Standards for Basic Research May Speed Clinical Tests
The concept of detecting cancer in DNA from bodily fluids has been around for more than a decade. But despite many promising feasibility studies, the strategy, which tests genes for a chemical change called methylation, has not yet made it to the clinic. New studies sponsored by NCI's Early Detection Research Network (EDRN) could change that.
Methylation can silence genes that normally suppress tumors, and methylated DNA has been detected in the saliva, sputum, urine, and blood of cancer patients. In the coming months, EDRN researchers will ask whether improperly methylated genes can be used to assess cancer risk and detect the disease early, when it may be treatable.
The studies will be the first to use a high-quality collection of samples and a semi-quantitative technology, real-time methylation-specific PCR. This technology generated reproducible results in a previous study sponsored by EDRN.
"The goal is to nail down a manageable set of markers that could be used in a clinical study for one or more cancers," says Dr. Sudhir Srivastava, a leader of EDRN in NCI's Division of Cancer Prevention.
"We are looking at molecular changes that put a person at high risk of developing cancer," he adds.
Methylation is an epigenetic change, which means that it can alter the activity of a gene without causing a change in the DNA sequence. Cells normally use methylation to regulate genes, but a breakdown in the system can lead to the silencing of genes that protect against cancer. This often occurs early in the disease.
The field of cancer epigenetics exploded in the mid-1990s when methylation was shown to inactivate genes involved in cancer. A few years later, researchers detected methylated tumor-suppressor genes in the urine of patients with prostate cancer and in blood from lung cancer patients. Tumor-suppressor genes are rarely methylated in normal cells.
Dozens of subsequent studies proposed panels of markers for diverse cancers. But few, if any, of these panels were validated, and the field has had difficulty replicating findings. There are good reasons for this, researchers say: Cancers are genetically diverse, laboratories use different procedures and technologies, and samples vary in quality.
To address these issues, EDRN has created standards for developing and validating methylated markers. Guidelines and recommendations for reagents, tools, and protocols for measuring methylation were discussed at a 2005 workshop between EDRN and the National Institute of Standards and Technology.
The meeting also produced protocols for the collection and processing of body fluids, which can influence results. A summary is to be published soon in Cancer Research.
With the standards in place, new studies are set to begin. Drs. David Sidransky of Johns Hopkins Medical Center and Adi Gazdar of the University of Texas Southwestern Medical Center and their colleagues will study markers for lung cancer in sputum.
Dr. Paul Cairns of the Fox Chase Cancer Center and his colleagues will focus on markers for kidney and bladder cancers in urine, as well as markers for breast cancer in blood.
"Only now has a consensus emerged so that investigators in different labs could test samples using the same technology," says Dr. Cairns. "We hammered out the main issues at EDRN meetings in 2005 and 2006, and we are now moving ahead."
Progress in the field has been slowed by practical issues. Profiling methylation requires more time and materials than DNA sequencing, and there are no tools for detecting methylation sites throughout the genome. Many of the genes being studied were selected because they appeared to be promising candidates.
"But these genes are not necessarily the best ones for our purposes," notes Dr. Cairns. The field needs an optimized panel of genes for screening because no single gene is methylated in 100 percent of cancers, he adds.
Reliable samples have been another issue. Most feasibility studies have been done using conveniently available sets of tumors. But validation requires high-quality samples from patients at various stages of cancer, individuals at high risk of the disease, and a carefully matched comparison group that is free of cancer.
The reference samples developed by EDRN and their collaborators meet these criteria. The samples are being collected prospectively and include detailed medical records.
Dr. William Rom of New York University Medical Center will provide the lung cancer samples. His collection includes individuals exposed to cigarette smoke and asbestos, which may affect methylation. The collection also has samples from individuals showing nodules and ground glass opacities in the lung. Both of these abnormalities increase the risk of developing lung cancer.
"Our goal is to pick the best genes among those we have identified as potential markers for lung cancer during preliminary trials and test them in a blinded set of samples," says Dr. Sidransky.
Clinical tests for individuals who are healthy must be sensitive enough to detect changes associated with cancer, but specific enough to ignore those that are not cancer. When the new results become available in a year or so, they will be evaluated by other EDRN teams.
Dr. Cairns is optimistic. "We need to go forward with today's best specimens, technology, and genes rather than just say, once again, that we're not ready for validation yet," he says.
Even failure, should it occur, could be productive by leading the field in new directions. "Getting the data out there will guide future research," says Dr. Srivastava.
Standards are critical to all areas of biomedical research, not just methylation markers, he stresses. The importance of standards is well known to the DNA microarray community, which developed guidelines to help researchers collect and share data.
"For anything we want to do in the future, such as proteomics, we need to have standards," Dr. Srivastava says. "This is the most important issue not just for NCI but also for NIH."
The EDRN collection of reference samples will be made available to researchers in other fields.
By Edward R. Winstead