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May 1, 2014 (Vol. 34, No. 9)

Discovering More Telling Biomarkers

  • Whether it’s mining the metabolome, probing for epigenetic hot spots, or dissecting tumor phenotypes, researchers are discovering new ways to advance biomarker identification and validation using modern tools.

    Although the search for biomarkers presents many challenges, researchers persist because biomarkers have enormous potential for bringing new drugs to market, enhancing molecular diagnostics, and tailoring therapeutics for improved patient stratification and outcome.

    Cancer is increasingly viewed as both a genetic and an epigenetic disease. “Aberrant DNA methylation is one of the most studied epigenetic mechanisms in colorectal cancer,” says Noel Doheny, CEO of Epigenomics, the U.S. subsidiary of the same-named German company. “Because aberrant methylation is an early-stage change, it can be used as a biomarker for cancer detection.”

    If detected early, colorectal cancer is curable in 80% of cases. Yet it remains one of the highest causes of cancer-related death in the industrialized world. One reason the current tests, colonoscopy and fecal occult blood testing, fail to prevent more deaths, Doheny suggests, is that they have low patient compliance.

    “We wanted to identify a blood-based biomarker that could serve as an in vitro screening diagnostic targeting the 40 million individuals who do not participate in current standard testing,” continues Doheny. “Our goal is not to replace colonoscopy, but rather to present a screening tool that, if positive, could be followed up with a colonoscopy.”

    Epigenomics used genome-wide discovery methods to identify and characterize hundreds of DNA methylation-based biomarkers for colorectal cancer. Subsequently, the company identified its strongest biomarker candidate—the Septin9 gene. (This gene, which codes for a GTP-binding protein, has a promoter that is hypermethylated in colorectal cancer.) Finally, using this gene as a biomarker, the company developed a blood-based kit.

    The test involves two steps. First, DNA is extracted from blood plasma and treated with bisulfite, which converts unmethylated cytosine residues to uracil. (Bisulfite is considered state-of-the art for sensitivity and throughput for molecular diagnostics.) Second, the bisulfite-converted DNA is assayed via duplex real-time PCR to detect if Septin9 DNA is methylated. The test is referred to as “duplex,” says Doheny, because it can support the simultaneous use of an internal control (beta-actin).

    “When we used the test and compared it to colonoscopy in a large prospective trial of 8,000 average-risk individuals, we found 67% sensitivity and 88% specificity,” asserts Doheny “The Epi proColon® test is already available in Europe and undergoing FDA evaluation in the United States.”

  • Epigenetic Immune Cell Markers

    Characterizing immune cell populations is an important aspect of applications ranging from infectious diseases to autoimmune diseases to cancer. Traditional methods can be cumbersome and inaccurate, according to Ulrich Hoffmueller, Ph.D., chief business officer and founder, Epiontis.

    “Often, immune cell profiling is accomplished via counting cells and obtaining ratios of different leukocyte subpopulations. This employs flow cytometry of peripheral blood and immunohistochemistry (IHC) solid tissue,” says Dr. Hoffmueller, who adds that each of these techniques is inexact. Flow cytometry results can be based on subjective gating protocols, and IHC lacks precision. “Another problem,” notes Dr. Hoffmueller, “is the stability of patient blood samples from clinical trials.”

    Epiontis utilizes an epigenetic format based on euchromatin structure of actively expressed and silenced genes. Moreover, the company focuses on the epigenetic markers that it has identified in healthy human immune cells. “We find unique epigenetic markers to identify single cell types and develop assays that can be utilized in clinical trials to quantify different types of immune cells,” explains Dr. Hoffmueller. “These data are complementary to flow cytometry and IHC.”

    To find unique biomarkers requires much work. First, Epiontis scientists perform genome-wide association studies to find potential genes. Next, they look for areas that are likely demethylated, such as CpG repeats, which represent active genes. Finally, they choose about 100 candidate amplicons and carry out bisulfite sequencing.

    “The challenge is to find the biomarker that can provide the best signal-to-noise ratio,” remarks Dr. Hoffmueller. “We’ve identified and developed assays for about a dozen immune cells including T regulatory cells, B cells, neutrophils, and CD4+ cells. The discovery of cell-type-specific epigenetic markers allows very precise and robust quantitation of immune cells in all types of human samples.”

    The PCR-based technology, comments Dr. Hoffmueller, presents several advantages: “The readout is stable, and samples, which can come from both blood and tissues, can be frozen, making this technology ideal for clinical trial samples.”

    “This is a great technology for developing countries that do not have access to immediate clinical testing,” continues Dr. Hoffmueller. “One could envision doing a simple pin prick and dotting the blood on a filter that can be sent to a lab for follow-up analysis.”

  • Mining the Metabolome

    Click Image To Enlarge +
    Metanomics Health, a subsidiary of BASF, offers a range of applications from its analytical toolkit, providing approaches from both schools of thought that are currently being used in metabolomics research: broad unbiased metabolite profiling and targeted profiling platforms.

    The metabolome, the complete set of human metabolites, may be assessed for biomarkers to create metabolite profiles. Such profiles may provide unique chemical signatures reflecting environmental influences and individual predispositions to a host of diseases. Since 2003, Metanomics Health, a subsidiary of BASF, has utilized both targeted and nontargeted metabolic profiling for biomarker identification and validation.

    “Metabolite profiling is a robust tool for predicting and explaining complex phenotypes,” says Tim Bölke, M.D., the managing director of Metanomics Health. “It is an efficient strategy that best reflects cellular metabolism on a functional level and thus can correlate phenotype with pathophysiology.”

    To derive a metabolic signature, we look at small molecules of less than 1,500 daltons such as amino acids, carbohydrates, and lipids. These are end products of a biological cascade triggered by DNA.”

    Metanomics Health is initially focusing on oncology applications, cardiometabolic diseases such as type 2 diabetes, and congestive heart failure. In collaboration with academic partners, the company has clinically validated several biomarkers. The company’s mass spectrometry platform allows the annual analysis of more than 100,000 metabolic profiles.

    The technologies employed include gas chromatography-mass spectrometry (GC-MS) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The company also applies bioinformatics and systems analysis for data validation, integration, and data mining.

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