Abstract
Anti-PTEN monoclonal antibodies (mAb) are arising as important tools for immunohistochemistry (IHC) and protein quantification routine analysis in clinical oncology. Although an effort has been made to document the reliability of tumor tissue section immunostaining by anti-PTEN mAb, and to standardize their IHC use in research and in the clinical practice, the precise topological and biochemical definition of the epitope recognized by each mAb has been conventionally overlooked. In this study, six commercial anti-PTEN mAb have been validated and characterized for sensitivity and specificity by IHC and FISH, using a set of prostate and urothelial bladder tumor specimens, and by immunoblot, using PTEN positive and PTEN negative human cell lines. Immunoblot precise epitope mapping, performed using recombinant PTEN variants and mutations, revealed that all mAb recognized linear epitopes of 6–11 amino acid length at the PTEN C-terminus. Tumor-associated or disease-associated mutations at the PTEN C-terminus did not affect subcellular localization or PIP3 phosphatase activity of PTEN in cells, although resulted in specific loss of reactivity for some mAb. Furthermore, specific mimicking-phosphorylation mutations at the PTEN C-terminal region also abolished binding of specific mAb. Our study adds new evidence on the relevance of a precise epitope mapping in the validation of anti-PTEN mAb for their use in the clinics. This will be substantial to provide a more accurate diagnosis in clinical oncology based on PTEN protein expression in tumors and biological fluids.
Highlights
Precise epitope mapping is relevant when the biosynthesis of the marker protein is influenced by mRNA alternative splicing or alternative translation mechanisms, or when the mature marker protein is targeted by dynamic post-translational modifications, such as phosphorylation.[11]
Precise epitope mapping of monoclonal antibodies (mAb) recognizing cancer biomarkers is substantial for the interpretation of IHC staining patterns in cancer research and to provide an accurate IHC diagnosis in clinical oncology
Rationally designed anti-BRAF, anti-EGFR, or anti-p53 mAb have been generated that recognize hotspot mutations in these cancer-relevant proteins, which could be highly valuable for IHC-based precision diagnosis and for novel potential precision therapies.[74,75,76]
Summary
Expression of biomarkers, as detected by standard immunohistochemistry (IHC), together with classic histological parameters, constitutes the first-line of diagnosis of most solid tumors.[1,2] IHC has arisen as a universal prognostic technique to assist in patient stratification and therapy decisions in oncology.[3,4] It is currently admitted that sensitivity, specificity, and reproducibility are essential factors to validate monoclonal antibodies (mAb) as IHC tools in research and in the clinical practice.[5,6,7,8] unless short defined synthetic peptides are used as immunogens in the mAb obtention, the precise topological and biochemical definition of the epitope recognized by the mAb is mostly overlooked. Many IHC marker proteins are frequently targeted for mutations in tumors, which could affect in several ways both the protein function and its recognition by specific mAb, with important prognostic implications.[12,13]
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