National Cancer Institute NCI Cancer Bulletin: A Trusted Source for Cancer Research News
July 28, 2009 • Volume 6 / Number 15

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A Closer Look

TechnologyThis is the first article in a new series of stories related to cancer technology. Look for the symbol on the left in an upcoming issue for the next article in the series.

Study Supports Use of Existing Technology for Clinical Proteomics Research

Image of test tubes and a pipette from a patient brochure produced by NCI's Clinical Proteomic Technologies for Cancer initiatives

A new study has found that an existing technology used in other aspects of biomedical research may play an important role in the long-sought quest to identify reliable protein biomarkers in blood samples that can signal the early presence of cancer or its return. The results represent major findings from NCI's Clinical Proteomic Technology Assessment for Cancer (CPTAC) Network—an effort to improve the science and technology underlying the field of clinical proteomics research—and enable the translation of discoveries from the field into the clinic.

The highly collaborative, team-based study that involved eight laboratories from the CPTAC Network (see sidebar) did not attempt to discover new biomarkers or prove that previously discovered biomarkers could accurately identify the early presence of an aggressive cancer. Rather, the study's chief aim was to determine whether the technology, multiple reaction monitoring with stable isotope dilution mass spectrometry (SID-MRM-MS), could reliably detect and quantify levels of prespecified proteins (using defined fragments of these proteins known as peptides), and whether the results would be consistent or reproducible across testing sites.

Published online June 28 in Nature Biotechnology, the study was successful on both counts, said lead investigator Dr. Steven Carr from the Broad Institute of the Massachusetts Institute of Technology and Harvard University. "The testing approach demonstrated that multiplexed protein assays based on SID-MRM-MS could be made to be highly reproducible within individual labs and from lab to lab," he said.

The reproducibility was a critical finding, he continued, because it had not been previously demonstrated and because some of the labs involved had no prior experience with SID-MRM-MS.

Participating CPTAC Labs

The eight laboratories which participated in this study were:
  • Broad Institute of the Massachusetts Institute of Technology and Harvard
  • Buck Institute for Age Research
  • Fred Hutchinson Cancer Research Center
  • Monarch Life Sciences
  • Skirball Institute at New York University
  • University of California, San Francisco
  • University of Victoria, British Columbia
  • Vanderbilt University School of Medicine

It's also important, added Dr. Henry Rodriguez, director of NCI's Clinical Proteomic Technologies for Cancer (CPTC) program, because although proteomics has generated optimism as a means of detecting fatal cancers in their earliest stages, the field has been plagued by failures to reproduce findings from individual studies on a given set of blood or tissue samples.

CPTAC is part of an effort, Dr. Rodriguez continued, to help address the areas of "variability" that have contributed to these reproducibility problems, such as differences in collecting and storing blood and tissue samples and applying different technologies and computer software in proteomic science. Improvements in these areas can also enhance the overall conduct of biomarker science, he stressed.

For example, the CPTAC study authors believe that their results indicate a testing approach based on SID-MRM-MS may be an ideal bridge between the discovery stage of protein biomarkers, where hundreds to thousands of potential leads are often turned up by a single study, most often using conventional mass spectrometry; and the clinical validation phase, in which highly sensitive, antibody-based tests are developed for use in large clinical trials to determine whether a biomarker or panel of biomarkers can reliably detect or rule out the presence of cancer. Although no firm figures are available, such antibody-based tests are thought to take more than a year to develop at a cost of up to several million dollars.

Culling through the thousands of potential markers generated in the discovery phase to identify the most promising ones to move forward into the validation phase has been the biggest rate-limiting step in proteomics biomarker research to date, Dr. Carr and his CPTAC colleagues wrote. Because of its targeted nature, high sensitivity (as much as 100 times higher than conventional mass spectrometry methods), and ability to assay tens of proteins simultaneously, the SID-MRM-MS technology "has the potential to become the critical filter used to assess candidate biomarker performance in a sufficient number of patient samples before committing the very substantial time and resources required to create clinical-grade immunoassays," the CPTAC research team wrote.

The study involved a rigorous, systematic approach to assess whether SID-MRM-MS could be an effective tool in this prevalidation—or verification—phase. "This highly important work, introduced by the CPTAC Network, can help move the field forward reliably and efficiently," Dr. Rodriguez said.

The study consisted of multiple sub-studies of increasing complexity to enable the investigators to define and understand sources of variability. The first involved the addition of "signature" peptides derived from the seven targeted proteins, as well as versions of these peptides that were tagged, or labeled, with radioactive tracer molecules, into a standardized plasma sample at multiple concentrations. ("Signature peptides" are unique to each protein in the genome and also provide high sensitivity for mass-spectrometry analysis.) The subsequent studies systematically introduced additional sources of variability, from added sample handling and processing steps up to the final study that simulated a real biomarker verification study.

The CPTAC team's study represents the "first critical step" in assessing SID-MRM-MS in this prevalidation phase, Dr. Carr said. The CPTAC group has already launched a follow-on study to evaluate reproducibility, using a 10-fold increase in the number of peptides and also enriched protein samples prior to SID-MRM-MS analysis to improve sensitivity (the ability to detect protein biomarkers present at very low concentrations) 10-fold over the current study.

The raw data from the study are available on the CPTC Web site. Interested investigators can obtain the reagents, study samples, and detailed study protocols there, as well. "The methods have been finely detailed, so as proteomics laboratories begin to explore the technology either for biomarker verification or for targeted detection of proteins or their modifications in basic biological studies they have a way to benchmark themselves," Dr. Carr said.

In addition, Dr. Rodriguez noted, CPTAC is working with the FDA to provide scientific support for potential future guidance that defines requirements for those developing multiplexed proteomics-based assays for use in clinical care.

Carmen Phillips

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