Immunohistochemistry and Targeted Therapy

Important application of monoclonal and polyclonal antibodies for the detection of specific antigens in tissue sections

An extraordinarily powerful tool in the armamentarium of the diagnostic surgical pathologist and research (signaling pathways-targeted therapy)

Important roles in health and disease

Diagnosis of cancers: specific tumor antigens are expressed de novo or up-regulated in certain cancers

Basic histologic examination of tissue: a useful and necessary component

1. Immunohistochemistry and Targeted Therapy Krisna Murti Department of Anatomic Pathology Faculty of Medicine University of Sriwijaya

2. Multi-disciplinary Tumor Board

3. Molecular Analysis PAS staining Imunohisto /cytochemistry TARGETTED THERAPY FISH Clonality analysis Identification of mutation Histo /cytochemistry

4. Targeted therapy Drugs or other substances that block the growth and spread of cancer by interfering with specific  molecules   ("molecular targets") that are involved in the growth, progression, and spread of cancer Disrupt the way cancer cells duplicate and interact with other cells Superior > conventional chemotherapy in the treatment of several tumor entities Can be used alone or in combination with other treatments, such as chemotherapy and radiation Side effects vary widely among the different types of medications, but milder and diminish at the end of treatment

5. Hanahan D. 2022 H a l l m a r k of C a n c e r MicroRNA regulation

6. Therapeutic targeting based on the hallmark of cancer Hanahan D and Weinber, 2011

7. Role of HER2 in breast cancer HER2 enriched subtype occurs in 25-30% of breast cancers HER2 proto-oncogen regulation of normal cell growth Gene amplification or HER2 overexpression activate HER2 receptors: stimulates cell growth HER2-positive tumors poor prognosis shortened disease-free survival

8. HER2 overexpression HER2 (+3)

9. Trastuzumab (Herceptin) on breast cancer cells

10. Epidermal growth factor receptor (EGFR)

11. EGFR expression in human tumors Arteaga C. Semin Oncol 2003 Saif MW. Expert Opin Investig Drugs 2010 SCCHN; squamous cell carcinoma of the head and neck NSCLC ; non-small-cell lung carcinoma Colorectal ca (CRC) Type of tumor Tumors with EGFR expression Colon 1 Up to 77 % Advanced CRC 2 Up to 90% SCCHN 1 95% Pancreas 1 Up to 50% Esophageal 1 Up to 88% Breast 1 Up to 91% NSCLC 1 Up to 80% Ovary 1 Up to 70% Prostate 1 Up to 100% Glioblastoma 1 Up to 60%

12. Immunohistochemistry for EGFR Non small cell carcinoma of lung cancer 95% tumor cells are positive Two classes of EGFR inhibitors Anti-EGFR mAbs : cetuximab, panitumumab Small molecule inhibitors of tyrosine kinase activity (EGFR-TKIs): erlotinib , gefitinib

13. Erbitux ® ( cetuximab ) Erbitux ® ( cetuximab ) is an IgG1 MAb targeting the EGFR Binding blocks EGFR signaling inhibits Proliferation Angiogenesis Metastasis Stimulates apoptosis and differentiation The main toxicity is an acne-like rash that generally improves during treatment, and usually does not preclude continued treatment

14. Monoclonal antibodies for lymphomas

15. Immunocytology of Bone Marrow Smear Lymphoma cells-immunopositive with anti - CD20 Lymphoma Anti-CD20 for lymphomas

16. Hydrophobes Phosphoprotein 35kD From mature B cell and > 90% all malignant B cell Anti-CD-20-Antibody Murine variable region Human constant region Complement binding Antibody-dependent cell mediated cytotoxicity (ADCC) B-cells

17. Diagnosis of Hodgkin lymphoma and Anaplastic large cell lymphoma based on immunohistochemistry HL worldwide incidence is 65,950, with 8,500 new cases estimated to be diagnosed in the US in 2016 (males and females) HL incidence typically manifests as two peaks in both genders, either in early (20–24 years) or late (75–79 years) adulthood Hodgkin lymphoma (HL) and Anaplastic large cell lymphomas (ALCL) are neoplasms of the lymphatic system

18. HL: Morphological characteristics CHL is characterized by the presence of Reed-Sternberg (RS) cells 1 NLPHL lacks RS cells and is instead characterized by the presence of popcorn cells, also known as lymphocyte predominant (LP) cells 2 Expert haematological review should be considered standard of care to avoid misdiagnosis IHC for CD3, CD15, CD20, CD30 and CD45 is recommended 3 1. Glass C. Am Fam Physician. 2008;78:615–22 2. Lichtman MA, et al. Lichtman’s Atlas of Hematology . Access Medicine:McGraw-Hill;2007 CHL, classical Hodgkin lymphoma NLPHL, nodular lymphocyte-predominant Hodgkin lymphoma RS cells in lymphocyte-rich CHL 1 Popcorn cells in NLPHL 2 RS cells are p athognomonic for HL but d efinitive d iagnosis is d ifficult

