A somatic EGFR mutation in the tumors of metastatic colorectal cancer (mCRC) patients has been identified and implicated in the development of resistance to the anticancer antibody cetuximab. Cell-based studies by scientists at the Hospital del Mar in Barcelona, and collaborators, initially found that a mutation in the cetuximab-binding domain of EGFR is acquired as a result of prolonged cetuximab treatment. This particular mutation effectively changes the EGFR protein sequence and structure of its extracellular domain, which stops cetuximab, but not panibumumab, from binding to the receptor and blocking EGFR activation and signaling.
Subsequent analysis of human mCRC samples showed the same sequence change in the EGFR epitope can occur as a result of cetuximab therapy in a clinical setting. Encouragingly, switching an eligible patient who carried this mutation and had also developed cetuximab resistance to another EGFR antibody, panitumumab, led to significant liver tumor regression.
Reporting in Nature Medicine, Joan Albanell, M.D., and colleagues, claim their findings have important clinical implications for mCRC, and other tumor types treated using cetuximab, and merit the development of tests to detect the mutation in mCRC patients treated using cetuximab. Their paper is titled “Identification of a mutation in the extracellular domain of the Epidermal Growth Factor Receptor conferring cetuximab resistance in colorectal cancer.”
The researchers started by treating an mCRC cell line with cetuximab continuously for five months to establish drug resistance. This cell line, DiFi, is wild-type for KRAS, BRAF, and PI3K, and has an amplification of EGFR.
Anayses of clones from the resulting cetuximab-resistant cells confirmed that they were unaffected by treatment with this particular drug in terms of proliferation, apoptosis, or EGFR signaling to exracellular-related kinase (ERK) and protein kinase B (AKT). They were, conversely, just as sensitive to the EGFR kinase inhibitor gefitinib as the cetuximab-sensitive DiFi parental cells, suggesting that cetuximab-resistance wasn’t associated with any less dependence on EGFR for growth and survival. The cetuximab-resistant clones also retained sensitivity to the antibody panitumumab, which blocked EGFR activation. The clones had acquired no mutations in KRAS, BRAF, and PIK3CA, or demonstrated any loss of PETN expression.
The results thus far indicated that the acquisition of cetuximab resistance may have been associated with a change in the EGFR cetuximab-binding epitope. Supporting this hypothesis, DNA sequencing of the EGFR coding region showed that all three cetuximab-resistant clones had acquired a C to A substitution at nucleotide 1,476 (C1476A), which wasn’t present in the antibody-sensitive parental cells. The C1476A mutation causes a substitution of serine to arginine at amino acid 492 (S492R), which is within the EGFR ectodomain: “a bulky side chain at this position could interfere with cetuximab binding,” the authors write.
Furthermore, in fibroblasts engineered to express the S492R mutant EGFR, treatment with the anticancer antibody panitumumab was capable of blocking EGFR activation, but cetuximab wasn't. Moreover, only panitumumab was able to bind to the mutant EGFR-expressing cells. “In vitro biochemical binding studies confirmed that the S492R mutant is selectively defective in binding cetuximab but not panitumumab,” the team adds.
When the investigators moved on to look more closely at EGFR in human mCRC, they couldn’t detect the S492R mutation in any tumors from chemotherapy-naive patients, or from those who hadn’t received a targeted agent. However, when they looked at pre- and post-therapy specimens from 10 patients who had experienced disease progression after prior response to cetuximab with chemotherapy, the S492R mutation was found in two subjects. One patient had the same C1476A EGFR mutation as the cetuximab-resistant DiFi clones, while the other had a different mutation that resulted in the same S492R change in the EGFR protein.
Encouragingly, while one of these two patients had already succumbed to the disease at the time of sample analysis, the other was offered panitumumab monotherapy, and after two months of treatment showed a more than 50% decline in the volume of liver lesions, and a marked decline in the carcinoembryonic antigen blood marker. This patient did subsequently demonstrate disease progression after five months of panitumumab therapy.
The Spanish researchers say their findings may provide a molecular basis for the clinical benefit of panitumumab in a subset of subjects with mCRC who don’t respond to cetuximab therapy. “These findings have substantial immediate clinical implications for persons with mCRC,” they write. “The specificity of the S492R mutation is expected to facilitate reliable testing to guide the clinical use of panitumumab after cetuximab failure and justifies prospective independent validation of the S492R EGFR mutation. It will also be of interest to determine whether this mutation contributes to the acquisition of cetuximab resistance in other tumor types.”