May 1, 2014 (Vol. 34, No. 9)
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
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.
“Biomarker discovery and validation requires a robust and highly reproducible metabolite profiling platform,” notes Dr. Bölke. “Aside from an untargeted (unbiased) metabolite profiling platform, access to targeted methods (such as lipidomics, energy metabolites, stress hormones, and eicosanoids) is also important.”
Dr. Bölke describes the company’s systematic approach to biomarker identification and validation: “First, we seek about 30 patients and matched controls and perform a fairly general analysis to see if the technology is a good fit. Then, in an identification phase, we take a couple hundred patients and matched controls to identify the ~5–20 crucial metabolites providing a unique signature for the target. Of these, we nominate ~5–10 biomarkers, and we perform clinical follow-up studies to validate the data. Finally, we translate our findings into standard diagnostic technologies for everyday clinical use.”
Obtaining signatures is arduous, but they may, suggests Dr. Bölke, provide better patient stratification and differential diagnostics: “Because of its broad coverage, metabolic profiling enables a deeper understanding of pathological mechanisms, more in-depth toxicity testing, and, of course, the identification of potential drug candidates.”
Pulmonary Disease Biomarkers
Diseases of the airways such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) result from a complex, subtle interplay of environmental and genetic susceptibility factors. To better understand the pathophysiological nuances of these diseases (and to improve the evaluation of new drugs), companies are focusing on biomarkers. One such company is Novartis, which is evaluating biomarkers through its research organization, Novartis Institutes for Biomedical Research.
“The complexity of respiratory diseases makes it difficult to develop new therapies targeting their underlying molecular pathology,” remarks Paul Whittaker, Ph.D., director, biomarkers, respiratory disease area. “Biomarkers help us identify groups of patients with the same dysregulated molecular pathway, or the same active disease process that our novel drugs are targeting. This increases the signal-to-noise ratio in our clinical trials by ensuring that the right drug is tested in the right patients and that the right outcome is measured.”
The identification of appropriate biomarkers for respiratory diseases presents several challenges. In Dr. Whittaker’s view, these challenges include 1) poor understanding of respiratory disease heterogeneity, 2) inadequate access to well-annotated clinical samples, particularly matched blood and lung tissue, and 3) difficulties in building and proving the link between the biomarker(s) and the disease process that is being targeted.
According to Dr. Whittaker, access to clinical samples is crucial for solving these problems. “The good news is that the proliferation of publicly funded biobanks (such as the Lung Tissue Research Consortium of the NIH’s Heart, Lung, and Blood Institute) as well as commercial suppliers (such as ProteoGenex and Tissue Solutions) has facilitated access to hard-to-get clinical tissue samples.”
Identifying biomarkers utilizing such available resources involves a number of approaches, observes Dr. Whittaker. “An area we’ve made good progress in is the identification of candidate biomarkers for remodeling of lung tissue, a process that is common to a number of respiratory diseases, including pulmonary arterial hypertension and IPF. The experimental approaches involve a combination of omics technologies, bioinformatics, literature mining, and pathway analysis. Use of human tissue is an integral part of this whole process. As we move steadily into the era of personalized medicine, these biomarkers will be the basis for companion diagnostics for our novel therapies.”
On average, only about 5 out of 10 patients receiving a given therapy benefit from it, while some may experience troubling side-effects. Identifying clinically meaningful patient subgroups to remedy this is a key element of the Roche Pharma Early Research and Development (pRED) oncology programs. Miro Venturi, Ph.D., site head for biomarkers and experimental medicine, oncology, says, “We are pursuing tailored biomarker strategies to identify mechanistic and pharmacodynamic as well as selection or stratification markers to improve patient benefit.”
To characterize tumors, Roche employs a broad range of platforms such as genetics, genomics, and protein and cellular phenotyping tools. “Our biomarker discovery efforts start very early in the research process,” explains Dr. Venturi. “First, we formulate predictive biomarker hypotheses by understanding the mechanism of action of our candidate drug and looking broadly at many targets. This requires the adoption of multiplexing technologies. Next, we look for confirmation of the key biomarkers as the most accurate descriptors of appropriate pharmacodynamics and clinical response. Finally, we identify biomarkers of resistance to the drug.”
Finding the appropriate biomarker is difficult. “Some of the challenges are deeply connected to the biology of cancer, especially for new compounds in onco-immunology that stimulate a patient’s immune system,” comments Dr. Venturi. “Because patient responses can vary, it is important to correctly identify both tumor- and patient-specific biomarkers. It is also critical to understand tumor heterogeneity.
“Often a small amount of tumor material—a patient biopsy—is removed and made available for further analysis. Since tumors can be heterogeneous, one needs to consider the limitations of such investigations and have a strategy at hand that allows conducting the analysis in more accessible patient samples or via imaging modalities.”
Biomarker identification also presents the opportunity for development of a companion diagnostic to predict or monitor patients’ response to treatment. As Dr. Venturi notes, “It’s important to start developing biomarkers for use as companion diagnostics as early as possible. Usually, this is done in Phase I trials or earlier. The more specific the biomarker, the more likely it will evolve into a diagnostic.”
Dr. Venturi sees some exciting new developments on the horizon. “In the future, there will be less invasive techniques. For example, it will be possible to characterize cell-free DNA in blood; that is, liquid biopsies will allow surveillance of DNA from tumors. We will still face challenges in identifying and validating companion diagnostics; however, we will have many more tools to do so and a progressively deeper understanding of the biology to build biomarker hypotheses upon.”