Institute says he and other ICBC members are using MS-based discovery in combination with transcriptional profiling, literature mining, and expert knowledge to enrich the pool of candidate biomarkers that the researchers will target in verification studies with immunological or MS methods. But other researchers say that some MS methods can be viable tools for discovery. “We are indeed undersampling with many [MS] approaches,” says Dick Smith, who is at the Pacific Northwest National Laboratory and is a member of ICBC. With multidimensional protein identification technology (known
On the first evening of the U.S. HUPO Second Annual Conference, Lee Hartwell of the Fred Hutchinson Cancer Research Center gave the featured talk on biomarker discovery. He said that proteomics scientists “are not discovering markers efficiently.” Going against popular opinion, he placed the bottleneck at the discovery stage, not at the validation or approval (by the U.S. Food and Drug Administration) stages of biomarker development. He explains, “The field has so few new candidate biomarkers to validate and approve that [researchers] are [testing] them one at a time. This is very expensive and inefficient. The way the field will move forward is if discovery is more effective, so that hundreds of biomarkers can be multiplexed and validated and approved on the same patient samples.” Hartwell says that to widen this bottleneck, researchers should broaden their scopes and use a combination of methods. He adds that instead of searching for biomarkers in blood, which is a highly complex mixture, researchers should focus their discovery efforts initially at the site of disease: at the In the blood? Some researchers say that initial bio tissue involved in the disease, or in marker studies should be conducted on tissue, not the fluid bathing the tissue of interblood, samples. est. Here, potential biomarkers are likely to be present in higher concentrations than in the blood. as MudPIT), for example, only a few of According to Hartwell, MS consisthe several peaks in an MS spectrum tently undersamples complex mix(generated after an LC separation) can tures such as plasma or serum, detectbe processed for a second round of ing only the most abundant species. Gil MS. “That’s really why, in our work, we Omenn at the University of Michigan stress the direct MS approaches,” says Medical School explains that MS is inSmith. His group generates a library herently a sampling technique that can of accurate mass and time (AMT) tags be improved with the addition of fracfor peptides by MS/MS, then uses the tionation methods. But fractionation tags to identify components observed takes time and decreases throughin single MS runs in subsequent experput. “So we have to have a combination iments. Unidentified features that apof discovery technologies and highpear promising are also investigated. throughput testing with other technolSmith says that MS approaches such as ogies,” says Omenn. He proposes that the AMT tag method, in combination the use of multiplexed ELISAs, antibody with fractionation steps, offer signifimicroarrays to detect tumor-associated cant promise. antigens, or methods to detect circulatAccording to Dave Speicher at the ing auto-antibodies against tumor proWistar Institute, the technology is just teins could help researchers discover lagging a bit. Like the Human Genome biomarkers. As a member of Hartwell’s Project investigators before them, proInternational Cancer Biomarker Conteomics scientists started with what sortium (ICBC), Steve Carr at the Broad was available at the time but must con-
tinue to improve the technology. “The tools we had when these studies were initiated are not capable of analyzing the plasma at [the low nanograms-permilliliter] level,” says Speicher. “So it’s not surprising that there’s been failure.” His group depletes most of the abundant plasma proteins, separates the flow-through by isoelectric focusing, and runs those fractions on a 1DE gel, which is cut into many slices. Each slice is then analyzed by LC/MS/MS. Although the method is currently lowthroughput, it can detect proteins at low nanograms-per-milliliter levels, which is the likely concentration range of cancer biomarkers. But maybe blood is not the place to start, says Hartwell. He points out that “the logical approach is to start with the tissue itself or its fluid,” because the highest concentration of potential biomarkers would be in the tissue where a disease develops. Later, once potential biomarkers are identified, one can look for them in the plasma or serum. Carr says that “proximal” fluids that bathe tissues could be a goldmine for biomarker discovery. But not every disease can be localized to one tissue or organ. In such cases, plasma or other biofluids may be a better starting point. Also, examining a tissue could lead one astray. “One drawback to that approach is that not everything that is present in the tumor [or tissue] is going to make it into the surroundings,” he cautions. Carr’s group is currently studying a “pseudo” proximal fluid for potential breast cancer markers that are shed by t umors. Immediately after being removed from a patient, a tumor is cut into several pieces and soaked in buffer. The tumor “is allowed to slough off whatever it’s shedding and secreting naturally [because] the tissue is still alive at this point,” he says. The resulting “shedome” is then analyzed for potential biomarkers. What about the fate of the HUPO Plasma Proteome Project (PPP) if tissue and nonblood biofluids are the way to go? According to Omenn, Hartwell’s proposition is not contrary to the goals of the PPP. The PPP “was always a combination of looking [at] what we could detect in plasma and coordinating all of the [HUPO] organ-specific or dis-