19. CD30 signaling trigger cell proliferation and suppression of apoptosis in HRS cells HRS cells CD30 overexpression Activation of NF-  B and MAP kinases Deregulated gene expression Anti-apoptotic effect Cellular proliferation Deregulated CD30 gene expression CD30 self-activation CD30L binding ? ? A cell membrane protein that belongs to the tumor necrosis factor receptor (TNFR) superfamily Lymphocyte activation associated antigen

20. CD30 expression defines HL and ALCL CD30 expression is typically upregulated in: Classical Hodgkin lymphoma (CHL) 1-3 Anaplastic large cell lymphoma (ALCL) 3 All four CHL subtypes express CD30 3 In CHL, a small proportion of cells (the cancerous HRS cells) stain positively for CD30 3 (brown stained cells in Figure A) In ALCL, all cells stain positively for CD30 3 (Figure B) Figure A. In HL, HRS (tumor) cells are CD30 positive Figure B. In ALCL, all malignant cells express CD30

21. Brentuximab vedotin (ADCETRIS) Antibody drug conjugate (ADC) consisting of 3 components: Antibody cAC10 specific for human CD30 Microtubule-disrupting agent monomethyl auristatin E (MMAE) -cell death Protease-cleavable linker that covalently attaches MMAE to cAC10 Phase 1 studies completed in patients with relapsed/refractory CD30-positive malignancies including Hodgkin lymphoma (HL) and systemic Anaplastic large cell lymphoma ( sALCL ) Phase 2 data published in 2012: relapsed or refractory HL post ASCT and relapsed or refractory sALCL Granted accelerated approval by the US FDA in 2011

22. Immune check point blockade

23. PD-L1 immunostaining in lung cancer

24. Milestone of selected immune check point blockade of breast cancer

25. Increasing understanding of molecular carcinogenesis: change paradigms in oncology Characterize key mutations and molecular pathways responsible for tumor development and progression Identification of a large number of potential targets for diagnostic and therapeutic intervention New challenges for surgical pathology Surgical pathology has adapted a number of molecular methods applicable for formalin-fixed paraffin-embedded (FFPE) tissue: Immunohistochemistry (IHC) Fluorescence in situ hybridization (FISH) PCR and its multiple variants Sequencing (pyro/Sanger), next-generation sequencing, DNA-arrays, methylation analyses, and so on Pathologists (and molecular biologists) roles: Able to read a patients tissue as 'deeply' as possible and to obtain information on morphological, genetic, proteomic and epigenetic background Molecularly targeted therapy

26. Immunohistochemistry (IHC) Important application of monoclonal and polyclonal antibodies for the detection of specific antigens in tissue sections An extraordinarily powerful tool in the armamentarium of the diagnostic surgical pathologist and research (signaling pathways- targeted therapy ) Important roles in health and disease Diagnosis of cancers : specific tumor antigens are expressed de novo or up-regulated in certain cancers Basic histologic examination of tissue: a useful and necessary component

27. Common methods of protein detection and protein quantification Immunohistochemistry (IHC): semi quantitative ELISA Western blot Immunoprecipitation Spectrophotometry Enzyme assays Immunofluorescence X-ray crystallography Nuclear magnetic resonance (NMR) spectroscopy of proteins

28. Immunohistochemistry C ombination histological-immunological-biochemical techniques I dentification of specific tissue components D etects proteins (antigens) in cells within a tissue section ( specific antigen-antibody reaction) using antibodies tagged with a visible label Immunological localization of the protein of interest in its natural environment • Simultaneous evaluation of morphology and staining of the localized protein provide very complex information IHC can visualize the distribution and l ocalization of specific cellular components within a cell or tissue

29. Immunohistochemistry Various system and labelling for protein detection Peroxidase base Fluorescence microscope Confocal microscope Flow cytometry system Confocal microscope Flow cytometry system Fluorescence microscope : detect the presence and localization of fluorescent molecules in the sample Confocal microscope : specific fluorescent microscope to obtain 3D images of the sample with good resolution

30. Antigen - antibody reaction + visible label Targeted therapy CD20 - anti CD20 Anti CD20 (Rituximab) Applications example Cancer diagnostics Therapy

31. Cellular antigens as targets

32. Cellular antigens as targets: locations? Locations Nuclear Cytoplasmic Cell membrane

33. Tumor types; Benign / Malignant Tumor origin; epithelial / lymphoid / soft tissue Tumor classification Predicts prognosis Targeted therapy Immunohistochemistry (IHC)

