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May 1, 2011 (Vol. 31, No. 9)

Cancer Detection Improved with Noninvasive Testing

Search for Novel Biomarkers Detectable in Accessible Bodily Fluids Proves Promising

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    Principle of SomaLogic’s multiplex SOMAmer affinity assay. (A) Binding. SOMAmers and samples are mixed in 96-well microwell plates and allowed to bind. Cognate and noncognate SOMAmer-target protein complexes form. Free SOMAmer and protein are also present. (B–H) Schematic sequence of assay steps leading to quantitative readout of target proteins. (B) SOMAmer-protein binding: DNA-based SOMAmer molecules (gold, blue, and green) have unique shapes selected to bind to a specific protein. SOMAmers contain biotin (B), a photo-cleavable linker (L), and a fluorescent tag at the 59 end. Most SOMAmers (gold and green) bind to cognate proteins (red), but some (blue) form noncognate complexes. (C) Catch-1: SOMAmers are captured onto a bead coated with streptavidin (SA) which binds biotin. Uncomplexed proteins are washed away. (D) Proteins are tagged with NHS-biotin. (E) Photocleavage and kinetic challenge: UV light (hn) cleaves the linker and SOMAmers are released from beads, leaving biotin on bead. Samples are challenged with anionic competitor (dextran sulfate). Noncognate complexes (blue SOMAmer) preferentially dissociate. (F) Catch-2 SOMAmer-protein complexes are captured onto new avidin-coated beads by protein biotin tag. Free SOMAmers are washed away. (G) SOMAmers are released from complexes into solution at high pH. (H) Remaining SOMAmers are quantified by hybridization to microarray containing single-stranded DNA probes complementary to SOMAmer DNA sequence, which form a double-stranded helix. Hybridized SOMA-mers are detected by fluorescent tags when the array is scanned. [Reprinted from PLoS ONE]

    The best approach to cure cancer is to detect it early, while a tumor is still localized, can be surgically removed, and is more readily treatable with radiation and/or chemotherapeutic and immunotherapeutic agents. Cure rates for early-stage cancers may exceed 80%, whereas once the cancer has spread, survival rates may drop precipitously.

    At the 2011 annual meeting of the American Association of Cancer Researchers (AACR) held last month in Florida, David Sidransky, M.D., professor, Johns Hopkins Medical Institutions, and Francisco Esteva, M.D., Ph.D., professor, University of Texas, M.D. Anderson Cancer Center, chaired a symposium that focused on the discovery and development of molecular biomarkers for solid tumors.

    The presentations described research aimed at identifying and validating various types of biomarkers that are detectable in readily accessible bodily fluids, such as blood and urine, and have diagnostic and prognostic value to help guide clinical decisions related to cancer risk, as well as the need for more invasive diagnostic procedures. The goal is to detect biosignatures that are more specific and sensitive than existing diagnostic modalities, can detect tumors earlier in the course of disease, and can reduce the need for more invasive and costly biopsies and imaging studies. Molecular biomarkers may also represent cost-effective screening tools in high-risk populations.

    Dr. Sidransky led off the session by outlining several challenges in developing and implementing a molecular cancer diagnostic once a tumor-related biomarker has been identified. These include the need for analytical validation of the biomarker to demonstrate high enough accuracy—sufficient specificity and sensitivity—and the importance of identifying the appropriate target population before moving the test forward into a clinical trial setting. Subsequent challenges relate to clinical validation and the need to define a biomarker’s (or panel of biomarkers’) intended use and implications and to understand what the results mean and how best to apply them.

    Harvey Pass, M.D., New York University Langone Medical Center and Cancer Center, presented his group’s experience working with SomaLogic to develop an aptamer-based diagnostic to detect malignant mesothelioma in asbestos-exposed individuals. About 27.5 million Americans were occupationally exposed to asbestos between 1940 and 1979.

    Mesothelioma has a developmental latency period of 10–40 years. About 3,000 new cases are reported each year in the U.S., but the incidence of pleural mesothelioma will not peak for another 20 years. Median survival of patients with late-stage disease averages 12 months, compared to 48 months for individuals diagnosed with stage 1 mesothelioma; an early-stage diagnosis is made in only about 10% of patients.

    The group used SomaLogics’ SOMAmer multiplexed proteomic assay to quantify about 850 proteins simultaneously in 15 µL blood samples. The assay utilizes high-affinity aptamers that bind selectively to a protein target, with slow dissociation rates. They applied the SOMAmer platform to analyze blood samples from three study sites in a prospective case/control study, comparing serum samples from 90 patients with malignant mesothelioma to 80 control samples from asbestos-exposed individuals who did not have mesothelioma.

    The technology utilizes fluorescently tagged, biotin-linked aptamers called SOMAmers that bind to the proteins in the sample, forming DNA/protein complexes. These complexes are isolated and processed to select for specific binding. The protein components are then labeled and immobilized. The aptamers are eluted off the proteins, applied to a microarray, and hybridized to complementary moieties for detection and quantification.

    The samples were divided into two groups: 75% were used as a training set and 25% as a blinded test set. The study led to the identification of 19 significant biomarkers.

    Dr. Pass presented data derived from the application of subsets of these biomarkers to the blinded test set, demonstrating 100% specificity and 80% sensitivity for their ability to distinguish asbestos-exposed controls from mesothelioma cases. The biomarkers were able to detect 15 of 19 stage I/II cases. Ongoing studies are aimed at analytical validation of the biomarker panel using comparative ELISA measurements.

    Among women with ovarian cancer, five-year survival rates vary widely from about 90% for stage I disease (localized to the ovary) to 5% for stage IV cancer. At present, about 70% of patients are diagnosed with stage III or IV disease. Measurement of CA125 in the blood is the test currently used to monitor ovarian cancer treatment, follow patients for recurrence, and in some cases screen high-risk individuals to detect early-stage disease.

    However, as Christine Coticchia, Ph.D., research fellow at Children’s Hospital Boston and Harvard Medical School, pointed out, CA125 is relatively nonspecific for ovarian cancer and uninformative in a substantial percentage of patients. Dr. Coticchia and colleagues are studying a combination of two matrix metalloproteases (MMPs)—MMP-2 and MMP-9—in urine for their utility as biomarkers to predict the presence of ovarian cancer in women with normal CA125 levels.

    Zymographic analysis of urine to measure MMP levels showed significant differences in MMP-2+MMP-9 levels in ovarian cancer versus control samples. Higher levels of each biomarker alone correlated with a greater likelihood of ovarian cancer, but the combination fingerprint of MMP-2+MMP-9 yielded greater diagnostic accuracy—which improved even more when multiplexed with age, with the three variables together yielding an area under the curve (AUC) of 0.82.

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