In the last decade, epidermal growth factor receptor has emerged as a primary area of interest in cancer research, with a number of drugs, including antibody-based therapeutics like Imclone's Erbitux – known generically as cetuximab – and Amgen's Vectibix – or panitumumab – targeting the protein.
EGFR's multiple isoforms have made it difficult to predict the effectiveness of these antibody-based drugs, however, raising the need for more reliable companion diagnostics for directing therapy and choosing patients for clinical trials, said Nita Maihle, a professor of Obstetrics, Gynecology, and Reproductive Sciences at Yale University.
To date, Maihle's team has reported the expression of three naturally occurring alternate EGFR isoforms, one of which – soluble epidermal growth factor receptor, or sEGFR – is the source of the major circulating EGFR isoform in human blood and, she notes, a potential confounding factor in predicting the efficacy of EGFR-targeted therapies.
"In the past decade people have studied EGFR expression by looking at the molecule's expression with antibodies targeting [its] ligand-binding domain," which is also the portion of the protein targeted by antibody-based drugs like cetuximab and panitumumab, Maihle told ProteoMonitor.
"But we know that there are other forms of the EGF receptor. We're looking at and choosing [just] one isoform for our diagnostic tests, and so it's not surprising that there's really a poor association between who responds to [EGFR-targeted treatment] and what we find by the [diagnostic] test," she said, noting that results from existing EGFR expression tests like Ventana's Confirm EGFR and Dako's EGFR pharmDx provide essentially no guidance as to who will respond well to anti-EGFR therapies.
"We've made drugs against [EGFR], we measure [EGFR expression] to determine who should get the drug, and then we give the drug based on who makes the receptor," she said. But "even though [with such tests] we find [EGFR] over-expression in a wide variety of tumor types, what we find is there's absolutely no relationship between what we report in those tests and response” to drugs like cetuximab and panitumumab.
This is likely because the antibodies used in such tests target both EGFR and isoforms like sEGFR, leading to misleading data on patients' EGFR expression. This is also a concern with regard to the effectiveness of antibody-based drugs, which may be binding to off-target isoforms, reducing their effective doses.
Maihle and her colleagues raised these issues in a paper published in the December 2010 edition of the journal Cancer, suggesting that sEGFR is an unanticipated first target for cetuximab and panitumumab in humans. With researchers from the University of Kentucky, the Mayo Clinic's Proteomics Research Center, and the University of Medicine and Dentistry, New Jersey, she published an article in this month's edition of Biochemistry detailing production of an antibody targeting a 31-mer peptide unique to sEGFR which the scientists used to show that the sEGFR isoform is co-expressed with EGFR in a variety of carcinoma-derived cell lines.
"We've got drugs that are FDA approved that are clearly targeting isoforms” of EGFR, Maihle said. "We know they're expressed in a variety of human tissues. Yet we don't distinguish between the isoforms at all in our diagnostic assays. It's kind of just ignored in spite of the biological understanding that we have."
The EGFR story, she said, is a replay of researchers' experience with tests for HER2 protein expression levels to determine which patients should receive Genentech's breast and stomach cancer drug Herceptin.
The questions surrounding HER2 are "over a decade old," Maihle said. "So we're saying that we have a very similar controversy and conundrum coming along for EGFR, and it's almost like we haven't learned anything from studying and using the HER2-directed antibody [Herceptin] in the clinic over the past decade."
Having good diagnostics to predict patient response to EGFR-directed antibodies is key to clinical trials for such drugs, Maihle noted. In the Cancer paper, she and her co-authors cited the failure of two recent phase 3 cetuximab trials in colorectal cancer patients selected for EGFR expression as examples of the need for such tests.
"We really need to rethink how we're stratifying patients for treatment with these drugs because we're wasting money and research time and [the participation of] people who take part in these trials," she said. "There's a depth of biology that we're simply not applying to the design of these clinical trials and the design of these diagnostic tests."
While isoforms like sEGFR are complicating factors, some research has suggested they could themselves be useful as diagnostic biomarkers, Maihle said, citing a presentation at the 2009 American Society of Clinical Oncology annual meeting by researchers from the Danish Colorectal Cancer Group indicating that sEGFR levels are correlated with responsiveness to cetuximab treatment.
"What we do know for sure is that the current diagnostic tests which we interpret as measuring one isoform are not predictive," she said. "So it's likely that we have to more comprehensively test and measure the various isoforms in order to be predictive."
There has been a growing appreciation among proteomics scientists of the importance of protein isoforms in clinical research, with companies like Tempe, Ariz.-based Intrinsic Bioprobes working on a variety of tests for diagnostically useful isoforms (PM 07/16/2010). Maihle and her colleagues have patented sequences and antibodies for several EGFR isoforms that they hope to either license or develop with commercial partners for use in guiding EGFR-directed therapies, she said.
In the future, Maile said, the researchers hope to partner with groups conducting clinical trials for drugs targeting EGFR in order to access the sort of biospecimens needed to develop better antibody-based drugs and companion diagnostics.
"The receptor isoform work is in its infancy, but certainly there's consensus that these isoforms exist and that they're important," she said. "But it's the implementation of this information, its application in the development of clinical diagnostics, that has only just started."
Broomfield, Colo.-based proteomics firm Biodesix is working on tests to guide EGFR-directed antibody therapies in non-small cell lung cancer patients. Rather than measure specific EGFR isoform levels via immunoassay, though, the company compares MALDI mass spectra of patients who do and don't respond to antibody treatment to develop spectral profiles that can be used to guide therapy.
The company's Veristrat diagnostic, which it launched commercially in May 2009, uses a similar approach to identify lung cancer patients likely to respond to small molecule kinase inhibitors like Roche's Tarceva – known generically as erlotinib – and AstraZeneca's Iressa – or gefinitib – targeting the EGFR signaling network.
Isoforms like sEGFR play a role in the function of small-molecule inhibitors as well, Maile said, but the presence of good gene-based companion diagnostics for these drugs allows for more effective patient stratification.
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