Researchers from Houston's Methodist Hospital Research Institute have identified a set of plasma peptides that could prove useful as biomarkers for the early detection of breast cancer.
Detailed in a study published this month in Clinical Chemistry, the work isolated six peptides generated by the zinc metallopeptidase carboxypeptidase N (CPN) that show measurably higher expression in patient plasma in early stage breast cancer.
Much protein biomarker work has revolved around efforts to identify plasma proteins whose expression changes in response to tumor development. The Methodist researchers took a different tack – aiming instead to identify plasma peptides generated by proteolytic activity associated with cancer.
CPN itself had not been associated with cancer outside of a 1983 study that found increased levels of the protein in the sera of lung cancer patients. However, previous work had linked one of the protein's substrates, the peptide C3f_S1304-R1320, to bladder, prostate, and breast cancer. Using MALDI-TOF mass spec to profile breast cancer mouse models and controls, the researchers identified five C3f peptides and one peptide from the peptide bradykinin – also a known CPN substrate – that showed increased expression in plasma from the cancer models.
Notably, said Ye Hu, a Methodist researcher and author on the paper, this increased expression was most evident in the first weeks of tumor growth, suggesting that the peptides might prove useful as markers for early detection of the disease.
To further validate their findings, the researchers measured them in 56 human plasma samples, including 10 healthy controls, 11 stage I breast cancer patients, 12 stage II patients, 15 stage III, and 10 stage IV. As in the mouse model work, they found that expression of the six peptides differed significantly in stage I and II patients compared to healthy controls.
Furthermore, Hu said, the researchers found that while there was increased CPN expression at the tumor site, levels of the protein in plasma remained unchanged between cases and controls. This, he noted, indicates that the increase in the plasma levels of the six peptides is, in fact, due to an increase in tumor-associated CPN activity, as opposed to the proteolytic activity of circulating CPN.
While the six peptide markers appear promising, analysis of these molecules required confronting a pair of key hurdles, Hu told ProteoMonitor.
The first, he said, was the technical challenge of actually measuring the target peptides in plasma. Such peptides, he noted, are typically present in relatively low concentrations, meaning some form of enrichment is required. Antibody-based enrichment can be used in some cases, but, Hu said, many peptides – especially smaller ones – can be challenging targets for antibody development.
Instead, the Methodist team used a nanopore-based enrichment approach they had previously developed that uses nanopore chips to capture low molecular weight peptides for mass spec analysis. According to Hu, using this method, the researchers are able to quantify targets at the sub-femtomolar level without immunoenrichment.
The second challenge involved in such peptide-based biomarker work, Hu said, is more of a conceptual issue — to wit, how to establish the specificity of the identified peptides for a particular disease?
"When you capture the peptides, if you sequence them you will often find that for most of them, the parent proteins are pretty common and have been studied very well and are associated with many types of diseases," he said.
Given this, it was necessary to demonstrate that the identified peptides were, in fact, specific to CPN, a process Hu and his colleagues undertook via a series of ex vivo peptide cleavage assays.
Incubating synthetic C3f peptides in tumor and normal interstitial fluid, they demonstrated that production of peptides with CPN-characteristic cleavages was six-fold higher in the TIF compared to NIF. They further demonstrated that addition of EDTA, which inhibits CPN, decreased production of such peptides as did removal of CPN via immunodepletion. Production of C3f peptides cleaved by chymotrypsin – which is not affected by EDTA – remained unchanged.
Having demonstrated the markers' potential, the Methodist researchers are now collaborating with MD Anderson Cancer Center and the New York University Cancer Center to put together larger sets of patient samples for further validation, Hu said. He noted that while he and his colleagues used MALDI-MS for their analysis in part due to the fact that they had easy access to such a machine, he anticipates that they will benefit from the technology's high throughput as they move to analyze greater numbers of samples.
In addition to the validation work, the researchers are following up with animal studies exploring the function of CNP in breast cancer and its relationship to progression of the disease, Hu said.
Hu and colleagues are also looking at matrix metalloproteases as a possible source of peptide biomarkers in pancreatic cancer. "Generally we're focused on the proteolytic events of cancer and finding blood-based biomarkers [resulting from these events]," he said.
They are in the process, as well, of commercializing their nanopore enrichment platform, Hu noted. They have patented the technology and are currently in talks with a vendor regarding a possible licensing deal, he said.