Groups of biomarkers tracked over time help to unravel diseases, and this requires multiple approaches to identify and validate new markers.

click to enlarge  click to enlarge With Luminex’s xMAP technology, 10 concentrations of two fluorophores—red and near infrared—produce 100 distinct bead regions (top). Color-coding enables each microsphere set to be classified individually and to be multiplexed with other microsphere sets. In the Luminex analyzer (bottom), fluidics line up the beads and pass them by two lasers: a red laser classifies each color-coded microsphere to determine which assay is carried on that particle, and a green laser measures the assay result on its surface. (Source: Luminex)

One measurement is not enough. That’s what Craig N. Giroux, PhD, associate professor at the Karmanos Cancer Institute of Wayne State University, Detroit, Mich., thought when a physician suggested a measurement of prostate-specific antigen (PSA). Giroux said, “Let’s collect PSA readings for a couple years to get a baseline, and I’ll plot it in Excel. When it moves, I’ll call you.” Without gathering that dynamic history of a biomarker, individual variation makes disease difficult to distinguish from health. “You can’t just sample once and know,” says Giroux. Moreover, many diseases might require tracking more than one marker.

Although many techniques identify new biomarkers, they are not always the best ones. “We hear complaints from biotech and pharma that not enough biomarkers are making it to the clinic,” says Barry Schweitzer, PhD, director of protein analysis R&D at Invitrogen, Carlsbad, Calif. “But there is no decrease in the volume of these customers who want biomarkers. They just want faster, cheaper solutions that will fit their particular needs.”

Picking out proteins
Many proteins, including PSA, can serve as biomarkers, but scientists must efficiently extract them from fluids and tissues. For example, Invitrogen’s ProtoArray works with cerebrospinal fluid, saliva, serum, tears, tumor extracts, urine, and more.

The ProtoArray includes 8,000 unique human proteins as probes—made specifically for biomarkers. “It’s mostly used to look in bodily fluids for the presence of autoantibodies,” Schweitzer explains. These antibodies recognize self-antigens, which can serve as disease signatures for autoimmune and other diseases, including cancer and neurodegenerative disorders.

“When a spot of protein lights up on the array that’s different in one group versus another,” says Schweitzer, “you know exactly what that protein is.” Then, a researcher can do the bioinformatics—using Invitrogen’s freeware or other analysis packages—to look at that protein’s function and see if it makes sense related to the disease being studied.

click to enlarge   Ingenuity Pathways Analysis (IPA) 6.3. looks for relationships between biomarkers and characteristics of diseases, such as expression levels of genes. (Source: Ingenuity)

Many researchers also want to quantify proteins. “We are moving from qualitative to quantitative methods,” says Mary Lopez, PhD, director of Thermo Fisher Scientific’s Biomarker Research Initiatives in Mass Spectrometry (BRIMS) Center, Cambridge, Mass. For example, Thermo Fisher Scientific will soon introduce instrumentation that greatly increases the sensitivity of quantitative, selective reaction–
monitoring assays, according to Lopez. “This will allow us to develop more accurate, targeted assays for validating biomarkers in complex matrices, such as blood,” she says.

Lopez and her colleagues at the BRIMS Center are also developing new ways to prepare biomarker samples. One example is a workflow that integrates KingFisher, a high throughput sample-preparation robot for processing affinity capture-type magnetic beads in 96-well plates. The processed samples can be introduced directly into one of the company’s triple-quadrupole mass spectrometers, such as the Quantum Ultra, which specifically quantifies the level of a targeted peptide biomarker. “This facilitates the rapid validation of biomarker candidates,” Lopez says.

Bring on the beads
“Lots of people are looking at microRNA, or miRNA, for new biomarkers,” says Keld Sorensen, PhD, executive director of research and development of Luminex Corporation’s Bioscience Group, Austin, Texas. Moreover, the telling miRNA biomarkers come in groups. “Among unusual miRNA patterns,” says Sorensen, “I can’t think of any that are single miRNAs. The numbers tend to vary from 5 to 25.”

click to enlarge   PerkinElmer’s AlphaLISA uses two beads and excitation light to detect analytes, including biomarkers. (Source: PerkinElmer)

To look for groups of disease-linked miRNAs, researchers can use multiplexing, such as Luminex’s xMAP technology, which is based on 5.6μ polystyrene spheres that contain red and infrared fluorophores. Using varying concentrations of these dyes, Luminex makes 100 different beads. Then, the surface gets coated with reagents for a specific assay, such as one to measure miRNA.

