Standardization of Epidermal Growth Factor Receptor (EGFR) Measurement by Quantitative Immunofluorescence and Impact on Antibody-Based Mutation Detection in Non–Small Cell Lung Cancer

Abstract

Challenges in measurement of epidermal growth factor receptor (EGFR) protein expression have led to conflicting data on its prognostic value and discontinuation of its use for prediction of response. Herein is described a quantitative standardized assay for EGFR and its use in a series of retrospective cohorts of patients with nonsmall cell lung cancer (NSCLC). The AQUA technology of quantitative immunofluorescence was used in conjunction with Western blot analysis to calculate the absolute concentration of EGFR in two independent NSCLC cohorts (170 from Yale New Haven Hospital and 335 from Sotiria and Patras Hospitals in Greece). EGFR and mutated EGFR were measured using D38B1 antibody and two mutation-specific antibodies. All patients positive or borderline for mutation-specific antibody were genotyped. A threshold for reproducible detection of EGFR was defined as 0.85 ng/ g total protein. EGFR expression demonstrated no prognostic value in either cohort. The mutation rate was 1.79% in the Yale cohort, and 1.52% in the Sotiria/Patras cohort, with no antibody detection-based false-positive cases. No mutations were detected for EGFR concentrations <1.46 ng/ g total protein. In summary, accurate measurement of EGFR still shows no prognostic value in NSCLC. In these two population-based cohorts, the antibody-based EGFR mutation rate was lower than has been frequently reported.

EGFRmutant-specific immunohistochemistry has high specificity and sensitivity for detecting targeted activatingEGFRmutations in lung adenocarcinoma

“…Mutation analysis identified six other cases of exon 19 deletions, and only one of these was detected by the E746-A750 antibody (p.Leu747_Ser752>Q). In concordance with our results, most other authors have found the antibody is generally inadequate at identifying variant exon 19 deletions 11 15 17 21…”

A diagnostic algorithm using EGFR mutation-specific antibodies for rapid response EGFR-TKI treatment in patients with non-small cell lung cancer

“…Other authors have reported that paraffin-embedded NSCLC tumor samples with these [7] 88.9% (8/9) 0% (0/12) 100% (14/14) Kato et al [8] 81.8% (9/11) 0% (0/7) 75.0% (9/12) Simonitti et al [10] 100% (17/17) 0% (0/12) 92.6% (25/27) 0% (0/2) Wu et al [13] 93.5% (29/31) 10% (1/10) 88.4% (38/43) Kawahara et al [14] 75.0% (9/12) 28.6% ( antibodies showed sensitivity and specificity of the immunohistochemistry assay of 47-92% and 96-99%, respectively [6][7][8][9][10][11][12][13].…”

“…Several authors have demonstrated mutation-specific antibodies recognizing the delE746-A750 and L858R mutations, which can be used to identify the EGFR mutation status of tumor samples and provide a simple immunohistochemical method for diagnosing EGFR mutations in human tissue [5][6][7][8][9][10][11][12][13]. We have previously demonstrated the use of EGFR mutation-specific antibodies in immunohistochemistry, and their application to the diagnostic screening of NSCLC patients [14].…”

The biology of personalized cancer medicine: Facing individual complexities underlying hallmark capabilities

“…Quantitative immunohistochemistry or immunofluorescence assays are being developed that can accurately validate on tissue microarrays (TMAs) the expression of DNA/RNA signatures identified by whole‐tumor profiling; these techniques have the advantage of being highly reproducible and easily applicable for standardized tumor typing/grading, inter‐patient comparisons, and the dissection of intra‐tumoral heterogeneity. Recent applications of such TMA‐based technologies have validated prognostic gene signatures for several tumor types, including prostate and lung cancer (Anagnostou et al., 2011; Dimou et al., 2011; Ding et al., 2011; Zender and Lowe, 2008). Furthermore, the development of sophisticated computer‐based methodologies for TMA analysis (Beck et al., 2011) may facilitate better standardization of the results and also quantitatively assess clinically significant morphometric parameters (e.g., epithelial–stromal ratio, multiple nuclear pleomorphisms, etc…) that would escape conventional pathological examination.…”