34. Histopatologic procedure

35. I H C P R O C E D U R E

36. Pre-analytic phase

37. Pre-analytic

38. Fixation is very important! The dangerous of p roteolytic enzymes

39. Excision/biopsy Loss of blood supply Fixation maintains tissue morphology Fixation Process for preserving of tissue in solution to retain the “life-like” state Maintain antigenicity of target molecule Under fixation Protein damage on the tissue Reduce specific immunoreactivity Over fixation: Close the epitope Damage antigenicity of targeted protein Tissue Lysis P roteol i ti c e nz ymes P rotein Nucleic acid (DNA d an RNA) Proteases Nucleases Lipases Saccharidases Fixation Diagnosis

40. Tissue lamellation Ideal thickness: 3 mm, no more than 4 mm (excellent fixation and processing) Medawar d = K√t , d: depth of penetration, K: coefficient of diffusion (specific for each fixative), 10% formalin K = 0.78 Need 25 hours to fix a 10 mm specimens Geoffrey Rolls, Leica Biosystems

41. Lamellation of Tissue Important step to prevent decay of tissue Ensure all tissue parts are perfectly fixed Lamellation thickness: 3 - 4 mm Ensure fixative enter and distribute to all tissue parts Sharp knife/cutter with parallel of section Prevent from structural damage of tissue and lesion inside the tissue (difficulty in analysis) Discontinuity in one side (orientation)

42. Lamellation

43. Lamellation of tumor parallel gut axis Give signs in the distal and proximal ends Open one side of gut Gut is opened in one side Lamellation

44. Wrong containers Preparation

45. „Cold ischemic time“ Delay to formalin fixation The time from the tissue is removed from the body to when the tissue is placed into fixative should be as short as possible ( one hour or less ) American Society of Clinical Oncology-College of American Pathologists (ASCO – CAP) The deleterious effects of delayed fixation 0 hour 1 hour 4 hours 2 hours HER2 Tissue excision/biopsy Fixation

46. Fixation standard procedure Neutral Buffered Formalin 10% pH 6.8 - 7.2 All tissue parts are immersed in fixative Volume of formalin: 10-20 X vol of tissue (Ratio 1: 10-20) Tissue 1x1x0,5 = 0,5 cm 3 = 0,5 ml, fixation volume = 5 – 10 ml Lamellation thickness is 3 – 4 mm Ensure fixative enters and distributes to all tissue Duration of fixation: 24 – 72 hours Identity of tissue Identity of tissue

47. Sub optimal fixation

48. Inadequate fixation impacts on tissue

49. Small intestine Correct fixation-formalin buffer 10% Wrong fixation – ethanol 95% Effects of short/under-fixation

50. Effects of short/under-fixation Well-fixed tissue Good nuclear and cytoplasmic morphology Minimal shrinkage showing clearly defined basement membranes and cell margins. Poorly-fixed tissue Inferior nuclear and cytoplasmic morphology Excessive shrinkage Poorly defined cell margins. Vacuolation and fragmentation of both nucleus and cytoplasm of cells of the distal tubule Retraction of the glomerulus due to shrinkage HE stained HE stained

51. Effects of short/under-fixation Well-fixed tissue in the peripheral part of lymphoma case Good nuclear and cytoplasmic morphology Minimal shrinkage showing clearly defined basement membranes and cell margins Poorly-fixed tissue in the centre of lymphoma case Inferior nuclear and cytoplasmic morphology Excessive shrinkage Poorly defined cell margins HE stained HE stained

52. Effects of short/under-fixation center peripher center A B HE stained IHC stained Tissue folding Tearing and losing of staining intensity in the center

53. Imunohistokimia A. Inadequate Estrogen receptor (ER)  of breast c arcinoma B. Correct fixation

54. IHC Inadequate fixation In tissue central Ki-67 / lymphoma Fixed pheripheral tissue

55. Uneven fixation (zonal fixation) has resulted in uneven staining in this section (breast tumor, ER) Not-o ptimal f ixation center pheripher

56. Effects of prolonged/over-fixation Tissue was fixed for 24 hours Tissue was fixed for 11 weeks

57. 48 hours fixation Anti-Plasma Urokinase Inhibitor 7 days fixation Effects of prolonged/Over-fixation

58. Analytic phase

59. General Immunohistochemistry Procedures

60. Formalin- f ixed p araffin- e mbedded (FFPE) and t issue a nalysis Hematoxylin-Eosin (HE) Imunohistochemistry/ Immunofluoresence DNA - RNA Isolation MicroRNA extraction PCR RT-PCR In situ hybridization Fluorescence Chromogenic Sequencing Whole-genome (WGS) Next Generation (NGS) M O L E C U L A R Paraffine block

