While new biomarker diagnostic techniques have shown promise, the results still frustrate doctors and researchers.
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Electron micrograph of nanoparticle molecular cages, which can trap small peptides in a serum sample for analysis. (Source: Emanuel Petricoin, George Mason University)
This year, millions of men will have their prostate-specific antigen (PSA) levels checked, in an effort to detect any budding prostate cancers as early as possible. Women will line up for mammograms and Pap tests, hoping to detect any breast or cervical tumors.
The good news is that so many patients have access to these routine exams, which represent the cutting edge of cancer diagnosis. The tests save lives. The bad news, however, is that these assays are still embarrassingly primitive, routinely producing false positive or false negative results. Thousands of patients will have benign tumors biopsied or even excised, while thousands of others will harbor malignancies long enough for them to metastasize. Indeed, prostate, breast, and cervical cancer are exceptions; many cancers are far more difficult to detect.
Finding better biomarkers for cancer has been a high priority for researchers for decades, and the molecular biology revolution promised a new era for this effort. Unfortunately, even the availability of whole genome sequences didn’t end the field’s long history of disappointments.
“In ‘98, everyone was in a genotypic hysteria. We were going to solve all the world’s problems by sequencing Craig Venter’s DNA, and of course that came and went and it hasn’t done much,” says Ralph McDade, PhD, strategic development officer for Rules Based Medicine (RBM), a biomarker testing laboratory in Austin, Texas. McDade adds that “Now all of a sudden people are realizing that the effector molecules in the blood, proteins, are the ones that, of course, are some of the first, earliest signs of disease.” Turning those protein patterns into practical diagnostic tests is the field’s new focus, and while the strategy still faces major hurdles, experts are optimistic that cancer biomarkers are finally on the right track.
A thousand points of light
Established by the same team that founded Luminex, RBM started with a conceptually simple strategy: automating and multiplexing Luminex immunoassays for biomarker tracing. The Luminex technology uses dyed microspheres covered with antibodies to detect specific antigens in solution. By combining different spheres into a single assay, RBM can monitor changes in large panels of proteins over time.
The company’s HumanMAP Antigen product, for example, now quantifies the levels of 89 different antigens, from adiponectin to von Willebrand factor, in a serum sample. “In the not too distant future, we’re going to be talking about 1,000 quantitative immunoassays run on a few hundred microliters of serum or plasma,” says McDade.
Currently, RBM performs the assays as a service for basic biomedical studies and clinical drug trials, but a $1.1 million grant from the National Cancer Institute (NCI) is helping to translate the strategy for diagnostic use. Under the grant, the company will expand the HumanMAP system, initially adding 50 new cancer biomarkers that have been discovered by NCI researchers.
The second wave of the project will be magnitudes larger. “We understand there are 1,050 more [biomarkers] in the pipeline coming behind those. It’s a huge consortium project where they’re building antigens and antibodies for 1,100 different proteins that [are] either up- or down-regulated pretty strongly in various cancer types,” says McDade.
In other work, the company is helping to test a new panel of markers for ovarian cancer. “We’re about to go to a multisite clinical trial for a diagnostic that we believe has about 85% sensitivity and specificity for Stage II ovarian cancer,” says McDade. Stage II cancers involve one or both ovaries and adjacent organs in the pelvis. If the cancer is detected at that point, five-year survival rates can exceed 75%; survival drops off significantly as the disease gets more advanced.
Drowning in diversity
To discover these new biomarkers, most researchers are relying on the field’s workhorse tool: mass spectrometry. As spectrometers have become more robust and sensitive, and also less expensive, many labs have added the technology and begun hunting for additional cancer biomarkers. Translating those findings into clinical tests, however, may require immunological assays such as the ones RBM are developing. “Mass spec is a wonderful discovery tool, it’ll help you see some of these proteins going up and down, but mass spec will never make it to the clinic—it’s not quantitative enough, it’s not reproducible enough,” says McDade.
Others disagree. “There [are] actually some really good … practitioners who are really starting to learn the value of using mass spec in a clinical sense,” says Randall Nelson, PhD, director of the molecular biosignatures analysis unit at Arizona State University in Tempe, Ariz. Nelson, who also founded Intrinsic Bioprobes in Tempe, says he is already discussing these types of assays with reference laboratory companies.
Besides speeding the adoption of new biomarker tests, clinical labs with mass spectrometers would also be able to deploy entirely new types of tests. That’s important for Nelson and his colleagues, who are working on a strategy they call “population proteomics.” Rather than simply screening patients and controls for changes in the levels of particular proteins, they are also looking for changes in the individual protein’s post-translational modifications, an assay that requires mass spectrometry.
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Gold nanoparticles (GNP) covered with antibodies form complexes that can be detected very sensitively by dynamic light scattering. (Source: Qun Huo, University of Central Florida)
Proletarian proteins and peptides
Finding that such a mundane protein can be a useful biomarker also undermines a major assumption behind many current sample preparation protocols. Especially for biomarker studies, researchers often start by removing abundant and seemingly uninteresting proteins with immunodepletion columns.
