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Ihc

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mainly about basics of IHC

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Ihc

  1. 1. Immunohistochemistry in Oncology Dr D. Ramu
  2. 2. Introduction • Immunohistochemistry (IHC) 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 • IHC makes it possible to visualize the distribution and localization of specific cellular components within a cell or tissue.
  3. 3. • Antibodies bind to antigen in specific manner • Gives a spatial location • Can be used to locate particular cells and proteins • Can be used to identify cellular events – e.g.apoptosis
  4. 4. History • The principle has existed since the 1930s. • Started in 1941 when Coons identified pneumococci using a direct fluorescent method. • Indirect method • Addition of horseradish peroxidase • Peroxidase anti-peroxidase technique in 1979 • Use of Avidin & Biotin complex in early 1980’s
  5. 5. What cellular antigens can we target? • Cytoplasmic • Nuclear • Cell membrane • Lipids • Proteins
  6. 6. Important considerations for IHC • Antibody selection • Fixation • Sectioning • Antigen Retrieval • Blocking • Controls • Direct method • Indirect method • Immunoenzyme • Fluorescence • Multiple labeling
  7. 7. General antibody structure
  8. 8. Monoclonal v. polyclonal • Monoclonal • Mouse or rabbit hybridoma • Tends to be ‘cleaner’ • Very consistent batch-to- batch • More likely to get false negative results • Polyclonal • Many different species • Tends to have more non- specific reactivity • Can have very different avidity/affinity batch-to- batch • More likely to have success in an unknown application
  9. 9. Fixation  Aldehyde  10% NBF  4% formaldehyde with PBS buffer  2% formaldehyde with picric acid and PBS  The paraformaldehyde paradox  24-72 hours  Many others  Best for good architecture  Frozen  With or without sucrose  OCT  Fix with acetone or methanol (fix by coagulation, also permeabilizes)  Best for cell membrane antigens, cytokines
  10. 10. Sectioning • Paraffin • Must heat and process through xylenes and alcohols – ruins some antigens • Most commonly used • BEST if not stored more than two weeks – lose antigenicity after that time • Frozen • Better survival of many antigens • Poor morphology • Poor resolution at higher mag • Special storage • Cutting difficulty
  11. 11. Antigen retrieval  HIER  Use MW/steamer/pressure cooker ~ 20 minutes, slow cool  Citrate 6.0  Tris-EDTA 9.0  EDTA 8.0  Must determine for each new antibody/antigen target  PIER  Proteinase K  Trypsin  Pepsin  Pronase,etc.  Destroys some epitopes  Bad for morphology
  12. 12. Improving antibody penetration  Need this for intracellular (cytoplasmic, nuclear) or membrane components when epitope is inside cell membrane  Detergents most popular  Triton-X  Tween  Also decreases surface tension – better coverage  Can’t use for membrane proteins  Acetone/Methanol  Precipitate proteins outside cell membranes- more accessible  Saponin  Punches holes in cell membrane – holes close up when removed
  13. 13. Blocking • Background staining • Specific • Polyclonal antibodies – impure antigen used • Inadequate fixation – diffusion of antigen – often worse in center of large block • Non-specific • Non-immunologic binding – usually uniform • Endogenous peroxidases • Endogenous biotin
