Immunohistochemistry Dr. Rikin Hasnani

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

Histogenetic diagnosis of morphologically non-differentiated neoplasias

• 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.

Subtyping of neoplasias (eg: lymphoma )

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

• No. of markers positive and 5 years survival

• 0-3 – 76 %

• 4-5 – 62%

• 6-9 – 48%

Targeted Therapy

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