“The rationale was to deplete the serum or plasma or any body fluid of the sort of high-abundance proteins, so you could kind of drill farther into the … lower abundance analytes,” says Emanuel Petricoin, PhD, co-director of the center for applied proteomics at George Mason University in Manassas, Va.
Besides the potential for the abundant proteins to be biomarkers in their own right, Petricoin says these molecules also bind many of the smaller peptides that cells routinely excrete: everything from small hormones and immune signals to ordinary cellular garbage. Because the molecules are small, they can diffuse easily into the bloodstream, providing a cross-section of information about every process currently underway in the body.
“We believe that the peptidomic archive may contain or represent all of the analytes in the body in one form or another,” says Petricoin, adding that “it’s kind of a combination of a continent of information that’s relatively unexplored, plus the potential that it may be one of the most rich archives for analytes that could be sensitive and specific for ongoing disease processes.”
To collect an unbiased sample of the peptidome, the researchers designed nanoparticles that consist of a porous molecular cage surrounding a “bait” molecule with broad affinity for peptides. The openings in the cage exclude large proteins, allowing only peptides inside. The peptides then stick to the bait. “It’s like a little nano-lobster trap if you will—things get in, they don’t get out,” says Petricoin.
In practice, the investigators can add a patient’s blood to a tube containing the nanoparticles, allow the peptides to accumulate in the particles for a few minutes, and then centrifuge the peptidome away from the rest of the sample. The nanoparticles also seem to protect the peptides from degradation.
One nanosphere sandwich, easy on the sample
Petricoin’s team is certainly not the only one using nanoparticles to track biomarkers. In a separate effort, for example, researchers at the University of Central Florida in Orlando, Fla., have borrowed a technique from polymer chemistry to boost the sensitivity of nanoparticle-based immunoassays.
“There are many other groups working on these as well. However, I think we’re the first ones to come up with using the dynamic light scattering method to detect the nanoparticle probe,” says Qun Huo, PhD, an associate professor in the university’s department of chemistry and the lead researcher on the new work. Dynamic light scattering consists of shining a beam of light through a sample, then measuring the scattered light on the other side to track the movement of particles within the solution. From that readout, researchers can determine particle sizes very precisely. “It actually monitors the particle, monitors the scattering intensity change during particle movement in solution—that makes it very sensitive,” says Huo.
In a typical experiment, the investigators construct two batches of nanospheres, each festooned with a different antibody against a particular biomarker. When exposed to patient serum containing the antigen, the two particles form a sandwich around the antigen. Dynamic light scattering easily quantifies the number of sandwiched nanospheres in the solution, indicating the antigen’s concentration.
One potential application for the method is in a cheap assay for free PSA. Free PSA is considered a much more sensitive and specific assay for prostate cancer than total PSA, but the limited sensitivity of clinical ELISA tests makes it much more difficult to measure. Huo’s nanospheres could fix that.
Technically, the procedure is quite simple—Huo routinely trains undergraduates to do it—and it also uses much smaller samples than a traditional ELISA assay. “Certainly I’m looking to replace ELISA with this technique,” says Huo, adding that “ELISA, as you know, is a multistep, very complicated process compared to this one, and also ELISA uses a lot more biological sample than this technique.”
The view from the ground
Regardless of an assay’s apparent merits in the research lab, ultimately it will have to please a much tougher audience: clinical laboratory technicians. While many of these professionals acknowledge the need for better methods, they must implement them without neglecting their often enormous workloads.
“We have over 100 different assays running in the clinical laboratory daily, and we report out about 10,000 patient results on 1,200 samples daily,” says Alex Rai, PhD, who helps oversee the clinical laboratory service at Memorial Sloan Kettering Cancer Center in New York, N.Y. Slipping a new method into that schedule is like rebuilding an interstate highway at rush hour, but Rai—who also does translational biomarker research—agrees that the need is acute. “The currently used markers in the clinical laboratory today are not always ideal; they’re the best we have at the current time, but there’s a lot of room for improvement,” he says.
Pointing to PSA tests, which are often used as a benchmark for new biomarker assay techniques, Rai says that increased analytical sensitivity would be a boon to the field. More sensitive PSA detection would be especially useful for tracking recurrence in patients who’ve had their prostates completely removed. “In the case of radical prostectomy, there should actually be no PSA—once you start seeing PSA increasing once again, that’s a sign that the cancer is coming back,” says Rai, adding that “you want to be able to have an assay that picks up very low levels of PSA.”
Rai points to assay validation as the biggest current problem. “We have to be cautiously optimistic. There are important candidate biomarkers that are being reported every week, [but] I think we really have to be careful to make sure that initial promising results from pilot studies are reproducible in larger populations, performed in other laboratories, and even across technologies,” says Rai.
About the Author
Originally trained as a microbiologist, Alan Dove has been writing about science and its interfaces with industry and government for more than a decade.
This article was published in Drug Discovery & Development magazine: Vol. 11, No. 8, August, 2008, pp. 16-20.
Filed Under: Drug Discovery