TEST BANK For Lewis's Medical Surgical Nursing in Canada, 4th Edition by Jane...
IHC in cancer diagnosis
1. Illustration by Smart-Servier Medical Art
PRESENTER- DR ZUBIYA SUHA (2ND YEAR PG)
MODERATOR- DR SUPREETHA MS- ASSOCIATE
PROFESSOR
DR SNEHA K – ASSISTANT PROFESSOR
DEPT OF PATHOLOGY
IHC IN CANCER
DIAGNOSIS AND
MANAGEMENT
2. ● PURPOSE OF IHC
● INTRODUCTION
● BASIC PRINCIPLE
● PROCEDURE
● COLD ISCHEMIC TIME
● APPLICATION
● CLASSIFICATION IHC
● SUMMARY-PANELS
● TAKE HOME MESSAGE
OBJECTIVES
3. ● The Immunohistochemistry test is used to diagnose cancer by detecting a
specific type of antibody attached to the antigen on the cell surface.
● The purpose of the special staining technique is to detect molecules which
are usually markers specifying whether the cancer is benign or malignant.
● IHC meaning and its application are predominantly found in diagnosing
cancer
Purpose of The IHC Test
4. ● IHC technique was successfully introduced in
routine formalin fixed paraffin-embedded (FFPE)
section by Taylor and Burns in 1974 .
● Subsequently the development of monoclonal
antibody introduced a new era in the
immunohistochemistry .
● However, it took another 10–15 years to have regular
routine use of IHC in pathology diagnostic
laboratory.
● Presently IHC is an essential technique in every
pathology laboratory
INTRODUCTION
5. Basic Principles
● The principle of IHC is to localize antigens in
tissue sections by the use of labeled antibodies as
specific reagents through antigen-antibody
interactions that are visualized by a marker such as
fluorescent dye, enzyme, radioactive element or
colloid gel
6. IHC PROCEDURE
Masked epitopes can be recovered using
either enzymatic/proteolytic antigen retrieval, or heat-
induced antigen retrieval methods.
ANTIGEN RETRIEVAL
PRIMARY ANTIBODY
SECONDARY
ANTIBODY
CROMOGEN
Based on Direct on Indirect method
To add colour
7. ● One of the most important preanalytical variables starts in the operating room (OR) and is
known as cold ischemia time—the period that elapses between removal of the tissue from
the body by the surgeon and the fixing or freezing of the tissue (processes known as
tissue stabilization) by the pathologist.
COLD ISCHEMIC TIME
8. ● During this time, the tissue is still viable and experiences major biological stress in
the form of
anoxia, nutrient deprivation
temperature change
desiccation with subsequent changes in gene expression, protein translation and
modification, and molecular degradation.
COLD ISCHEMIC TIME
9. ● Stabilization halts further biological activity or molecular degradation and represents
a critical and time-sensitive step in tissue processing.
● Should always be as short as possible, but a maximum of one hour would be a
reasonable goal
● At present, the only enforced cold ischemia time in pathology practice comes from the
American Society of Clinical Oncology/College of American Pathologists (ASCO-
CAP) human epidermal growth factor receptor 2 (HER2) testing in breast cancer
guidelines with a strong recommendation of time to fixative within one hour
COLD ISCHEMIC TIME
10. ● An example of a non-
refrigerated case. The
tumor was strongly
positive for estrogen
receptor at 0.5 h of
delayed fixation
● but demonstrated
significant reduction at 3 h
11. CHALLENGES - FIXATION
OVERFIXATION
1.Tissue Hardening: Prolonged exposure to
formalin can lead to excessive cross-linking
of proteins, resulting in tissue hardening.
This can make it challenging to cut thin
sections for microscopic analysis.
2.Masking of Antigens: Overfixation may
mask or alter the antigenic sites, making it
difficult to detect specific proteins during
immunohistochemistry (IHC) studies.
3.Nucleic Acid Degradation
4.Altered Morphology
UNDERFIXATION
1.Incomplete Preservation: incomplete
preservation of tissues
2.Autolysis: more prone to autolysis
3.Loss of Antigens: Inadequate fixation may
result in poor retention of antigens,
impacting the success of
immunohistochemical studies.
4.Inadequate Cross-Linking: affecting the
stability of tissue structures.
