Biomarkers are increasingly recognized for their potential not only as biologic harbingers that provide early warnings of disease but also biochemical monitors of patient responses to therapies. However, the field is hampered by a lack of reliable, quantifiable, easily measured biomarkers that correlate well with biochemical measures of disease progression.
Companies are making strides to close the gap between bench and bedside. New advances in the field include identifying and quantifying biomarkers within tissue samples, designing image analysis tools that model human thought, and generating panels of biomarkers that provide a specific molecular signature.
Immunohistochemistry provides a powerful means to obtain fundamental insights into biological processes and disease states. Detection of protein biomarkers in tissue can be a vexing problem due to concentration variability and subjective scoring methods done one sample at a time by a pathologist. A new system developed by HistoRx (www.historx.com), called AQUA (Automated Quantitative Analysis), provides rapid quantitative readings of tissue microarrays and other samples, according to the company.
The AQUA system allows high-throughput, high-resolution analyses in a quantitative and reproducible fashion, states Paul Stroobant, Ph.D., vp head of research. It is unique because it isnt morphology-based but is linked to a fluorescent microscope that detects expression of multiple biomarker proteins by measuring the intensity of antibody-coupled fluorophores in molecularly specified subcellular compartments, such as the plasma membrane, cytoplasm, and nucleus.
Dr. Stroobant reports that a key capability of the technology is its Rapid Exponential Subtraction Algorithm. We view a multicolor slide, take two pictures in and out of focus, and then subtract the out-of-focus version to get a crystal clear picture similar to a confocal image, which allows rigorous quantification. We generate standard curves for quantification by calibrating the fluorescence signals with the quantities of antigens expressed by various cell lines and determined by standard ELISA.
Using the AQUA technology, HistoRx cofounder David Rimm found a disturbing pattern in conventional histological testing. He determined that test results for quantifying some tissue antigens with conventional histological assays can vary widely depending on the dilution of the detecting biomarker antibody used. This could have a profound effect on the interpretation of studies using biomarkers as predictors of outcome or as hallmarks of response to therapy, says Dr. Stroobant.
Although scientists can automate image acquisition from biological samples, interpreting those images is another matter. Noisy or low contrast data, especially when combined with complex images, require tedious human scrutiny, one image at a time. Definiens (www.definiens.com) says it is changing that paradigm with their Cognition Network, a technology designed to model human thought.
We are developing image-analysis tools in which we embed the decision-making processes into software that scientists use to analyze tissue, reports Zachary W. Pitluk, business manager, life sciences North America. Customers provide their own biomarkers and our software helps them assess morphological changes in tissues. This allows validating the distribution of biomarkers as well. One of the unique features of our software is that it is a scalable platform that represents a totally different approach to image analysis.
Cognition Network can extract, represent, and store knowledge from a complex input, such as images, by maintaining a hierarchy of structures in which multiple changes are represented as a pattern. Defining a pattern gives it power and can faithfully mimic human cognition of the observed phenotype.
Some companies outsource various aspects of biomarker discovery and analysis. Indiana Centers for Applied Protein Sciences (INCAPS; www.indianacaps.com) is a CRO with expertise in protein biomarker collaborations. Capital costs for instrumentation and methodology development can be considerable, notes James R. Ludwig, Ph.D., president and CEO. An alternative approach is to outsource to a company with these capabilities.
We began INCAPS with the idea to provide needed services, develop consortia for basic science problems, and validate new technologies. We also decided not to hold intellectual property. Its really a win-win situation. The company developing biomarkers gets to try the latest technology without the expense of buying it, the manufacturer gets their instrumentation independently validated, and we get to help customers.
INCAPS utilizes both untargeted and targeted approaches for protein biomarker discovery/validation. Some researchers are looking for broad protein changes. We are able to identify all proteins in a certain concentration range and determine which ones change. Since the range of protein concentrations in plasma is 1011, we cannot see them all in this way. High-abundance proteins can mask important low-abundance proteins. That is why for other clients we provide a targeted approach in which the sample is enriched and evaluated by more selective mass spectrometry approaches.
Dr. Ludwig points out that finding a biomarker is just the tip of the iceberg. Once identified, biomarkers must be validated and shown to correlate to the disease. Just finding a marker doesnt mean that it will translate well to the clinic. A researcher must ask many questions. Is the biomarker specific? Is it sensitive? Is it detectable in an appropriate way? Can it follow the course of disease or treatment?