Immunohistochemistry for Therapeutic Target Identification: The Total Test System

1. Immunohistochemistry for Therapeutic Target Identification: The Total Test System Timothy J. O’Leary Armed Forces Institute of Pathology

2. Armed Forces Institute of Pathology

3. Armed Forces Institute of Pathology

4. Tissue Processes in Pathology { Excision T i m e Nucleases Proteases Cell Death Lipases Saccharidases Fixation Dehydration Embedding The time between excision and fixation can vary widely, as can the conditions of fixation. Examination and Diagnosis Ancillary Studies

5. Proteases • Destroy protein antigens • Not all protein antigens are destroyed at the same rate

6. Nucleases • Destroy nucleic acids • RNA destroyed much more quickly than DNA • Found in different levels in different tissues. For example, pancreas and eosinophils have extremely high levels of ribonucleases

7. Stability • In general DNA>Protein>RNA • Some proteins are essentially as stable as DNA, however.

8. Fixation • Stops degradation of cellular components by enzymes • Preserves cellular morphology

9. Ideal Fixative An inexpensive substance that rapidly penetrated any tissue and inactivated destructive enzymes, preserving cell and tissue structure, without reducing antigenicity, the ability to extract cellular components for other analyses, or the ability to use cellular nucleic acids as substrates for polymerase reactions….

10. Fixatives • Cross-linking, such as formaldehyde • Precipitating, such as alcohol In general, cross linking fixatives stop enzymatic degradation more quickly and completely than to precipitaing fixatives. Formaldehyde exists in solution as methylene glycol, so certain properties resemble those of precipitating fixatives

11. Cross-Linking Fixatives • Intramolecular and intermolecular cross-links in proteins • Cross links between DNA bases, but only in non-double stranded areas • Adducts between RNA and formaldehyde (methylol), proteins, nucleic acids • Intermolecular bonds in some lipids

13. Fixatives and the Pathologist • Most pathologists prefer formaldehyde for most tissues - relatively “crisp” tissue morphology, with relatively good preservation of antigenicity. • Other fixatives include alcohols, acetone, Zenkers, Bouins, B5, zinc-formalin, etc….

14. Formaldehyde Fixation and Protein Antigens • Fixation may reduce antigen reactivity in immunohistochemical reactions by masking epitopes or by making the tissue inpenetrable by antibodies. • Fixation does not destroy secondary structure. • Fixation partially reversible, but….

15. Antigen Retrieval • Generally some variant on heating a slide in solution to a high temperature and incubating it. • Removes formaldehyde, which forms a relatively weak bond with proteins. • When the formaldehyde comes off, the protein becomes denatured.

16. Protease Digestion of Tissue • Has been virtually completely replaced by antigen retrival approaches for immunohistochemisty. • Still a major component of many in-situ hybridization protocols.

17. Immunohistochemistry • Incubate with primary antibody. • Wash off primary. • Detect primary antibody • Dako

18. Immunoperoxidase Methods • Direct immunoperoxidase • Indirect immunoperoxidase • Peroxidase-antiperoxidase (PAP • Avidin-biotin complex (ABC) • Biotinylated tyramide amplification • Envision • Etc., etc., etc.

19. Method Comparison http://www.hmds.org.uk/histology.shtml

20. Antigen Detection in Tissue by ABC Immunohistochemistry SPECIMEN :Specimens are 4-6 micron sections of formalin-fixed, paraffin-embedded tissue that have been placed on coated glass microscope slides. Two slides are required at a minimum. MATERIALS and SOUTIONS Oven set at 600C Water bath set at 35-400C Magnetic stir plate, stir bars Gloves Pipettes Xylene 100% ethanol 0.05% protease VIII (Sigma, St. Louis) in 0.1M sodium phosphate buffer, pH 7.8 100% methanol with 3% hydrogen peroxide (H202) 0.01M sodium phosphate, pH 7.40, 0.15M NaCI (PBS) Normal serum (specific for secondary antibody species) Primary antibody Biotinylated secondary antibody Avidin-Biotin Complex (ABC) reagent 0.016% diaminobenzidine tetrahydrochioride (DAB), 0.24% H202 in PBS Gill's hematoxylin 2% conc. ammonia in deionized water Permount (Fisher Scientific)

21. Antigen Detection in Tissue by ABC Immunohistochemistry CONTROLS Positive Control Slide - This is a slide known to demonstrate the antigen of interest; it should be weakly or moderately (not strongly) positive. A positive control is run with every patient specimen. This control assures that the reagent system is functioning properly. Negative Control Slide – Patient slide with phosphate-buffered saline substituting for the primary antibody, to control for nonspecific binding of the detection system. Sections should not give a signal. PROCEDURE - STEPWISE 1. Paraffin sections mounted on pretreated slides are heated in an oven for 30 minutes. 2. Deparaffinize sections using 4 changes of xylene for 5 minutes each, followed by 4 changes of 100% ethanol for 3 minutes each. For sections that do not require digestion, go directly to Step 6. 3. For sections requiring digestion, rinse in deionized water for 3 minutes. 4. Incubate sections in buffered protease VIII at 370C for 1 to 30 minutes. The standard time for digestion is 3 minutes. 5. Rinse sections in 2 changes of deionized water for 2 minutes each, then 3 changes of 100% ethanol for 3 minutes each. 6. Block endogenous peroxidase activity with 3% H202 methanol for 30 minutes.