  14. 14. Controls • Positive control • Best is tissue with known specificity • Negative control • Best is IgG from same species immunized against non-biologic molecule – e.g. BRDU when no BRDU is present in tissue • Can also use non-immunized serum from same species
  15. 15. Direct method- primary antibody only Goat anti-actin labeled with 594
  16. 16. Indirect method – primary and secondary antibodies Goat anti-actin Donkey anti-goat labeled with 488
  17. 17. Enzyme linkage indirect method Goat anti-actin Flourochrome (488) conjugated streptavidin Biotinylated donkey anti- goat
  18. 18. Enzymatic detection methods Brightfield microscope sufficient for analysis of specimens Suitable for tissue analysis at low magnification Resolution of subcellular structures not as good as with fluorescence methods, but can be combined with electron microscopy Unimited shelf life of labelled specimens Substrate reagents often toxic/carcinogenic
  19. 19. Usefulness of IHC tests. • Poorly differentiated tumors and mixed carcinomas • Undifferentiated tumors of unknown origin • Treatment based on sub-type of cancer: Personalized Medicine eg., Breast Ca • Monitoring progress of cancer (Predictive and Prognosis) • In Malignant Lymphoma, • In identifying Carcinoid (Neuroendocrine) tumors • In Cytologic specimens
  20. 20. Histiogenic Dx of Neoplasm A) Expression of cytokeratin AE1/AE3 in lung carcinosarcoma ; B) chromogranin expression in gastric neuroendocrine carcinoma ; C) HMB 45 immunostainning in melanoma . Epithelium NeuroEndocrine Melanocyte
  21. 21. Metastatic Adenocarcinoma of unknown origin PSA +(-) (-) (-) (-) (-) (-) (-) (-) + + + + + + + Prostate Lung Stomach/Pancreas Breast Colon Colon Stomach / Pancreas Breast Ovary Pancreas,(Ovary serous) Stomach / Pancreas Breast / Stomach / Pancreas Source: www.clincancerres.aacrjournals.org/egi/content/full/11/10/3766 TTF-1 GCDFP15 CDX2 / CK20 ER CA125 Mesothelin Lysozyme + + + (-) (-) (-) CDX2 CK 7 Mesothelin MC5+ (98%)
  22. 22. Endocervical Ad.Ca & Endometrial Mucinous Ad.Ca ECA • MUC-1(-) • ER(-) • PR(-) • P16(+) EMMA • MUC-1 (+) • ER(+) • PR(+) • P16(-) Khoury T et al.BMC Clin Path 2006;6:1
  23. 23. Prostate Ca or Benign ? • Prostate Cancer • EpCam + • ATM + • AMACR + • PSA + / (-) • 34ßE12 (-) almost all • p63 (-) almost all • Prostein + • NKX3.1 + • Benign Prostate • EpCam (-) • ATM (-) / + • AMACR (-) • PSA + / (-) • 34ßE12 + • p63 + • Prostein + • NKX3.1 + ATM=ataxia-telangiectasia mutated;AMACR=alpha-methylacyl-CoA racemase;Ep- Cam=epithelial transmembrane glycoprotein
  24. 24. Urothelial Ca vs Prostate Ca • EMA • CK7 • P63 • CK5/6 • EpCam • CD57 • PSA • PAP • NKX3.1 • Prostein + - + - +/- 0 +/- 0 +/- 0 -/+ +/- 0 + 0 +/- 0 + 0 + 0 + Urothelial ca Prostate ca Hammerich KH.Archives of Pathology and Laboratory Medicine;132(3):432-440 Urothelial ca Prostate ca
  25. 25. Risk Biomarkers (or screening biomarkers) Describe risk of cancer occurrence or cancer progression because they are implicated in neoplastic progression and include: 1. Genetic predisposition (e.g., BRCA1/2) 2. Over expression of genes (e.g., BCR-ABLTyrosin Kinase Inhibitor in CML, HER-2/neu in Br Ca,PTEN, RAS,Colo Rectal Cancer AKTin Pancreatic cancer) 4.Environmental factors and lifestyle (e.g., HPV or HBV infection http://www.esmo.org/content/download/8713/176680/file/ The-use-of-Biomarkers-for-Treatment-Sessa-Fasolo.pdf