All of the Luminex xMAP assays come in 96-well plates, with each well containing up to 100 types of beads. “So you can get 100 results from one well, which translates to 9,600 results for an entire plate,” Sorensen says.

Scientists at PerkinElmer, Waltham, Mass., also developed a bead-based approach to finding biomarkers. The company’s AlphaLISA is a homogeneous ELISA that works with two beads, which are 200 nanometers across. Both beads, covered with antibodies, connect to the target analyte, and then excitation at a wavelength of 680 nanometers causes generation of singlet oxygen that travels from the first bead to the second, causing it to emit light at a wavelength of 615 nanometers, which indicates the presence of the target. “The emission peak is sharp and intense, and the signal is highly amplified by the singlet oxygen as well as the chemical reaction in the acceptor bead. This leads to an excellent sensitivity of the assay, detecting some analytes in the femtomolar range and needing only very small amounts of precious sample,” says Martina Bielefeld-Sevigny, PhD, director of molecular pharmacology at PerkinElmer. In addition, the 615 nanometer emission experiences no interference from hemoglobin.

PerkinElmer offers a variety of kits covering known targets. “For rare or unknown targets,” says Bielefeld-Sevigny, “we offer a custom development service. If someone has in-house antibodies, they can send them to us, and we will couple them to beads and develop the assays.”

C-Path Leads New Kidney Safety Drug Tests  The Critical Path Institute (C-Path) announced in June the first results of a collaboration to identify improved methods to test the safety of new drugs in development. More than 200 scientists from pharmaceutical companies, non-profit research organizations, and advisors from the Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) participated in the Predictive Safety Testing Consortium (PSTC). The proposal submitted by the consortium has been evaluated by the FDA and EMEA and seven new tests have been deemed by the agencies as “qualified” markers of drug-induced kidney injury in animal studies. The tests measure the levels of seven key proteins or biomarkers found in urine that can provide additional information about drug-induced damage to kidney cells, also known as renal toxicity. “The PSTC is proving that government and industry scientists can–and should–work together to improve the methods for testing the safety of new drugs,” says Dr. Raymond L. Woosley, C-Path’s president and CEO. “The qualification of these seven new tests is the critical first step towards their use in clinical drug development.” “Using current kidney tests that were developed over 100 years ago, 70 percent of kidney function must be lost before damage can be detected. The newly approved biomarkers are far more sensitive and specific for drug-induced kidney damage,” says Dr. Wm. Mattes, C-Path’s director of the PSTC.

Searching with software
Simply identifying new biomarkers does not guarantee that they are actually useful in the clinic. That depends on validation. “Part of the challenge is sample size,” says Brigitte Ganter, PhD, director of product management at Ingenuity Systems, Redwood City, Calif. “You need a good number of samples for identification and validation.” Once scientists collect that data, they can interrogate it with a pathways-analysis application, such as Ingenuity Pathways Analysis (IPA) 6.3.

“This tool helps you understand how relevant identified biomarkers are in the context of a specific disease,” Ganter says. IPA includes information on known drug targets and information about a molecule’s pathways. “It lets you filter down a list of biomarkers to the more-relevant ones,” Ganter says. The filtering offers many parameters, including expression levels of genes and proteins, tissue distribution of a molecule, detection of the protein in a specific body fluid, and disease association—all linked directly to the supporting literature.

“We’ve gone into the literature,” says Ganter, “and extracted biological findings, like: Mutant mouse Mekk4 (Map3k4) gene (homozygous knockout) in mouse decreases synthesis of mouse Ifng protein in mouse T lymphocytes expressing mouse CD4 protein that involves IL-12 and IL-18 protein in culture medium.” Ingenuity’s analysis software includes a specific tool for biomarkers.

Eventually, researchers want to build a complete picture of individuals. “Every person has an identity that reflects their history and exposure in the environment,” says Giroux. This identity comes from a person’s collection of genes, how they interact with the environment, and how the environment interacts with them. As a result, future “biomarkers” could consist of networks.

Giroux and Ganter are already looking at such networks. We get a collection of genes from our parents, and connections—biochemical pathways, neuronal connections, and so on—get made during development and early in life. “Your environment in the first two years of life impacts how strongly you react to agents, including disease,” Giroux says. “So biomarkers should not be the individual building blocks—genes and proteins and such—but the connections, the networks.”

Today’s biomarker technology could help tomorrow’s scientists apply such biomarker networks.

About the Author
May is a publishing consultant for science and technology based in Minnesota.

This article was published in Drug Discovery & Development magazine: Vol. 11, No. 7, July, 2008, pp. 30-32.