The EGF Receptor Conundrum

Burt Vogelstein is quoted as saying “…if mutations (in proto-oncogenes) really are driving the neoplastic process, then they have to be among the best markers one could think of for detecting specific tumors ¹.” While this concept has become axiomatic, it has not held true for the epidermal growth factor receptor (EGFR/HER1) or EGFR’s related HER family members. The essence of this paradox as it applies to HER2 recently has been reviewed by M. Allison in an article entitled ‘The HER2 Conundrum’ ². EGFR expression, similarly, has proven to be an unreliable positive predictive marker for ‘EGFR-driven’ malignancies. Extensive analyses suggest that more than just technical issues confound the clinical utility of these two receptor biomarkers.

As one case in point, the FDA has approved panitumumab (Vectibix) and cetuximab (Erbitux), two EGFR-directed monoclonal antibodies, for the treatment of patients with advanced colorectal cancer (CRC). Paradoxically, tumor EGFR expression does not predict responsiveness to either of these antibodies, whereas mutations in genes encoding downstream effectors (i.e., PTEN, BRAF, KRAS) are negative predictors of efficacy ³. The failure of two recent phase III cetuximab trials in CRC patients selected for both EGFR and wtKRAS expression (N0147, COIN ⁴) illustrates the urgent need for improved methods to select patients for treatment with these drugs.

In this regard, we previously have reported the expression of three alternate EGFR isoforms, which arise from naturally occurring alternative transcripts (reviewed in ⁵). One of these transcripts encodes a 90/110-kDa cell surface protein designated sEGFR, which is expressed in human normal human tissues and tumors, and also is the source of the major circulating isoform of soluble EGFR in human blood ⁶.

It has been speculated that this circulating sEGFR isoform may serve as a sink or even as an alternate target for antibody-based EGFR-directed therapeutics ⁷, in a manner similar to trastuzumab binding to shed HER2 in blood ⁸. This proposal is supported by sequence inspection of sEGFR, which contains the epitope (subdomain III) recognized by both cetuximab and panitumumab. As shown in Figure 1, here, we directly demonstrate that both cetuximab and panitumumab recognize sEGFR using antibody concentrations far below therapeutic dosages. Since serum sEGFR concentrations range between 0.0125 to 5 μg/ml (~100 to 50,000 fmol/ml) in healthy adults and may be even lower (<10 fmol/ml) in some cancer patients ^{9, 10}, we propose that this interaction likely occurs in vivo in patients treated with either cetuximab or panitumumab. Given that trough and peak cetuximab treatment concentrations range between 41 to 235 μg/ml (241,176 to 1,382,353 fmol/ml IgG), circulating cetuximab concentrations would be predicted to range between 140,000-fold to only a 5-fold molar excess relative to circulating sEGFR concentrations in treated cancer patients. Since serum sEGFR arises from a cell surface precursor that is ubiquitously expressed in normal tissues^{11, 12} the effective concentration of these antibodies for the presumed (single) therapeutic target (i.e., EGFR) may be even lower in most patients. In contrast, some cancer patients exhibit baseline serum sEGFR concentrations below 10 fmol/ml ¹⁰; these patients would be predicted to receive a substantially greater effective dose of cetuximab. While such antibody/sEGFR interactions may be relevant in calculating the effective dose of these therapeutic antibodies in cancer patients, we would not predict a similar interaction between sEGFR and small molecule inhibitors such as gefitinib or erlotinib; previous studies examining serial serum sEGFR concentrations in gefitinib treated breast cancer patients are consonant with this prediction¹³.

Figure 1. Cetuximab and panitumumab immunoprecipitate sEGFR.

Chinese hamster ovary (CHO) cells were chosen for these studies because they lack endogenous EGFR expression. Conditioned medium (CM; 5 ml) from CHO cells stably expressing human sEGFR was incubated with protein A/G-conjugated agarose beads alone (ln 1), or with 4μg panitumumab (ln 2), 40μg panitumumab (ln 3), 2μg cetuximab (ln 4), or 10μg cetuximab (ln 5). Lane 6 was loaded with 100 μl CM alone (arrowhead indicates mobility of sEGFR; molecular weight markers are as indicated). All incubations were performed overnight (4°C), and were followed by SDS PAGE and immunoblot analysis with anti-EGFR monoclonal antibody (clone 15E11¹⁷).

Together these observations suggest that serum sEGFR may be an unanticipated “first target” of EGFR-directed antibodies in humans, where it may interfere with accurate pharmacokinetic/dynamic measurements, and also may moderate therapeutic efficacy. Moreover, the co-expression of sEGFR with EGFR on the surface of tumor cells may contribute to the lack of concordance between tumor ‘EGFR expression’ (as it is currently assayed), and responsiveness to cetuximab or panitumumab. It is clear, however, that tumor cells are not likely the major source of circulating sEGFR (unlike circulating HER2), since several studies have shown that serum sEGFR concentrations actually increase following tumor resection and/or chemotherapy ^14–16.

While the study of alternate EGF/HER receptor isoforms is in its infancy, we propose that the expression of these intrinsic regulators of EGF/HER receptor signaling adds a new level of complexity to our understanding of EGFR/HER1 signal transduction that is perhaps analogous to the role of IGFBP’s in insulin/IGF-1 family signaling. Despite our limited understanding of the function(s) of these naturally occurring EGFR isoforms in blood and other normal tissues, the potential of these proteins to interfere with the measurement of EGFR in human tumors/tissues, as well as the therapeutic targeting of EGFR in cancer patients should be considered in the design of future clinical trials using EGFR-directed immunotherapeutic drugs.