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PNNL Team Searching Triple-Negative Breast Cancer Samples for Diagnostic Protein Markers


A proteomics team from the Pacific Northwest National Laboratory is using an $8.6 million research grant from the Department of Defense to scour samples of triple-negative breast cancers from the Walter Reed National Military Medical Center for diagnostic signatures and new drug targets for the aggressive disease.

The team, led by PNNL's Richard Smith, will profile several hundred matched plasma and preserved tissue samples from the Walter Reed Clinical Breast Care Project cohort. The group will use genomic and transcriptional information matched to the samples to narrow down the list of candidate protein markers associated with triple-negative breast cancer for further analysis in plasma using selected-reaction monitoring MS.

Karin Rodland, a member of the PNNL team and a cancer biologist at the lab, told ProteoMonitor that the researchers are confident the study will allow them to collect enough "rich proteomic data to provide insight into how the proteome reflects the changes that have been observed at the mRNA level, [which] will inform insights as to the actual mechanisms of unregulated proliferation in triple-negative breast cancer cells."

The team hopes that information will eventually lead to the identification of therapeutic targets and the isolation of diagnostic signatures, though the study's initial 20-month funding period may only be a first step.

"In 20 months we're not going to get there," Rodland said. "But we're hoping some of those changes we see at the protein level are significantly robust that they show up in the plasma with enough sensitivity to potentially be diagnostic markers."

Because triple-negative breast cancers are difficult to detect using mammography, there is clinical need for a plasma-based test that could either be used as a companion to mammography screening, or as a standalone screening tool.

According to Rodland, PNNL has previously partnered with Walter Reed to develop proteomic technology to deal with OCT-embedded tissue for investigations of proteome differences between ER-positive node-positive and node-negative tumors.

The triple-negative cancer study is an extension of this earlier work, Rodland said, taking advantage of PNNL's "technical strengths" to examine a disease that has been a "really difficult nut to crack."

Triple-negative cancers are not only more aggressive, and more difficult to diagnose, than other types of breast cancer, but they are also disproportionately frequent in African-American women, who themselves represent a higher percentage of women in the military. "So this is clearly a question of interest to Walter Reed and the army medical folks," Rodland said.

In the upcoming study, PNNL will be looking at proliferation-associated pathways that are "not steroid receptor dependant and not EGFR axis dependant," according to Rodland.

"We are also going to be looking at some pathways known to cross-talk with [EGFR] for activating the MAP kinase pathway," she said. Additionally, the group may incorporate new data on pathways and targets from analyses of triple-negative samples performed by the Cancer Genome Atlas project, which are expected to be published soon.

According to Rodland many of the samples have already gone through exome and RNA sequencing. For any that haven’t, the Windber Research Institute, which houses the Walter Reed samples, will provide new exome and RNA-seq analyses.

The researchers plan to map the changes in abundance at the mRNA and protein level against gene amplification and deletions on the genomic level to narrow down the list of candidate biomarkers based on pathway analysis and concordance of expression.

The project will start with an initial discovery phase using the Walter Reed tissue samples. Rodland said in an e-mail that the group will use a "custom-built automated 2D LC and/or metal-free HPLC system coupled to [a Thermo Fisher Scientific] LTQ-Orbitrap Velos hybrid MS instrument" for global proteomic and phosphoproteomic analysis.

Rodland said there are about 200 or 300 triple-negative samples within the Walter Reed Clinical Breast Care Project sample bank, but exactly how many the PNNL group analyzes will depend on the amount of tissue available in each.

The group will use SRM assays to measure the levels of selected proteins in the matched blood plasma samples to try to isolate proteins that can distinguish triple-negative cancers.

"We'll initially look with a standard SRM, antibody-free strategy," she said. "It's an immunodepletion strategy to remove the top 21 high abundant and moderately abundant proteins from plasma."

"Using that, we can get down to the low nanogram-per-milliliter range without further enrichment," she explained.

If the researchers find they need to look at a biomarker with lower than 5 ng/mL concentration they may also use a PNNL targeted selection strategy called PRISM (High Pressure High Resolution Separation with
Intelligent Selection and Multiplexing), Rodland added.

The study's DOD funding is for a 20-month period, and the team plans to use the first 12 months for the discovery work and the next eight for blood plasma analysis.

Rodland said that by the end of the study, the group hopes to have "an answer on whether we see proteins in the plasma that are unique to triple-negative breast cancer with sufficient robustness to justify going into a pre-clinical trial."