PEMERIKSAAN NEOPLASMA AKIBAT VIRUS DARI ASPEK PATOLOGI ANATOMI

Transcription

1 PEMERIKSAAN NEOPLASMA AKIBAT VIRUS DARI ASPEK PATOLOGI ANATOMI DR. H. DELYUZAR, SP.PA (K) DR. BETTY, SP.PA DEPARTEMENT ANATOMIC PATHOLOGY MEDICAL FACULTY UNIVERSITY OF NORTH SUMATERA MEDAN

2 Flow chart depicting a simplified scheme of the molecular basis of cancer

3 Immunohistochemistry

4 Immunohistochemistry Merupakan suatu teknik untuk menentukan lokasi antigen (protein target) dalam jaringan/sel dengan menggunakan reaksi antigen-antibodi. Visualized by a marker such as : Fluorescent dye Enzyme Radioactive element Colloidal gold

5 Albert H. Coons et al. (Coons et al. 1941, 1955; Coons & Kaplan 1950) : 1 st to label antibodies with a fluorescent dye Used : to identify antigens in tissue sections Expansion & development IHC technique, enzyme labels have been introduced such as : Peroxidase (Nakane & Pierce 1966; Avrameas & Uriel 1966) Alkaline phosphatase (Mason & Sammons 1978) Faulk & Taylor (1971) : Colloidal gold label has Used : to identify immunohistochemical reactions at light & electron microscopy. Other labels include : Radioactive elements Immunoreaction can be visualized by autoradiography

6 Immunofluorescent Demonstration of anti-pemphigus vulgaris antibody.

7 Immunohistochemistry Involves : specific antigen-antibody reaction Advantage > traditionally (used special enzyme staining techniques) identify only a limited number of : Proteins Enzymes Tissue structures Crucial technique & widely used in many medical research laboratories as well as clinical diagnostics.

8 Antibody types The antibodies used for specific detection can be : Polyclonal Antibodies Monoclonal Antibodies

9 Polyclonal Antibodies Made by : injecting animals with peptide Ag After a secondary immune response is stimulated, isolating antibodies from whole serum. Heterogeneous mix of antibodies that recognize several epitopes. Monoclonal Antibodies Generally considered to exhibit greater specificity

10 Immunohistochemistry techniques Direct technique Indirect technique

11 Antibodies reagents Can also be classified as: Primary reagents Secondary reagents

12 Primary reagents Raised against an antigen of interest Typically unconjugated (unlabelled)

13 Secondary reagents Raised against primary antibodies Recognize immunoglobulins of a particular species Conjugated to either biotin/a reporter enzyme such as alkaline phosphatase/horseradish peroxidase (HRP). Some secondary antibodies are conjugated to fluorescent agents, such as the Alexa Fluor/Dylight Fluor family, are also frequently used for detection of proteins in IHC procedures Protein concentration is generally measured by densitometry analysis, where the intensity of staining correlates with the amount of the protein of interest

14 Immunohistochemistry Several procedures are avaiable The 2 most commonly used : 1. Peroxidase-antiperoxidase immune complex method 2. The biotin-avidin immunoenzymatic technique

15 Immunoperoxidase procedures A. Peroxidase (PX) antibody conjugate, direct. B. Peroxidase antibody conjugate, indirect. C. Labeled antigen method. D. Enzyme bridge procedure. E. Peroxidase antiperoxidase (PAP) immune complex method. Solid semicircle indicates antigen. (From Falini B, Taylor CR. New developments in immunoperoxidase techniques and their application).

16 Biotin-avidin immunoenzymatic techniques. Solid semicircle, antigen; PX, peroxidase; *, biotin; shaded open cross, avidin. A. Biotinylated primary antibody method. B. Biotinylated peroxidase method. C. Avidin biotin peroxidase complex method. (From Falini B, Taylor CR. New developments in immunoperoxidase techniques & their application).

17 Various methods sensitivity of the procedure The aim : To expose antigenic sites (epitop) that may unexposed ( masked ) antigenic unmasking / antigen-retrieving techniques They include : Digestion with a variety of proteolytic enzym (e.g. enzim trypsin) Treatment with microwave Exposure to the combined action of heat & pressure in a pressure cooker

18 The advent of monoclonal technology: Antibodies have become available for which the antigenic determinant is : Chemically poorly defined, or Totally unknown

19 . The advent of monoclonal technology: Marker located in: Nucleus Cytoplasm Cell membrane Extracellular space The advantages offer is that Generally higher degree of specificity Practically no diffusion If the marker location is nuclear stain can be combined with another with different chromogen aimed at cytoplasmic/cell membrane marker

20 Predictive markers A. ER is detected in the nucleus by IHC studies. PR has the same appearance. B. HER2/neu overexpression is detected on the cell membrane by IHC C. Amplification of the HER2/neu gene, detected by FISH analysis with a fluorescent probe for the gene. A normal cell has 2 copies of the gene. These tumor cells have over 25 signals, indicating amplification of the gene for HER2/neu.

21 Hormone receptors The effect of hormones in target organs is medicated by intracellular (largely intranuclear) peptides known as hormons receptors. Monoclonal antibodies are available for : Estrogen receptors Progesterone receptors Androgen receptors Originally the technique worked reliably only in frozen section material but now : the results in paraffin-embedded material They compare favorably with those obtained with the conventional biochemical assay.

22 HER2/neu (ERBB2) Protein membran sel Berhubungan erat dengan EGFR Over-expression : Acts as an oncogene Breast ca (22%) Lung adenoca (28%) Colorectal ca (17%) Lung SCC (11%) Gastric adenoca (11%) Useful for predicting response to Herceptin in breast ca & other organs.

23 Hepatocyte paraffin 1 (Hep-Par1) This antibody recognizes an unidentified mitochondrial antigen present in: Normal & neoplastic hepatocytes. It has a coarsely granular cytoplasmic quality Negative in : Bileducts Stromal cell. It is utilized to identify : Hepatocellular carcinomas Hepatoblastomas, and Hepatoid carcinomas, and Seems to have a high degree of specificity.

24 CD117 This antibody identifies c-kit (a transmembrane tyrosine kinase) Normal expressed by : Cajal s interstitial cells GIST tumor family Melanocytes melanoma Mast cells mastocytosis Germ cells germ cell tumors

25 Desmin This muscle type intermediate filament (MW ) is found in cells of : Smooth muscle smooth muscle tumor Striated muscle skeletal muscle tumor Primarily used for the identification tumor of : Smooth muscle Skeletal muscle Desmin (+) associated with Actin (-) is a feature of : Subset cells of myofibroblastic appearance Hormone dependent stroma (vagina, breast0 Desmoplastic small cell tumor

26 Keratin / cytokeratin A family of water-insoluble, intracellular fibrous proteins Present in : almost all epithelia ± 20 sub-classes have been identified on basis : MW Isoelectric ph value (5-8) Keratins represent an excellent marker for epithelial differentiation regardless of whether the tumor is of: Endodermal Neuroectodermal Mesenchymal, or Germ cell derivation

27 Keratin stain in a type B1 (lymphocyte-rich) thymoma

28 Keratin stain. Anaplastic carcinoma of pancreas

29 MBI-1 (Ki-67) An antigen corresponds to a nuclear nonhistone protein expressed by cells in proliferative phases G1, G2, M and S In general, there is a good correlation between Ki-67 staining & mitotic count

30 Strong immunostaining for MBI-1 in germinal center of hyperplastic lymph node

31 Latent membrane protein 1 (LMP1) An EBV membrane protein expressed in latent infection Is considered to be EBV oncoprotein

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