modifications in genes relevant to tumorigenesis and tumour progression. Their potential clinical utility is indicated by the launch of commercial tests for cancer screening (4,5). In addition to DNA and its modifications, blood provides an easily accessible source for cellular (leukocytes) as well as cell-free stable microRNAs. The latter two already provide complementing information on molecular processes at distinct sites of the body. microRNAs play crucial roles in the regulation of virtually all aspects of living cells. These small (17-27nt) non-coding RNA molecules exhibit their post-transcriptional regulatory function by binding to their mRNA targets, thereby either inhibiting translation or poising the bound target for degradation (6). microRNAs have also been described to exhibit pivotal regulatory functions in the development and progression of numerous diseases. These properties reveal the enormous potential of these molecules to serve as versatile biomarkers. This is further substantiated by the rapidly growing number of successful pilot studies on microRNA blood tests for diagnosis and prognosis of diverse pathological conditions.

The role of microRNAs in pathogenesis and the possibilities to associate expression changes with disease states underscore their value as molecular biomarkers. The expression of microRNAs is not only highly specific for tissues and developmental stages, but has also been demonstrated to allow for the molecular classification of tumours. Moreover, microRNAs detected in FFPE tissue and human blood, both in mononuclear blood cells and as free circulating nucleic acids, have been identified as stable potential blood-based markers for the detection of numerous cancers and a variety human diseases (7-16). As an example for the broad range of different diseases which has been successfully analysed for diagnostic blood-derived microRNA biomarkers, Figure 1 provides an overview of selected diseases which have been successfully addressed by a biomarker discovery centre (BDC), either by customer’s projects or within a research consortium of academia and industry sponsored by federal funds, collaborating to identify and characterise novel biomarkers.

Excellent Performance of microRNA Signatures from Blood

Discovery, development and application of comprehensive microRNA biomarkers tailored to the specific application of interest are promising approaches to novel diagnostic tests. Standardised protocols for the integrated utilisation of hypothesis-free discovery and quantitation by next-generation sequencing, highly sensitive (custom-content) microarrays and quantitative real-time polymerase chain reactions, enable highly sensitive analyses from limited amounts of noninvasive clinical sample material such as serum, whole-blood or other bodily fluids. Screening for microRNA profiles on wellestablished microarrays has been shown to allow the comparably inexpensive highthroughput development of microRNA biomarker signatures for accurate early diagnosis, detection, assessment of progression and prognosis of several diseases (see Figure 1) (7-16).

A research group led by Professor Eckart Meese identified differential microRNA biomarker signatures from whole blood that allowed for the accurate discrimination of lung cancer patients from healthy controls as well as of lung cancer patient from patients suffering from chronic obstructive pulmonary disease (COPD) (9, 10). The study compared the expression of 863 human microRNAs in blood cells of 28 lung cancer patients, 24 COPD patients and 19 healthy controls. The microRNA profi les from patients with lung cancer and COPD were more similar to each other than to the healthy controls. Nevertheless, a microRNA signature comprising of only six differential microRNAs discriminated lung cancer and COPD patients with 90.4 per cent accuracy, 89.2 per cent specifi city, and 91.7 per cent sensitivity demonstrating that blood microRNA signatures are suitable to distinguish lung cancer from COPD (see Figure 2). The differentiation between COPD and lung cancer might be of high relevance since COPD is not only a common co-morbidity of lung cancer, but also precedes tumours in many cases (10).

A similar approach has been successfully used by Dr Benjamin Meder and co-workers (11). This study focused on the early diagnosis of acute myocardial infarction (AMI). Blood sample analyses of 20 patients with acute myocardial infarction and 20 control patients identifi ed 121 microRNAs as deregulated in AMI patients as compared to healthy controls. Unique signatures of one to 20 microRNA have been identifi ed to discriminate AMI patients from healthy control with up to 96 per cent specifi city, 90 per cent sensitivity and 93 per cent accuracy (see Figure 3). Microarray results have been confi rmed by qRT-PCR and signatures have been validated on independent patient cohorts. This study shows that a single microRNA and especially microRNA signatures derived from peripheral blood could serve as novel biomarkers for cardiovascular diseases (11).

Specific signatures identifi ed for several diseases comprising as few as two, up to 200 differentially regulated microRNAs yield comparably accurate classifi cation results (7-16). Recently, a high-throughput multi-centre study reporting on a global comprehensive approach to defi ne the human disease miRNome in blood has been published in Nature Methods (16). This study emphasises the enormous potential of easily accessible microRNA signatures to serve as biomarkers and paves the way to a pathway and target annotated disease miRNome. This adds up to the growing knowledge of function and relevance of microRNAs for an increasingly large number of diseases deposited in the human microRNA disease database (HMDD) (more than 100 diseases). Combined with bioinformatic prediction this enables the assessment of relevant pathways and potential targets and adds yet another powerful feature to microRNAs as universal biomarkers. Moreover, due to their regulatory functions and inherent stability, microRNAs themselves are considered potential therapeutic targets as well as candidate therapeutic agents.