12. Sample of Tissues for Immunocytochemistry
Formalin-fixed paraffin-embedded tissue Frozen section Cytology
13. Sample of Tissues for Immunocytochemistry
Cell block tissue
Smear from liquid-based
cytology/imprint smear
14. • Diagnosis should be based on H&E morphology, with confirmation by
immunohistochemistry or molecular testing
• A stain / result is not just positive or negative; focus on the types of cells that are
immunoreactive and determine if they are tumor cells, inflammatory cells, normal cells or
stromal cells; comparing the results to an H&E stained section or a negative control of the
same block should be done
• After you identify the type of cell staining, it is helpful to note the percentage of these cells
staining, the intensity of staining (weak, 1+, 2+, 3+, 4+) and the pattern of staining
(membranous, cytoplasmic, nuclear, dot-like)
IHC
CAP Laboratory Improvement Programs: Principles of Analytic Validation of Immunohistochemical Assays
(Am J Surg Pathol 2007;31:1627, J Clin Pathol 2011;64:466)
15. IHC
Nuclear staining pattern: Transcription factors and
steroid hormone receptors.
Cytoplasmic staining pattern: vimentin, actin,
desmin, and cytokeratins.
16. IHC
Membrane staining patterntypical examples are the
majority of CD antigens.
Extracellular staining pattern: this pattern is
characteristic for extracellular and tissue matrix
antigens in addition to the cell secretion products such
as collagens and CEA.
17. Multicolor Detection:
Immunofluorescence allows for the simultaneous detection of multiple antigens labeled with different
fluorophores. This is particularly useful for studying complex biological processes involving multiple proteins
or cellular structures.
IHC vs IMF
18. Applications of Immunohistochemistry
(IHC) in Anatomic Pathology
(a) classification of poorly differentiated neoplasm:
-carcinoma(cytokeratin)
-Lymphoma (CD45+)
-Melanoma (SOX10/S100/Melan A/HMB-45)
1- Diagnosis of tumors
Cytokeratin 7
HMB-45
19. Applications of Immunohistochemistry
(IHC) in Anatomic Pathology
1- Diagnosis of tumors
(b) Diagnosis of carcinoma of unknown primary:
- Lung (TTF-1,Napsin A)
- Thyroid (PAX 8/TTF-1)
- Prostate (PSA/NKX3.1)
- Colon (CDX2)
Lung (TTF-1)
20. Applications of Immunohistochemistry
(IHC) in Anatomic Pathology
1- Diagnosis of tumors
(c) Diagnosis of invasion:
- Loss of myoepithelial cells (breast cancer)
- Loss of basal cells(prostate cancer)
- S100-Colon cancer invasion
21. Applications of Immunohistochemistry
(IHC) in Anatomic Pathology
- Ki67/MIB1 (general proliferation
marker)
- HER2 (adverse prognosis in breast and
gastric cancer)-also a predictive marker
2- Assessment of markers reflection
prognosis-Prognostic marker
22. Applications of Immunohistochemistry
(IHC) in Anatomic Pathology
3- Assessment of markers reflecting a
Therapeutic Response( “Predictive” or
“Theranostic Marker”)
- ER/PR (Tamoxifen in breast cancer)
-HER2 (Herceptin for breast and gastric
cancer)-Trastuzumab
- ALK- crizotinib, ceritinib
- PD-L1 (NSCLC)-pembrolizumab
23. Applications of Immunohistochemistry
(IHC) in Anatomic Pathology
- Melanoma
- Breast cancer (Cytokeratin)
- Viruses (HSV, CMV.Adenovirus, SV40
for polyomavirus)
- Bacteria H,(Pylori, anti-treponemal
antibody for syphilis)
- Toxoplasma
4- Detection of Micrometastases:
5-Identification of Infective Organism:
24. IMMUNOMARKERS CAN BE
BROADLY CLASSIFIED
INTERMEDITAE FILAMENT MARKERS
EPITHELIAL RELATED MARKERS
GERM CELL TUMOR MARKERS
NEUROENDOCRINE MARKERS
MESENCHYMAL MARKERS
BASEMENT MEMBRANE MARKERS
HORMONE RECEPTORS
CELL PROLIFEARTION MARKERS
25. ● Cytokeratins are the most important markers used for the diagnosis of epithelial neoplasms.