22. Procedure (cont.) 7. Rinse sections 3 times with deionized water. 8. Reduce nonimmunologic binding of antiserum by placing sections in 10% normal serum-PBS derived from the same species as the biotinylated (secondary) antibody (e.g., if biotinylated antibody is goat anti-rabbit antiserum, use normal goat serum) at 40C overnight. 9. Shake off excess normal serum and place slides in leveled staining trays. Distribute the slides according to the primary antiserum that is to be applied. Care should be taken so that the sections do not touch during incubation with primary antisera. The need to separate the slides into groups in order to preclude any chance of contaminating a section with the wrong antibody cannot be over emphasized. Cover section with primary antiserum, e.g. rabbit antiserum specific for desired antigen, for 30 minutes at room temperature. Make sure the chamber lid is on to prevent air-drying during the incubation. 10. Wash sections with PBS 3 times. 11. Flood sections twice with 10% normal serum for 10 minutes each, and then rinse in PBS. 12. Shake oft excess buffer and cover sections with biotinylated secondary antibody. Incubate for 30 minutes at room temperature, making sure the chamber lid is on to prevent air-drying of sections. 13. Wash sections 3 times with PBS. 14. Shake off excess buffer and cover sections with ABC reagent, prepared according to the recommendations of the manufacturer, for 30 minutes at room temperature, making sure the chamber lid is on to prevent air-drying of the sections.

23. Procedure (cont.) 15. Wash sections 3 times with PBS. Place sections in staining racks for a final 10-minute soak in PBS. 16. Develop in 0.016% DAB and 0.24% H202 in PBS solution for 10 to 25 minutes at room temperature. The usual time for optimal development of DAB is 15 minutes, but this may vary with the lot of DAB being used. 17. Wash sections 1or 2 minutes 3 times with deionized water. 18. Counterstain with Gill's hematoxylin. 19. “Blue" sections with ammonia water. 20. Wash in deionized water for 5 minutes. 21. Dehydrate and mount sections with Permount. REPORTING RESULTS Positive antibody-antigen reaction is brown, with nuclei blue. The assay is indeterminate if the assay is negative, and the positive control section shows no reactivity. The negative control should be carefully examined to control for nonspecific staining.

24. Procedure Notes • Throughout the procedure, keep drying of the sections to a minimum in order to prevent destroying epitopes on the cell membrane. Drying also causes nonspecific background staining. • In Step 2, it is important to complete deparaffinization because paraffin can mask epitopes from the primary antibody. • In Step 16, special precautions should be taken when handling DAB because of its possible carcinogenic properties.

25. Therapeutic Utility Studies Studies on the therapeutic utility of estrogen receptor IHC demonstrates both that the quality of therapeutic decision making is influenced by the performance characteristics of IHC tests , and that there is a high level of interlaboratory variability in their performance .

26. Immunohistochemistry for ERBB2 The Herceptest ® assay differs substantially from the immunohistochemical assay used to select patients in clinical trials.

27. Immunohistochemistry for ERBB2 (cont.) It is likely that therapeutic decisions regarding the use of Herceptin ® are being made based on results of “home brew” assays that haven’t even gone through the “indirect validation” that was used for Herceptest ®!

28. Immunohistochemistry for ERBB2 (cont.) These “home brew” assays may differ substantially in their performance characteristics; in many cases the differences may not be known to either the laboratory performing the test, nor the clinician making use of the test result.

29. Standardization in IHC • National Cancer Institute workshop (1977). • Biological Stain Commission workshop. • FDA workshop. • NCCLS Guidelines (1999) • EORTC Workgroup (Dr. Hammond)

30. Variation in Immunohistochemical Results • Dako Different amounts of antigen? Different amounts of antigen degradation? Different effectiveness of antigen retrieval? Different assay for same analyte? Complete or partial assay failure? What is 2+ anyway?

31. CliveTaylor: • Immunohistochemistry is a total test system. It includes everything from the time the tissue is excised, up to and including the pathologist interpretation. • Pre-analytic issues are often much more important than the analytic procedure itself, or variations in assay execution.

32. Needed Improvements • Development of certified reference materials for common immunohistochemical stains. • Development of “reference methods.” • Validation of commercial staining instruments and test systems against these reference materials and methods.