Conclusion

Applying expert standardised procedures covering all aspects from pre-analytic considerations to comprehensive data analyses pipelines facilitated the presented examples on successful microRNA biomarker development and demonstrate the excellent performance of these biomolecules as potent genomic biomarkers. microRNA signatures comprising two to 200 differentially regulated microRNAs can be readily exploited for diagnostic/prognostic laboratory testing in larger studies or as fast (co-)diagnostic tools with clinical utility. Integrated approaches combining widely used classical protein biomarkers with microRNA biomarker signatures have clinical utility and can increase the diagnostic opportunities for the benefits of the patients. The growing number of microRNAs with annotated defined pathways and specific targets add yet another valuable feature to the broad range utilities of these molecules. The reported features of genomic biomarkers in general and microRNA signatures in particular qualify (circulating) nucleic acids from easily accessible bodily fluids as ideal candidates for virtually all biomarker applications at academic and clinical institutions up to companion diagnostics in industrial drug development. Individually tailored pilot studies performed by expert custom research organisation offer the opportunity to cost-effectively and specifically explore the potential benefits of blood-derived nucleic acid biomarkers for the study objective of interest.

Multivariate integration of other genomic biomarkers (for example DNA methylation, SNPs and mRNA profiles) into the context of classical proteomic and metabolomic biomarkers and imaging data from advanced imaging techniques will even potentiate the high value of microRNA signatures alone and will probably significantly contribute to improved clinical disease management and the delivery of cheaper and safer drugs.

References

  1. Frank R and Hargreaves R, Clinical biomarkers in drug discovery and development, Nature Reviews Vol 2: pp566-580, 2003
  2. Tizon X, Imaging Cancer Biomarkers, European Biopharmaceutical Review 54: pp32-36, 2011
  3. Kroh ME, Parkin RK, Mitchell PS and Tewari M, Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR), Methods 50: pp298-301, 2010
  4. Schwarzenbach H, Hoon DSB and Pantel K, Cell-free nucleic acids as biomarkers in cancer patients, Nature Reviews Cancer 11: pp426-437, 2011
  5. Antony P, Rose M, Gaisa NT, Alkaya S, Heidenreich A, Knüchel R and Dahl E, Charakterisierung von Methylierungs-Biomarkern für das Harnblasenkarzinom, Pathologe 31(Suppl 2): pp244-250, 2010
  6. Cortez MA, Bueso-Ramos C, Ferdin J, Lopez-Berestein G, Sood AK and Calin GA, MicroRNAs in body fluids-the mix of hormones and biomarkers, Nat Rev Clin Oncol 8(8): pp467-477, 2011
  7. Keller A et al, Stable serum miRNA profiles as potential tool for non-invasive lung cancer diagnosis, RNA Biol 8(3), 2011
  8. Fichtlscherer S et al, Circulating MicroRNAs in Patients With Coronary Artery Disease, Circ Res 107(5): pp677-684, 2010
  9. Keller A et al, miRNAs in lung cancer: studying complex fingerprints in patient’s blood cells by microarray experiments, BMC Cancer 9: p353, 2009
  10. Leidinger P et al, High-throughput miRNA profiling of human melanoma blood samples, BMC Cancer 10: p262, 2010
  11. Meder B et al, MicroRNA Signatures as novel biomarkers for acute myocardial infarction, Basic Res Cardiol 106(1): pp13- 23, 2010
  12. Roth P et al, A specific miRNA signature in the peripheral blood of glioblastoma patients, J Neurochem Volume 118(3): pp449-457, 2011
  13. Häusler S et al, Whole blood-derived miRNA profiles as potential new tools for ovarian cancer screening, Br J Cancer 103(5): pp693-700, 2010
  14. Leidinger P, Keller A, Borries A, Reichrath J, Rass K, Jager SU, Lenhof HP and Meese E, High-throughput miRNA profiling of human melanoma blood samples, BMC Cancer 10: p262, 2010
  15. Keller A et al, Multiple sclerosis: MicroRNA expression profiles accurately differentiate patients with relapsing-remitting disease from healthy controls, PLoS One 4(10): pe7440, 2009
  16. Keller A, Leidinger P, Bauer A, ElSharawy A, Haas J, Backes C, Wendschlag A, Giese N, Tjaden C, Ott K et al, Toward the blood-borne miRNome of human diseases, Nature Methods, 2011

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Ioannis Amarantos, Senior Field Application Scientist at febit biomed GmbH, is responsible for scientific support and managing of customer projects. Prior to joining febit, Ioannis held a postdoctoral position on functional genomics at the European Molecular Biology Laboratory (EMBL), Heidelberg. Having successfully completed his studies at School of Chemistry, University of Patras, Greece, Ioannis received his MSc and PhD at the Department of Biochemistry of the University of Patras, Greece in the field of protein synthesis. Email: ioannis.amarantos@febit.de

Thomas Laufer, Application Development Scientist at febit biomed GmbH, is responsible for the development and application of innovative DNA- and RNA-based assays. Thomas successfully finished both his diploma studies of biomedical engineering as well as his master studies of biotechnology (MSc) at the University of Applied Sciences in Münster. Email: thomas.laufer@febit.de

Thomas Brefort is febit’s Director Application Development & Genomic Services. He is responsible for both the development of innovative methods in nucleic acid biomarker research. Prior to joining febit, Thomas worked as a post-doctoral fellow, project leader and head of lab on functional Phytopathogenomics and secretomics at the Max Planck Institute for terrestrial microbiology where he had successfully completed his PhD studies beforehand. A molecular biologist by training, Thomas received his diploma at the Department of Microbiology of the University of Marburg. Email: thomas.brefort@febit.de

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