● Cytokeratins are intermediate filament proteins building an intracytoplasmic network between
the nucleus and cell membrane of epithelial cells.
● AE1/AE3 - First line epithelial marker in screening of CA.
• HCC is AE1/AE3– (Cam5.2+; CK903–, EMA–).
• RCC is variably reactive with both AE1/AE3 and Cam5.2 (EMA is best).
• NE CAs, including SmCC, show variable reactivity with both AE1/AE3 and Cam5.2.
MARKERS FOR EPITHELIAL DIFFERENTIATION
27. MARKERS FOR EPITHELIAL DIFF
EMA-EMA is used in conjunction with CKs as
a “CK helper”.
• DDx of RCC (EMA+) vs. adrenocortical
neoplasms (EMA–)
• ID of meningioma and a few other EMA+
non-epithelial neoplasm
28. MARKERS FOR EPITHELIAL DIFF
MARKERS FOR EPITHELIAL DIFF
Ber-EP4 and MOC31 are primarily used to differentiate lung adenoCA (Ber-
EP4+ and MOC31+) from mesothelioma (Ber-EP4– and MOC31–)
29. ● Smooth muscle differentiation- Myogenin and
myoD1
● Myofibroblasts- desmin –ve SMA +ve
MARKERS FOR MUSCLE DIFF
Skeletal muscle differentiation- Desmin and Smooth
muscle actinin
31. ● HMB-45 (+ve in melanoma, pecoma, -ve in nevi, resting melanocyte)
● Melan A (+ve in nevi, resting melanocyte and desmoplastic melanoma)
● MIFT
● Thyrosinase
● S-100 (high sn for melanoma)
MARKERS FOR MELANOTIC DIFF
32. ● The classic list of “NE markers” includes synaptophysin (SYN), chromogranin A
CD57 (Leu-7) and neuron specific enolase (NSE) are second-line NE markers, and are
rarely used in practice.
● CD99
● CD56
● NB-84 (neuroblastoma)
NEUROECTODERM AND
NEUROENDOCRINE MARKERS
33. MARKERS FOR VASCULAR DIFF
CD31 immunostain of cutaneous angiosarcoma showing strong
vascular marker expression
CD34 immunostain of cutaneous angiosarcoma showing strong
vascular marker expression
41. PANEL FOR CNS
Astrocytic neoplasm GFAP+, AE1/3CK
cocktail
Oligodendroglioma S100, Leu7+
Ependymoma GFAP, S100, Vimentin+
Meningioma EMA, S100/+, CK20-
42. TAKE HOME MESSAGE
Immunohistochemistry (IHC) is a potent method for visualizing protein
distribution in tissues, providing key insights for disease understanding and
targeted therapy development.
As a vital ancillary technique in pathology and molecular biology, IHC aids
clinicians in disease diagnosis, prognosis, treatment guidance, and patient
response monitoring, enhancing overall healthcare
Immunohistochemistry (IHC) is an important application of monoclonal as well as polyclonal antibodies to determine the tissue distribution of an antigen of interest in health and disease.
IHC is widely used for diagnosis of cancers; specific tumor antigens are expressed de novo or up-regulated in certain cancers
Schematic representation of direct (A) and indirect (B) IHC detection. S: Chromogenic substrate. P: Colored insoluble product. Reporter enzyme is indicated by the red star.
In the direct method, the primary antibody is labeled and applied to the tissue in a quick one step process; however, this method is not commonly used due to lack of signal amplification and thus the requirement for a higher concentration of antibody as well as the need to label each primary antibody. In the indirect method, the secondary antibody is labeled, allowing for signal amplification and use with many different primary antibodies.
Antigen retrieval enables an antibody to access the target protein within the tissue
Block with sera or a protein to prevent non-specific antibody binding and reduce background and potentially false positive results. Antigen Retrieval: Tissues may undergo antigen retrieval, a process to enhance the exposure of target antigens. This can involve heat-induced or enzyme-induced methods.
Blocking: Non-specific binding sites on the tissue are blocked to prevent false-positive results.
Primary Antibody Incubation: A primary antibody, specific to the target protein, is applied to the tissue sections. This antibody binds to the target antigen.
Washing: Excess primary antibody is washed away to reduce background staining.
