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.T he identification of molecular markers that can help guide treatment decisions in cancer is central to improving the therapeutic index of the current arsenal of chemotherapeutic drugs. Platinum drugs, such as cisplatin and oxaliplatin, are part of standard chemotherapy regimens in several cancer types, including non-small cell lung cancer (NSCLC) and colorectal cancer (CRC). CRC is one of the leading causes of cancer death, accounting for approximately 8% of total cancer deaths worldwide 1 . In metastatic CRC, response rates to oxaliplatin-based chemotherapy are as low as 34% 2 . Therefore, to improve upon patient survival and quality of life, the identification of a predictive biomarker profile for platinum-based chemotherapy is essential, so that only patients that are likely to respond receive platinum chemotherapy.Oxaliplatin and other platinum compounds inhibit tumor cell proliferation and induce cell death due to the formation of intracellular platinum-DNA adducts. These adducts consist of platinum-DNA monoadducts, platinum-DNA intra-and interstrand crosslinks, as well as DNA-protein crosslinks 3,4 . Platinum-DNA monoadducts and intrastrand crosslinks can be processed and repaired by nucleotide excision repair (NER) 5 . Interstrand crosslinks (ICL) are repaired through the activation of ICL repair, which involves several repair systems, such as homologous recombination, translesion synthesis, as well as NER 6 . The ERCC1-ERCC4 (XPF) heterodimer plays a critical role as a structure-specific endonuclease involved in both NER and ICL repair 7,8 , making it an interesting target for study in relation to platinum sensitivity/resistance.A possible link between ERCC1 and platinum se...

Emerging concepts on drug resistance in bladder cancer: Implications for future strategies

“…Immunohistochemistry with monoclonal antibody 8F1 is the standard method to evaluate ERCC1 protein expression, although novel antibodies have been recently evaluated [15]. Presently, it is still debated if a scoring system based on the relative expression of ERCC1, without accounting for the proportion of tumor cells with each expression level (e.g.…”

Development and Validation of an ERCC1 Immunohistochemistry Assay for Solid Tumors

“…The staining intensity of ERCC1 was judged relative to the intensity of the internal stromal fibroblasts Similar to previous studies, the median H score per tumor type was used as a cutoff to define tumors ERCC1 positive or negative results. 4,6 A sample was considered ERCC1 þ if the above internal control criteria were met and the assigned H score was greater than or equal to the median of the H scores for that specific tumor type. Negative staining of adjacent stromal cells resulted in a sample being scored not evaluable, regardless of tumor cell staining seen in the sample.…”

“…16 This antibody has also been used to develop an ERCC1 assay specific for colorectal tumors. 4 Additionally, some groups have examined the isoforms targeted by the 4F9 epitope to confirm that the isoforms relevant for DNA repair were included. 4 The development of an antibody that would target only the isoforms responsible for DNA repair is currently a high priority for researchers interested in ERCC1; however, because none of the isoforms have isoform-specific sequences, that is challenging.…”

“…4 Additionally, some groups have examined the isoforms targeted by the 4F9 epitope to confirm that the isoforms relevant for DNA repair were included. 4 The development of an antibody that would target only the isoforms responsible for DNA repair is currently a high priority for researchers interested in ERCC1; however, because none of the isoforms have isoform-specific sequences, that is challenging. 6 The objective of this study was to test the performance of 2 anti-ERCC1 antibody clones, 4F9 and D6G6, to develop and validate an ERCC1-specific IHC assay for formalinfixed, paraffin-embedded tissue (FFPE) that could be used for clinical trial samples.…”

“…These adducts consist of platinum-DNA monoadducts, intrastrand and interstrand cross-links, and/or DNA-protein cross-links. 4 These DNA lesions can be repaired by the nucleotide excision repair pathway. …”