Measuring ERCC1 protein expression in cancer specimens: validation of a novel antibody

Abstract

Platinum chemotherapy remains part of standard therapies in the management of a variety of cancers. Severe side effects and a high degree of resistance to platinum drugs have led numerous researchers to search for predictive biomarkers, which could aid in identifying patients that are the most likely to respond to therapy. The ERCC1-ERCC4 endonuclease plays a critical role in the repair of platinum-DNA damage and has widely been studied in relation to sensitivity to platinum chemotherapy. The standard method to evaluate ERCC1 protein expression is through the use of immunohistochemistry with monoclonal antibody 8F1, an antibody that was recently found to bind an unrelated protein. The present study determines the specificity of a novel antibody, monoclonal antibody 4F9, and presents a method to evaluate ERCC1 expression in colorectal tumor specimens. Using relevant cell lines as controls, the specificity of antibody 4F9 was tested by immunoblotting, immunohistochemistry and immunofluorescence. Scoring guidelines to aid in the evaluation of ERCC1 tumor expression were developed and evaluated in archival formalin-fixed paraffin embedded colorectal cancer specimens. Antibody 4F9 was found to be specific by all methods applied and it was possible to evaluate the ERCC1 expression in the majority (85%) of colorectal cancer tumor specimens.

Figure 1. Testing the specificity of anti-ERCC1 antibody 4F9.

(A) Cropped western blot of protein lysates from Colo-205 and XP2YO. (B) Cropped western blot of protein lysates from Colo-205 with and without 8 μM oxaliplatin. (C) Immunohistochemical staining of paraffin-embedded Colo-205 and XP2YO cells. (D) Detection of ERCC1 and γ-H2AX in Colo-205 chamber slide cultures by immunofluorescence. (E) Immunohistochemical staining of parallel tonsil sections in presence and absence of peptide corresponding to the antibody 4F9 epitope. A germinal center in the absence (left) and presence of epitope peptide at 1 μgml⁻¹ (middle) and 10 μgml⁻¹ (right).

Figure 2. Graphical representations of results from fixation study.

Comparisons between selected cell populations with an external tonsil reference.

Figure 3. Immunohistochemical staining of a stage III CRC tumor specimen.

(A): Ganglion cells. (B): Tumor cells. (C): Crypt epithelium. The applied scoring guidelines require a comparison of tumor cells (B) with ganglion cells (strongest intensity is reference for score 3) (A) and crypt epithelium (weakest intensity is reference for score 1) (C). The tumor consists of cells producing staining intensities similar to ganglion cells, as well as cells that stain weaker, but stronger than crypt epithelium (i.e. moderate). Therefore, the final score of this tumor was 2–3, which was reported as ‘ERCC1 high – homogenous'.

Figure 4. ERCC1 IHC Scoring System.