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Introduction
• Definition : Immunohistochemistry (IHC) refers to the process of detecting antigens (e.g. proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.
• Immunohistochemistry Combines histological, immunological and biochemical techniques for the identification of specific tissue components by means of a specific antigen/antibody reaction tagged with a visible label
• It was invented by Albert Coons in1941.
• It is widely used for diagnosis of cancers because specific tumor antigens are expressed de novo or up-regulated in certain cancers.
Principle
• The principle of immunohistochemistry is the localization of antigens in
tissue sections by the use of labeled antibodies as specific reagents
• Antigen-antibody interactions that are visualized by a marker such as
fluorescent dye, enzyme, radioactive element or colloidal gold.
Procedure
• Sample preparation - tissue collection, fixation and sectioning
• Sample labelling – direct , indirect, PAP method
Reporter molecule • These are molecules that report the presence or absence of Ag-Ab
reaction.
• It can be chromatogenic or flouroscent.
• With chromogenic reporters, an enzyme label is used that reacts with a substrate to yield an intensely colored product that can be analyzed with an ordinary light microscope.
• Alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two most commonly used enzymes.
• Fluorescein isothiocyanate (FITC), Rhodamine are commonly used fluoroscent molecules.
Uses of IHC
• The most important are:
• 1) histogenetic diagnosis of morphologically non-differentiated neoplasias
• 2) subtyping of neoplasias (such as lymphomas, for example);
• 3) characterization of primary site of malignant neoplasias;
• 4) prognostic factors and therapeutic indications of some diseases;
• 5) discrimination of benign versus the malignant nature of certain cell proliferations
• Immunohistochemistry stains in squamous cell carcinoma and adenocarcinoma of lung. H&E: hematoxylin and eosin; CK: cytokeratin; TTF-1: thyroid transcription factor 1. Squamous carcinomas are typically positive for CK5/6 and P63, and negative for CK7 and TTF-1, with the reverse profile for adenocarcinoma although this case of squamous cell carcinoma demonstrates focal weak staining for CK7.
Characterization of primary site of malignant neoplasias• Metastatic Adenocarcinoma of
unknown originwith site specific markers
Prognostic factors and therapeutic indications of some diseases• 1. Genetic predisposition (e.g., BRCA1/2, p53)
• 2. identification of genes involoved in carcinogenesis (e.g., BCR-ABL TyrosinKinase Inhibitor in CML, HER-2/neu in Breast Ca , RAS in Colo Rectal Cancer)
• 3.Environmental factors and lifestyle (e.g., HPV or HBV infection)
Role of Immunohistochemistry in Lung Cancer
• It is used to confirm the diagnosis of undiagnosed lung cancer
• To prognosticate the disease
• To evaluate the therapeutic potential of targeted therapy
• CK5 is a very sensitive marker for squamous cell carcinoma and also a more specific marker than P63. The combination of CK5 and P63 was not more informative than CK5 alone.
• TTF1 and NAPA are very sensitive markers for adenocarcinoma. • Furthermore, the combination of both markers was even more sensitive. • The sensitivity of TTF1 and NAPA is lower for large-cell carcinomas.• Mucinous adenocarcinomas and poorly differentiated adenocarcinomas
are known to be positive less often as well, although only a limited number of cases have been specifically reported
• Apart from lung cancer, NAPA is mainly positive in renal cell carcinoma and a few cases of thyroid cancer.
• 15% were positive for ER.
• The goals of this study were to evaluate a panel of molecular biologic markers in patients with stage I NSCLC after resection and to develop a prognostic model based on the involvement of these molecular variables, independent of other variables.
• The factor with the strongest independent prognostic value was p53. 52 vs 70
• angiogenesis factor viii was associated with significantly decreased survival.56vs 70
• The presence of the protooncogene erb-b2 was also a strong negative prognostic factor.47 vs 67
• The metastatic adhesion protein CD-44. 54 vs 67
• absence of rb was associated with significantly decreased survival 55 vs 63
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