George Mason University researchers led by Lance Liotta and Emanuel Petricoin have used laser capture microdissection combined with reverse phase protein microarray technology to help them identify three proteins interconnected within a single pathway that may determine whether conventional chemotherapy will be efficacious against a childhood form of cancer called rhabdomyosarcoma.
The scientists said that this is the first time that proteomics researchers have combined these technologies to arrive at a clinical outcome. They also said that the identified proteins — 4EBP1, phosphorylated eIF4E ser209, and AKT — may eventually be used by drug makers and physicians to separate responders from non-responders, and to act as potential therapeutic targets. If validated, these proteins may become components in diagnostic/therapeutic companion products, also known as a 'theranostics.'
At present, there is no way to prospectively identify the 30 to 40 percent of patients who will not respond to therapy for rhabodomyosarcoma, noted Petricoin and Liotta, who along with Lee Helman, the chief of pediatric oncology at the National Cancer Institutes, presented the findings at the annual Americian Society of Clinical Oncology meeting held two weeks ago in Orlando, Fla.
By analyzing clinical samples from patients with the disease, which affects connective tissues such as muscle and fat, the scientists found that if the pathway containing the three identified proteins was suppressed, then treatment was successful and prognosis was good. However, if the protein pathway was active, treatment response was poor.
In his oral presentation at the ASCO meeting, Helman presented data from mice studies that showed that when the three proteins in the pathway were inhibited, the growth of tumors in mice models of rhabdomyosarcoma was inhibited. The studies are currently in the review process for publication.
"This is a very good finding. It's an exciting application of this technology to discover potential leads [to therapy]," Liotta told ProteoMonitor. "We intend to follow up on this further and anticipate that in a year or so, this could morph into [human] clinical trials."
Rhabdomyosarcoma, a highly aggressive form of cancer, is the cause of more than 50 percent of soft-tissue cancers in children, and accounts for 5-8 percent of all childhood cancers.
Liotta said that he and his research group are planning on setting up a CLIA laboratory for proteomics at Inova Fairfax Hospital. The laboratory, to be established later this year, will be a place to perform research relating to clinical trials, including the one anticipated for rhabdomyosarcoma.
Liotta and Petricoin first developed their method for mapping molecular networks using reverse phase protein microarrays about five years ago. A discussion of the technology was published in the May 20 issue of the Journal of Clinical Oncology.
How It Works
Reverse phase protein microarray technology takes thousands of cells and produces a detailed quantitative analysis of more than a hundred signaling proteins at once. An analysis of the resulting diagram indicates whether the various proteins are linked together in pathways.
"From a tiny piece of tissue we're measuring so many different phosphorylation events together," said Petricoin. "If two different phosphorylation events went up and down together simultaneously, it's strong evidence that they're physically linked together."
To identify the three proteins associated with rhabdomyosarcoma, Liotta and Petricoin's group first analyzed 34 rhabdomyosarcoma patient samples using laser capture microdissection. To analyze the samples, the researchers employed a panel of 13 specific antibodies selected based on ongoing work pointing towards specific cell signaling pathways playing a role in the biological behavior of rhabdomyosarcoma.
Analysis showed a clear clustering of samples into two groups. The clustering had a strong correlation between treatment response and activation/suppression of a signaling pathway called the mTOR pathway. Further analysis showed that three proteins — 4EBP1, phosphorylated eIF4E ser209, and AKT — completely segregated responders from non-responders.
"This is the first time this technology is being used for clinical outcomes," said Petricoin. "Hopefully, this will serve as an illustration of what can be served with this approach, not just with cancer, but with diabetes, heart disease, and other metabolic disorders."
Petricoin noted that the new technology is different from conventional proteomic technologies in that it offers a more direct line toward therapy by identifying a faulty pathway.
"It's not enough to just tell if a patient is a responder or non-responder," Petricoin pointed out. "What happens if you're in the non-responder category?"
Petricoin said he envisions a targeted treatment approach for rhabdomyosarcoma in which patients would first be screened to see if they are responders for conventional chemotherapy. If they are found to be non-responders, then they would be treated first with a therapeutic to suppress their mTOR pathway, then treated with conventional therapies.
"Proteomics can go beyond prediction and help us understand which pathways to pursue in patients who, based on that prediction, are destined to fail conventional therapies," said Petricoin. "For the first time, we can profile the ongoing state of protein pathways in a biopsy specimen. We are hopeful that this new information may ultimately lead to patient-tailored treatment, new clinical trial designs, and effect a positive impact on public health."
— Tien-Shun Lee ([email protected])