Abstract CN09-03: The EGFR conundrum: EGFR-based diagnostics in the era of precision medicine
Authors: Wilken Jason A. , Baron Andre T. , Maihle Nita J. 
Published: January 2011

Abstract
The epidermal growth factor receptor (EGFR) is a clinically validated target for the treatment of diverse cancers, as demonstrated by the FDA's approval of five drugs directed against this receptor (cetuximab, panitumumab, erlotinib, gefitinib, and lapatinib), which are used for the treatment of metastatic breast cancer (MBC), colorectal cancer (CRC), non-small cell lung cancer, head and neck cancer, and pancreatic cancer. Yet the clinical utility of measurements of EGFR expression has been limited. Assessment of EGFR expression in tumors is insufficient to stratify cancer patients for therapy, even with EGFR-targeted drugs. Here we propose many of the limitations associated with measurement of EGFR expression result from failure to independently assess all four isoforms of EGFR.
In addition to full-length EGFR, three naturally occurring EGFR isoforms are synthesized from alternative EGFR transcripts that arise from either read-through of an exon-intron boundary (1.8 kb EGFR), or by incorporation of one of two alternate exons, i.e., 15A (2.4 kb EGFR) or 15B (3.0 kb EGFR). The best studied of these alternate isoforms is the product of the 3.0 kb EGFR transcript, which we have designated sEGFR. sEGFR is a 90/110-kDa protein consisting of most of the EGFR extracellular domain followed by a unique carboxy-terminal cysteine-rich 78-amino acid sequence.
While the biosynthesis of sEGFR is predicted to follow a classic secretory fate, sEGFR is unexpectedly a cell surface protein. How sEGFR associates with the cell surface is unclear, but recent studies demonstrate that sEGFR is proteolytically released from the cell surface through a regulated process (1). This process is important since it gives rise to serum sEGFR, as demonstrated by mass spectrometric analysis (2). Quantification of both serum and tumor sEGFR has been shown by our laboratory and others to have diagnostic and prognostic utility in cancer patients, as outlined below.
Ovarian cancer: EGFR is a controversial tumor marker in epithelial ovarian cancer (EOC). In ∼40 published studies, only half report that EGFR is a prognostic marker (3). IHC studies using antibodies specific for sEGFR vs. full-length EGFR (Yale cohort: n=196) demonstrate that sEGFR and EGFR are expressed independently in both normal and malignant ovarian tissues.
Moreover, while neither sEGFR nor EGFR are independent prognostic markers, combined tumor sEGFR and EGFR expression is a powerful negative predictor of overall survival (4).
In addition, four laboratories have demonstrated the diagnostic utility of serum sEGFR in EOC (2, 5-7). Serum sEGFR is characteristically lower in women with EOC, even early stage EOC, compared to healthy controls of similar age and menopausal-status. Serum sEGFR concentrations remain stable or rise in EOC patients immediately following surgery (8). The fact that low serum sEGFR concentrations are negatively correlated with tumor burden in EOC, whereas high tumor sEGFR expression is negatively correlated with survival suggests that the regulation of sEGFR release into the bloodstream is intricately associated with the progression of this disease.
Breast cancer: A similar reciprocal pattern between serum sEGFR and tumor EGFR expression has been reported in MBC patients: high tumor EGFR expression and low baseline serum sEGFR are both correlated with shorter overall survival (9-12). Paradoxically, however, serum sEGFR decreases in response to chemotherapy (12). Baseline serum sEGFR also has been evaluated as a theragnostic marker for MBC patients treated with aromatase inhibitors and SERMs (9, 13, 14), chemotherapy (10, 12), and trastuzumab (15), however, these studies report mixed results, perhaps because of differences in trial design or methods of sEGFR assessment. Strikingly, however, increased concentrations of serum sEGFR can predict the development of breast cancer 17 months prior to clinical diagnosis of this disease (16).
Endometrial cancer: We have completed the first analysis of serum sEGFR in patients with endometrial cancer (17). In this GOG (229C) study baseline serum EGFR expression (n=23) was the only significant prognosticator of overall patient survival in patients treated with gefitinib.
Lung cancer: Baseline serum sEGFR concentrations have not been associated with improved survival in patients treated surgically (18, 19), or in patients treated with either chemo- or radiation therapy (20). High baseline sEGFR, however, has been correlated with response to gefitinib treatment (21). Serum sEGFR also is a leading candidate diagnostic marker in lung cancer patients, where age and gender affect the ability of this biomarker to discern lung cancer patients from healthy controls (22). In preliminary studies both human and mouse circulating sEGFR levels can be used to detect lung cancer (22, and S. Hanash, personal communication); the future use of this biomarker in animal models of cancer may, therefore, provide an excellent opportunity for exploring the limits of early detection using this new serum biomarker.
Colorectal cancer: Two EGFR-directed antibodies, cetuximab and panitumumab, are FDA approved for the treatment of patients with CRC. Recent studies have shown that tumor expression of downstream effectors such as PTEN, BRAF, and KRAS are negative predictors of the efficacy of these drugs, whereas EGFR expression does not predict responsiveness to either of these antibodies (23). In contrast, high baseline serum sEGFR is strongly associated with best objective response in patients (n=42) treated with gefinitinb/FOLFOX6 for 10 cycles followed by gefitinib maintenance (24), and also with improved survival in patients (n=118) with third-line irinotecan and cetuximab treatment (25).
While the basis for this association is not yet understood, we recently have demonstrated that both cetuximab and panitumumab bind to sEGFR with high affinity (26). We estimate that peak cetuximab concentrations are likely in only 28-fold molar excess relative to circulating sEGFR concentrations in treated patients. Since baseline serum sEGFR concentrations are characteristically decreased in many cancer patients (27), some patients would be predicted to receive much greater effective doses of cetuximab than others; to date, however this effect has not been considered in tailoring treatment for lung cancer patients.
Baseline serum sEGFR also has been reported to have diagnostic utility in CRC; Abdel-Aziz and coworkers have shown that sEGFR can differentiate between CRC patients (n=48) and normal subjects (n=20) with a sensitivity of 71% at 100% specificity (28), and Spindler and colleagues have shown a significant difference between pretreatment sEGFR concentrations in controls (n=126) vs. patients (n=118) (25).
Conclusions: Quantification of EGFR expression in human tumors has proven problematic, in part, because of the coincident expression of multiple EGFR isoforms in human tissues, a phenomenon that has been overlooked in the development of diagnostic assays. The independent assessment of tumor sEGFR vs. EGFR in a large EOC cohort as reported here, is clinically informative, and provides the first proof of principle of the benefit of independently measuring individual EGFR isoforms. Tissue sEGFR is a precursor of serum sEGFR, and serum sEGFR has been demonstrated to have independent diagnostic, prognostic, and theragnostic utility in diverse malignancies. In addition, serum sEGFR appears to be an unanticipated “first target” for at least two EGFR-directed therapeutic antibodies. While study of variant EGFR isoforms is in its infancy, these early studies demonstrate a previously unrecognized level of complexity in EGFR-based diagnostics that may account for current paradoxes associated with the clinical utility of EGFR-based diagnostic assays, similar to the ‘HER2 Conundrum’ described by Allison (29).
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Citation Information: Cancer Prev Res 2010;3(12 Suppl):CN09-03.