DNA methylation is critical for a variety of biological processes that range from imprinting to stem cell formation, making it a potential biomarker for both prognostic and diagnostic applications. “Methylation of DNA is a well-known and important regulatory mechanism as well as a signal for certain cancers,” noted Cathy Lofton-Day, Ph.D., vp of molecular biology, diagnostics at Epigenomics (www.epigenomics.com).
Dr. Lofton-Day presented her work at the “Biomarker World Congress” last month. “In the last 20 years, scientists have realized that gene promoters become methylated and turn off. When the promoter is for a tumor suppressor gene, this has been linked to development of cancers.”
Because changes occur early in the neoplastic process, detection of methylation alterations can allow early detection of cancer. In particular, Dr. Lofton-Day has been studying early detection of methylation in colorectal cancer (CRC). “Our goal is to develop a noninvasive test for colorectal cancer. When detected in its early stages, survival is greatly increased. The problem is that current noninvasive tests are based on detection of blood in stool and people do not use them.
“Colonoscopy is invasive, expensive, and can be associated with some morbidity and mortality. Our strategy is to detect free genomic DNA in blood that is derived from tumors. To achieve this, we identified a region of the Septin 9 (SEPT9) gene that is methylated in more than 90% of colorectal cancer tissues with little or no methylation in normal colon tissue or other controls. This suggests it can be used as a biomarker in blood indicating the presence of colorectal cancer.”
The technology initially employed to identify the methylation of SEPT9 involved a methylation-sensitive, restriction enzyme-based detection method. “Once we identified SEPT9 as a suitable biomarker, we developed a highly sensitive real-time PCR-based assay in which the DNA sample is treated with bisulfite allowing us to identify methylated from nonmethylated DNA,” Dr. Lofton-Day reported. “We verified the marker performance in blood in case control studies of more than 3,000 patient samples.”
Now the challenge is to make the assay “clinic friendly” and perform clinical trials. To do that, Dr. Lofton-Day has geared the assay for high-throughput applications and has launched a multicenter clinical trial targeting the CRC screening population. “We are excited about the potential of the Septin 9 marker and especially this new and emerging technology.”
Poor reproducibility of the methods used for qualitative and quantitative proteome analysis can severely restrict biomarker discovery as well as validation, according to Peter Schulz-Knappe, Ph.D., CSO, Proteome Sciences (www.proteomics.com).
“Currently, there is no universal technology for proteomics. It is segmented into many different technologies and workflows, because there are a myriad of proteins and peptides each possessing different profiles as to biochemical activity, size, form, etc. Reproducibility of studies is poor and comparability between labs is often not possible. A comprehensive and sensitive technology is needed in order to improve this situation.”
One of the newest and best technologies for biomarker discovery and development is to use mass spectrometry combined with quantitative tags for analysis, noted Dr. Schulz-Knappe. “At Proteome Sciences, we have patents covering the field of isobaric mass tags and have introduced Tandem Mass Tags® (TMT).
“All tags have the same overall mass and identical physico-chemical properties. After individual labeling of up to six different patient samples, the samples can be mixed and processed together. Labeled proteins behave identically during all steps such as sample handling and chromatography, but since the mass tags have individual fragmentation patterns, the patient proteins can be quantified specifically during mass spectrometry. TMT’s only prerequisite is that you must be able to label the sample and that the analyte can be detected with MS.”
The idea behind the TMT technology is that the quantitative ratio between individual proteins remains constant after mixing of samples. “This conservation is key for subsequent separation and analysis. It allows much better control, precision, and accuracy to study hundreds to thousands of proteins.”
Proteome Sciences also made advances in the development of reference standards for proteomics. “Clinical laboratories typically use reference materials to calibrate and certify assays. Unfortunately, proteomics lacks such materials to benchmark performance. We decided to address this need and now provide TMT-labeled reference materials for blood plasma, urine, and other samples. These materials serve as a defined proteome reference. Using this as control sample, it is now possible for scientists to have quality control and share their results worldwide. We expect reference materials to help speed up discovery and development of biomarkers.”