RESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Endonucleasas/metabolismo , Neoplasias/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos , Especificidad de Anticuerpos/inmunología , Línea Celular Tumoral , Estudios de Cohortes , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Endonucleasas/genética , Endonucleasas/inmunología , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias/diagnóstico , Neoplasias/genéticaRESUMEN
AIMS: The availability of specific antibody-based test systems is essential to testing of HER2 protein expression. Here, we mapped epitopes recognized by three pharmacodiagnostic HER2 antibodies and investigated their specificity towards peptides and fusion proteins homologous to the intracellular domains of HER1, HER2, HER3 and HER4. The investigated antibodies were PATHWAY(®) HER2 (clone 4B5; Ventana Medical Systems Inc., Tucson, AZ, USA), HercepTest™ (Dako Denmark A/S, Glostrup, Denmark), and Oracle(®) HER2 (clone CB11; Leica Microsystems GmbH, Wetzlar, Germany). METHODS AND RESULTS: Epitopes were mapped using the alanine scanning method. Specificity was investigated in immunohistochemical stainings, competitive enzyme-linked immunosorbent assay (ELISA) and immunoblotting. All three antibodies reacted with HER2 proteins and peptides in immunohistochemical stainings, ELISA and immunoblotting. PATHWAY(®) HER2 also stained HER4-expressing cells, reacted with HER4 peptide in ELISA and detected HER4 fusion protein in an immunoblot. Oracle(®) HER2 weakly detected HER4 in immunohistochemical stainings, whereas the HercepTest™ antibody showed no cross-reactivity with other HER proteins. CONCLUSION: Our study shows that the PATHWAY(®) HER2 antibody can bind HER4 peptides and fusion proteins in three different experimental settings. This should be investigated further to determine whether binding of HER4 also occurs in tissue samples and if such binding would have implications for therapy decisions for breast cancer patients.