61. Deparaffinization and Rehydration Antigen Retrieval Frozen section does not need this step

62. Blocking of Endogenous peroxidase Primary antibody Hydrogen Peroxide 3% in TBST Blocking – 20 min Rinse with Buffer Wash in Buffer x 3 Wash in Buffer x 3

63. Secondary antibody/Envision Rinse in DW Wash in Buffer x 3

64. Diaminobenzidine/DAB Rinse in DW Wash in distilled water

65. Background staining Rinse in tap water X 3 (5 min each) Coverslip with aqeous medium

66. Procedure notes Throughout the procedure, keep the sections wet in order to prevent destroying epitopes on the cell membrane Drying also causes nonspecific background staining Paraffin can mask epitopes from the primary antibody a complete deparaffinization is important DAB has carcinogenic properties -

67. Analytic Pre-treatment Proteolysis Sectioning Antigen Retrieval Methods Blockings Controls Validation Platform Manual Automated Primary antibody Antibody selection Antibody clone Dilution Buffer Time Temperature Counterstain Time Color Visualization system Sensitivity, specificity Enhancement Chromogen Sensitivity Localization

68. Monoclonal vs. 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 Varies avidity/affinity batch-to-batch More likely to have success in an unknown application Primary antibody selection

69. Antigen retrieval After fixation, the epitopes in tissue can be cross-linked and covered that makes it difficult for antibody-binding I n many cases the fixation and processing steps involved in the preparation of tissue results in loss of antigen immunoreactivity Several methods/techniques Microwave antigen retrieval: protease-induced antigen retrieval (PIER) Proteolytic digestion: trypsin A number of buffers may be used for this purpose The precise choice is usually recommended on product specific datasheets, or determined experimentally

70. Complex Antigen-Antibody is not visible with standard microscopy and must be labeled but their binding specificity is not interfered Common labels include fluorochromes ( eg , fluorescein, rhodamine ), enzymes demonstrable via enzyme histochemical techniques ( eg , peroxidase, alkaline phosphatase), and electron-scattering compounds for use in electron microscopy ( eg , ferritin, colloidal gold) Labeling antibodies

71. Direct immunoenzyme method The second layer antibody may be labelled with an enzyme such as peroxidase, alkaline phosphatase or glucose oxidase Methods of labelling Indirect immunofluorescence method The second layer antibody can be labelled with a fluorescent dye: FITC, rhodamine or Texas red Systems of labelling PAP Method (peroxidase anti-peroxidase method Avidin-Biotin Complex (ABC) Method Labeled Strept -Avidin Biotin (LSAB) Method

72. Ensure correct application of antibody Immunofluorescence (IF) Immunohistochemistry (IHC ) Western Blott (WB) ELISA Immunoprecipitate (IP) etc. Antibody is validated for ??? Check the data sheet

73. Blocking Non-specific staining Before block After block

74. High background staining Background staining No background staining

75. Positive Control A positive result from the positive control, even if the samples are negative, will indicate the procedure is optimized and working Verify that any negative results are valid Ideally use the tissue of known positivity Negative Control Ideally use tissue of known not to express the protein you are detecting Check for non specific binding and false positive results Controls Antigen controls Reagent control ....negative control Ensures staining is produced from primary antibody, staining the antigen and not from detection system or the specimen Using the detection system with diluent alone and no primary antibody

76. Post-analytic phase

77. Control Internal/external Critical stain quality indicators Interpretation Positive/negative criteria Localization Cut-off point Panels, algorithms Quantification-software Post-analytic Reporting Diagnostic context Research Data analysis

78. Results Negative control Positive in Nuclei & Cytoplasm Positive in Cytoplasm Positive in Membrane Cell Positive in Nuclei

79. Analysis-quantification

80. Standardization in IHC National Cancer Institute workshop (1977) Biological Stain Commission workshop FDA workshop NCCLS Guidelines (1999) EORTC Workgroup (Dr. Hammond) NordiQC Immunohistochemical quality control

81. IHC and procedure Important of correct procedure of pre-analytic, analytic and post-analytic phases Appropriate and correct protein expression

82. IHC and preparation Preparation is important before taking samples Adequate fixation is critical Good fixation, good paraffin block and sectioned can give great data Bad fixation, bad paraffin block and sectioned, can give misleading data and waste money Molecular methods applicable for formalin-fixed paraffin-embedded (FFPE) tissue: Immunohistochemistry (IHC) Fluorescence in situ hybridization (FISH) PCR and its multiple variants Sequencing (pyro/Sanger), next-generation sequencing DNA-arrays, methylation analyses, and so on

83. Take Home Message Accurate diagnosis helps patient therapy Specimen handling has important roles in ensuring the accurate diagnosis Optimal process of pre-analytical phase Support the molecular examination to be run optimally Support the accuracy of diagnosis and therapy Quality Research Begins with Quality FFPE

84. Thank you