  26. 26. Undifferentiated Tumors Use of IHC to differentiate broad lineage Pan CK AE1/AE3 CD45 (LCA) HMB45 or S100 VIM Carcinoma Positive Negative Negative Negative Melanoma Negative Negative Positive Positive Sarcoma Negative Negative Negative Positive Lymphoma Negative Positive Negative Negative
  27. 27. Carcinoma Use of CK7 and CK20 CK7 + CK20 + CK7 + CK20 - CK7 – CK20 + CK7 – CK20 - Urothelial Ca Pancreatic Ad Ca OvarianMucinous Ca Ad Ca of Bladder Gastric Ad Ca Cholangio Ca subset Breast Ca EndoMetrial Ad Ca EndoCervical Ad Ca Ovarian Cerous Ca Lung Ad Ca Cholangio Ca LungSmCC Mesothelioma Thyroid Ca SCC of Cervix SalivaryGland tumors Urothelial Ca subset Pancreatic and Gastric Ad Ca subset Colorectal Ad Ca Markel Cell ca Gastric Ad Ca subset Prostate Ad Ca SCC RCC HCC Mesothelioma AdrenoCortical ca NonSeminoma LungSmCC minorSubset Gastric Ad Ca subset http://www.pathinformatics.com/department/documents/SusanE%20Lecture.pdf
  28. 28. 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. SqCC AdCa “Ancillary Testing in Lung Cancer Diagnosis” Dublinski et al. Openi.nlm.nih.gov
  29. 29. Primary and Additional Markers For teaching purpose only For teaching purpose only
  30. 30. Tumors from Unknown Primary Site Metastatic AdenoCarcinoma from Unknown Primary For teaching purpose only
  31. 31. CUP Diagnosis (Cancer of Unknown Primary) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2631214/figure/f1-gcr1_6p0229/ For teaching purpose only
  32. 32. Hepatocellular Colon Biliary Pancreas Ovary /EM Sarcoma Hepar + - - - - - AFP + - - - - - CEA - + + +/- +/- - CK7 - - + + + - CK20 - + ? ? - - AE1 AE3 +/- + +/- + + - CA 19-9 - - - + - - ER - - - - + - (Not for Renal Cell Carcinoma). (?) suggests could be positive 35% to 65% or negative); and +/- dependent on subtype of histology from that organ. Immunoperoxidase Panel for Liver Lesions
  33. 33. Examples of common panels of Antibodies Used Generic T-cell Vs B-Cell : CD3, CD20, CD45 Folicular Lymphoma Vs Hyperplasia: Bcl2, Bcl6, CD3, CD10,CD20 Low Grade B Lymphoma: CD3, CD5, CD10, CD20, CD23, CD43, Bcl2, Bcl6, MALT Lymphoma: CD3, Cd5, CD20, Bcl2, ISH Kappa and Lambda Hodgkin’s Lymphoma: CD3, CD15, CD20, CD30, CD45 Myeloma: CD138, ISH Kappa and Lambda Carcinoma Vs Lymphoma: CD3, CD20, CD45, PanCK Metastatic Carcinoma: CK7, CK20, TTF-1 GIST: CD117, CD34, S100, Desmin, SMA Mesothelioma: PanCK, CK5/6, Calret, TTF-1, CEA, CD15 If male add PSA/ if female add BRST2
  34. 34. Antibodies commonly used • Breast ER, PR, gross cystic fluid protein, CK7,CK20, E-cadherin • Colon and other GI tract CEA (monoclonal), CK7, CK20 • Germ cell PLAP, -fetoprotein, -HCG, AE1/3 • Hepatocellular Hepar, -fetoprotein, CK7, CK20, AE1 and AE3 (separately) • Lung TTF-1, CK7, CK20, CEA, Ber-EP4, chromogranin, synaptophysin, S100 • Lymphoma LCA (CD45RB monoclonal), CD3, CD20, CD30, ALK-1, myeloperoxidase, and light chains, Bcl-2
  35. 35. Antibodies commonly used contd. • Mesothelioma: Calretinin, AE1, AE3 • Melanoma: S100 (when spindled cells), HMB-45 (when epithelioid), MART1 • Neuroendocrine : Chromogranin, synaptophysin, NSE • Pancreas : AE1/3, CK7, CK20, CA 19-9, CEA, chromogranin, synaptophysin, -antichymotrypsin, CD10, PR, Ber-EP4 • Prostate :PSA, CK7, CK20 • Renal: EMA, CD10, HMB-45, inhibin- (to exclude adrenal /cortical) • Sarcoma : Vimentin, S100, CD117 (c-Kit), CD34,SMA, myogenin, CD31, CD68, desmin,CD1a, CD99 • Thyroid: TTF-1, thyroglobulin, calcitonin, CEA • Urothelial : CK7, CK20,Uroplakin

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