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Speeding up biomarker discovery
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MEETING NEWS ease-specific proteome studies with the collection of plasma,” he says. Specifically, even if other projects find potential biomarkers in an organ, clinicians wouldn’t want to perform biopsies every time a disease is suspected; instead, they could look for those potential biomarkers in the plasma to make a diagnosis. Having a baseline list of proteins that are present in plasma is just one step of a comprehensive plan of attack. But plasma may still be valuable for discovery. Smith says, “I think there may be things that we can’t predict from [studying tissues]. There could be some biology we don’t understand.” For example, modifications could be added to or removed from proteins after they leave a tissue and enter the circulation. If those changes are not taken into account, a candidate biomarker could be missed when a researcher shifts his or her focus from the tissue to the blood
for diagnostics. Biomarker discovery could also be accelerated by forming large collabo rative groups, says Hartwell. “We need a ‘genome’-type project to tackle this big problem of biomarker discovery,” he says. “We need data standards, reagents, databases, algorithms, fund ing, and a large-scale organized activity.” Hartwell leads the ICBC, which is a multinational effort to find biomarkers for breast, liver, nasopharyngeal, ovarian, pancreatic, and stomach cancers. He points out that the U.S. National Cancer Institute recently formed the Clinical Biomarker Program, which will also coordinate a large biomarker discovery project. Another limiting step in the biomarker pipeline is assay development, says Hartwell. “If one can discover hundreds of candidates at the tissue level or the blood level, the problem
is going to be assaying the hundreds of candidates in hundreds of samples,” he explains. Although ELISA is the standard clinical assay for lowabundance proteins, it is too expensive to implement on such a large scale. Therefore, Hartwell suggests using the method of stable isotope standards and capture by anti-peptide antibodies (known as SISCAPA) and multiplereaction-monitoring MS. Some ICBC members are investigating this method to develop multiplexed assays for several potential biomarkers. Researchers say that no one correct answer exists to the biomarker discovery problem at the moment. Different approaches and perspectives are all necessary to move the field forward. “I don’t think we’re at the point of predicting which [method] is going to be the most effective yet,” says Smith. —Katie Cottingham
GOVERNMENT AND SOCIETY
U.S. FDA: Critical projects i nclude biomarker and bioinformatics development On March 16, 2006, the U.S. Food and Drug Administration (FDA) released an initial group of research topics that could help accelerate medical product development and approval. The Critical Path Opportunities Report and List (www.fda.gov/oc/initiatives/ criticalpath/reports/opp_report.pdf ) suggests 76 research topics, including some ideas that are geared toward various aspects of biomarker and bioinformatics development. The agency prepared the Critical Path Opportunities List after receiving comments from public and private stakeholders in response to its Critical Path Report, which was released in 2004. In the report, the FDA acknowledged that the rate of medical product submissions was slowing down and suggested that a long-term agency initiative was necessary to turn the tide. As part of that initiative, the FDA developed the Critical Path Opportunities List, which provides investigators with research ideas for developing better tools to evaluate and generate medical products and therapies. The list is divided into six broad ar-
eas: animal models and tools for biomarker identification and assessment, clinical trials, bioinformatics, manu-
Ideas for the taking. After consulting with scientists, the FDA has developed a list of critical research topics.
facturing, public health, and pediatrics. Of special interest to proteomics researchers are the sections on biomarkers and bioinformatics. According to the list, criteria for determining the usefulness of a biomarker should be developed. In addition, the scientific community needs standards for the DNA microarray- and proteomicsbased identification of biomarkers.
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Other biomarker topics include assessing disease-specific biomarkers and advancing the use of imaging methods to discover biomarkers. Suggested bioinformatics research topics include the development of biomarker assessment strategies, clinical trial simulations, and device performance models. To make headway on these topics, the FDA plans to support various research projects. Some of the projects will require the formation of large-scale collaborations coordinated by the FDA. One such collaboration, called the Predictive Safety Testing Consortium, was recently announced. The consortium consists of representatives from five major pharmaceutical companies that pledge to share methods derived in-house to determine the safety of new therapies. Companies will also test each other’s methods. According to the FDA, the Critical Path Opportunities List should stimulate scientists to investigate many crucial research topics that must be addressed to accelerate the approvals of medical devices and therapies. Updates on the Critical Path Initiative and funding opportunities can be found at www. fda.gov/oc/initiatives/criticalpath. —Katie Cottingham
