2. 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.
3. 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.
4. Procedure
• Sample preparation - tissue
collection, fixation and
sectioning
• Sample labelling – direct ,
indirect, PAP method
5. 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.
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9. 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
12. • 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.
14. Characterization of primary site of malignant
neoplasias
• Metastatic Adenocarcinoma of
unknown origin
with site specific markers
15. 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 Tyrosin
Kinase 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)
16. 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
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19. • 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.
20.
21. • 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
22. • No. of markers positive and 5 years survival
• 0-3 – 76 %
• 4-5 – 62%
• 6-9 – 48%
