In situ techniques for protein analysis in tumor tissue

Overview of the analytical techniques Assessment of protein expression is a vital part of diagnostic pathology routinely used in addition to morphologic evaluation for establishing a definite diagnosis and histological subclassification of tumors. Immunohistochemistry (IHC)-based technologies have expanded the spectrum of clinical applications beyond histological diagnosis towards identifying prognostic biomarkers and guiding selection of patients for personalized treatment. This chapter reviews the current analytical techniques for protein detection. Chromagenic immunohistochemistry IHC selectively detects specific proteins in tissue and provides complementary diagnostic information to morphological observations; IHC has thus become a routine technique for diagnostic purposes (a subset of popular IHC-assessed proteins contributing to tumor diagnosis, characterization, and prognosis are summarized in Table 9.1). In contrast, it has also been used as a companion diagnostic test to predict response to specific therapies. The classic examples of this usage are estrogen receptor (ER; 1) and human epidermal growth factor receptor 2 (HER2; 2) in breast cancer. Advantages of IHC include application in formalin-fixed paraffin-embedded (FFPE) tissue, wide availability, low cost, and simplicity when coupled with a pathologist's assessment at the light microscope. However, conventional IHC can be restricted by low sensitivity or differences in sample preparation and fixation (3,4). These issues have been addressed by developing signal-amplification techniques and antigen retrieval methods, respectively (5). In principle, IHC requires at least an antibody detecting the antigen of interest that is linked to an enzymatic system (most commonly peroxidase or alkaline phosphatase), used to visualize the site of antigen–antibody reaction.