Although high levels of prostate-specific antigen (PSA) can provide evidence of cancer, its use as a screening tool is controversial as normal prostate cells also shed this antigen into the blood. Prostate cancer gene 3 (PCA3) is being touted as a promising biomarker for prostate cancer. Gen-Probe has developed a PCA3 test that is currently on the market in Europe, initial clinical trials are set to start this quarter in the U.S.
According to the company, this first-generation test is semiautomated and semiquantitative and provides good clinical data. An endpoint assay, the PCA3 test uses chemiluminescence via the company’s hybrid-protection assay technology. A specific DNA probe hybridizes with a nucleic-acid target to emit a chemiluminescent signal. This is followed by target capture and amplification.
A prototype of the second generation of this assay is being developed as a real-time assay, combining amplification and detection in one step. “You can measure the kinetics of the reaction and that’s how quantitation is achieved,” said Norman Nelson, Ph.D., director of biochemistry. Fluorescence also allows measuring multiple signals in one tube. It’s amenable to the types of probes needed with real-time assays. “These are typically self-reporting probes, which are homogenous in that the fluorescence is quenched in a molecule with a stem-loop structure in the absence of target, but is unquenched in the presence of target.”
The multiplex format is more convenient, uses less reagent, and provides faster results, Dr. Nelson reported, adding that the biggest challenge when multiplexing nucleic acids is interference between oligonucleotides needed to build the test. “We solved this with a universal tag approach that’s unique to our methodology that makes amplification cleaner, with less interference.”
This technology has the potential for use in other cancers and disease areas, as well as in other markets driven by nucleic acid molecular testing, Dr. Nelson said. “These tests are all about oligos, and that’s one of the things that’s so powerful about DNA chemistry—it’s exquisitely precise and to make it work for you, you have to design it properly.”
Collaborating with researchers at the University of Michigan, Metabolon has discovered a number of markers indicative of the aggressiveness of prostate cancer and involved in the transition of noninvasive cancer to invasive.
“One of these compounds is sarcosine, a small molecule that is a methyl glycine,” explained John Ryals, Ph.D., CEO. “Glycine is used within cells to monitor or buffer levels of ethyl-densyl methionine, the methyl donor for methylation of DNA and other methylation reactions. When that goes too high, glycine is converted to sarcosine, which is mechanistically involved in this transition”.
Researchers have found that sarcosine levels are substantially increased during progression to metastasis. This small molecule, and several other discovered markers, can be detected in urine to differentiate aggressive versus nonaggressive cancers. “That’s the holy grail of prostate cancer, because most prostate cancers are not aggressive and won’t metastasize,” added Dr. Ryals.
The company has a platform built around its Metabolyzer™ data processor, which converts raw mass spec data into biomarker data. The biggest challenge in developing biomarkers, said Dr. Ryals, is obtaining source samples from existing studies. “Most of the time the studies aren’t designed in ways most efficient for discovering biomarkers, so you’re always trying to bridge an existing study to get the markers you want. In addition, banked samples are not always taken and stored the same way, causing large variation in analysis.”