For several types of common cancers, e.g., lung cancer, patient survival rates have not improved in the last decade (33
). Response to therapy is highly variable, and in some cases, e.g., ovarian cancer, does not improve overall survival (33
). This is particularly true for platinum-based therapies to which patients frequently become refractory (36
). Therefore, a major goal in oncology is to define biomarkers that can stratify cancer patients according to their likelihood of responding to chemotherapy. A highly active current focus of this effort is to measure expression of ERCC1 in tumors as a potential biomarker of DNA repair and therefore, resistance to genotoxic therapeutic agents (37
). To infer conclusions about the mechanism of therapy failure, it is imperative to rigorously standardize methods to accurately measure biomarker expression.
Immunohistochemistry (IHC) is an extremely valuable method for measuring tumor biomarkers. Unlike other immunological methods, IHC is applied to fixed specimens, which are the most abundantly available from surgical resections of tumors, allowing the large-scale screens necessary to validate the biological significance of novel biomarkers. Small amounts of tumor tissue, such as those obtained by needle biopsy, are sufficient for a semi-quantitative measurement of the antigen of interest. IHC is therefore used in decisions regarding diagnosis, prognosis and therapy of malignancies. However, there are multiple variables in the processing of samples in IHC and data analysis that need to be addressed prior to the widespread use of IHC as a quantitative immunoassay (40
). The staining intensity is affected significantly by the choice of fixative, the time of fixation (41
), the extent of deparaffinization (42
), thickness of the tissue section (41
), the antigen retrieval technique (43
), sensitivity and specificity of antibodies, and inter-observer inconsistencies in sample analysis (40
). Furthermore, scoring of samples as positive or negative for a particular biomarker can be based on a subjective scale of staining intensity or percent of positively staining cells. Therefore, the importance of validating and standardizing every IHC protocol used in clinical trials cannot be overstated. In this manuscript we have critically analyzed the reagents available for measuring ERCC1 expression in tissue samples and developed a standardized method for ERCC1 IHC that could be applied to tumors.
In order to develop an IHC protocol, the first step is to identify an antibody that is specific for the target antigen. This is accomplished by immunoblotting using protein extracts from human cells or tissue and demonstrating that the antibody detects a single band of the appropriate molecular weight (44
). It is imperative to include positive and negative controls in which the antigen is known to be expressed or depleted (either genetically or by shRNA), respectively (). Another excellent positive control is recombinant tagged protein included as a molecular weight control on immunoblot or overexpressed in a cell line (). Of eleven screened antibodies that detect ERCC1-XPF, ten were specific for the repair complex (Supplemental Table 1
The second step in developing an IHC protocol is to validate that an antibody retains its specificity for an antigen in fixed samples (44
). This can be accomplished directly in tissue samples only if negative controls (tissues in which the antigen is not expressed) are available. For many of the tumor biomarkers this is not feasible because the antigens of interest are key regulators of cell cycle control and genome maintenance, and thus are ubiquitously expressed. Alternatively, the specificity of an antibody to detect a biomarker in fixed material can be validated using positive and negative control cell lines processed according to the IHC protocol () (44
). In our experiments, normal human fibroblasts (C5RO) were used as a positive control and XP-F patient fibroblasts (XP2YO), deficient in ERCC1-XPF, were used as a negative control due to the absence of human tissue samples missing this protein (). Such internal controls must be included in every IHC analysis (41
). One important step in validating proper controls is establishing that the antigen staining has a proper subcellular localization, for example, ERCC1-XPF is a nuclear antigen (). A second step that can provide internal validation of an immunostaining protocol is to differentially label the positive and negative control cell lines and co-culture them, creating a sample with both immunoreactive and unreactive cells ().
Using the above methods with stringent controls, we discovered that antibody 8F1 is not suitable for measurement of ERCC1 expression because it detects a second antigen (20
). It has been reported that 8F1 could discriminate between HeLa cells and an isogenic strain in which ERCC1 expression was knocked down by siRNA (32
). Based on this, it was argued that 8F1 is suitable for measurement of ERCC1 by IHC. However, HeLa cells do not have appreciable amount of the non-specific antigen () and are thus inappropriate for use as either positive or negative control for validating this antibody. In the present work, using formalin-fixed paraffin-embedded normal and ERCC1-XPF deficient cell lines, it was confirmed that 8F1 is unable to differentiate between normal and ERCC1 deficient cells by IHC (). Since antibody 8F1 is the most widely used antibody for IHC (16
), it is important to emphasize that it is not specific for ERCC1 and that validated alternative antibodies exist to reliably measure ERCC1 by IHC. The extensive literature on 8F1-IHC does however indicate that the antibody may have prognostic value. This warrants further investigation to identify the unknown antigen recognized by 8F1.
After testing a panel of antibodies raised against ERCC1 and XPF, antibodies suitable for a variety of immunodetection techniques were identified (Supplemental Table 1
). Most of these are unsuitable for IHC, primarily because they do not discriminate between positive and negative controls in fixed material. Those that do work (FL297, 4H4, 3F2) should facilitate the intense interest in measuring ERCC1 expression in tumor samples by IHC. These are validated alternatives to 8F1 that can be used to reliably measure ERCC1 and XPF by IHC. FL297 and 3F2 were used to measure ERCC1 and XPF, respectively, in lung tumor sections (). Variable levels of cytoplasmic staining were seen in paraffin embedded cell lines (). This staining does not correlate with the level of ERCC1-XPF in cells, nor their sensitivity to genotoxic stress. It should therefore be noted that when using these antibodies, grading of expression levels should be based on the extent of nuclear staining only. The levels of both proteins vary between specimens, ranging from intense staining to virtually no staining, but parallel one another (). This indicates that either ERCC1 or XPF might serve as a biomarker of DNA repair in tumors. There was also differential expression within a tumor depending on the cell type. These results indicate that measuring ERCC1-XPF may be of value for stratifying patients for responsiveness to therapy.