Secondary Antibody Incubation: A secondary antibody, conjugated with a detection molecule (e.g., enzyme or fluorophore), is applied. This binds to the primary antibody.
Washing: Excess secondary antibody is washed away.
Detection: If an enzyme is used in the secondary antibody, a substrate is applied, leading to a visible color change. If a fluorophore is used, the sample is examined under a fluorescent microscope.
Counterstaining (optional): Additional dyes may be applied to provide contrast and aid in visualizing cell structures.
Cold ischemia time affects different classes of biomolecules in different ways, depending on the relative lability or stability of the molecular entity.
that is both data-driven and practicable in the clinical setting.
Nucleic Acid Degradation: Overfixation can degrade nucleic acids (DNA and RNA), affecting the quality of molecular studies such as PCR and in situ hybridization.
Altered Morphology: Cellular and tissue morphology can be distorted, making accurate histopathological interpretation more challenging.
UNDERFIXATION
Incomplete Preservation: Insufficient formalin exposure may lead to incomplete preservation of tissues, compromising the structural integrity of cells and tissues.
Autolysis: Underfixed tissues are more prone to autolysis, which is the natural degradation of tissues by their own enzymes. This can lead to distorted cellular structures and inaccurate pathological interpretation.
Loss of Antigens: Inadequate fixation may result in poor retention of antigens, impacting the success of immunohistochemical studies.
Inadequate Cross-Linking: Underfixation can result in inadequate cross-linking of proteins, affecting the stability of tissue structures.
Formalin-fixed paraffin-embedded (FFPE) tissue:
- This is the most popular sample for IHC. Presently most of the antibodies work on FFPE tissue section
- Cell Block The cell block from the cytology material is preferable in case of cytology sample. • Any specimen can be used for cell block except the specimen with very low cellularity. Advantages of cell block: 1. Multiple sections available. 2. The tissue can be further used for archival use. 3. Minimum background staining
Immunohistochemistry (IHC) is a tool for surgical pathology and research
The pattern of immunoreactivity should follow the anatomic distribution of the antigen before it is called positive / immunoreactive
Some antigens display a further restricted cytoplasmic staining pattern and stain-specific organelles, as, e.g., mitochondria (leading to a granular cytoplasmic staining) or the Golgi apparatus (unilateral perinuclear pattern).
Type IV collagen and laminin immunohistochemical staining of patients with end-stage heart failure pre- and post-LVAD support
characteristic for antigens located in the cytoplasm.
: characteristic for antigens located within the cell membrane,
Immunohistochemical staining was performed on serial sections of ovarian carcinoma using PD-L1 (E1L3N®) XP® Rabbit mAb #13684 (left), B7-H4 (D1M8I) XP® Rabbit mAb #14572 (middle), and Pan Keratin (C11) Mouse mAb #4545 to evaluate the pattern and level of expression of these two immune checkpoint proteins using fluorescent and chromogenic detection systems.
CD-Cluster of differentiation
HMB- Hydroxymethylbutarate
TTF1- Thyroid transcription factor
Human ovarian carcinoma stained with anti-Keratin 7 antibody using peroxidase-conjugate and DAB chromogen. Note the cytoplasmic staining of tumor cells.
Invasive cutaneous melanoma: strong cytoplasmic positivity on HMB45 stain
ALK- Anaplastic lymphoma kinase
HER2 (Human Epidermal Growth Factor Receptor 2):
Diagnostic Role: Indicates overexpression/amplification of HER2.
Treatment: Trastuzumab (Herceptin) and other HER2-targeted therapies are used in breast cancer cases where HER2 is overexpressed.
ER (Estrogen Receptor) and PR (Progesterone Receptor):
Diagnostic Role: Indicates hormone receptor status in breast cancer.
Treatment: Hormone therapy, such as tamoxifen or aromatase inhibitors, may be used in hormone receptor-positive cases.
ALK (Anaplastic Lymphoma Kinase):
Diagnostic Role: Indicates ALK rearrangement, often found in non-small cell lung cancer (NSCLC).
Treatment: ALK inhibitors like crizotinib, ceritinib, and alectinib are used in ALK-positive NSCLC.
PD-L1 (Programmed Death-Ligand 1):
Diagnostic Role: Indicates PD-L1 expression in tumor cells.
Treatment: Checkpoint inhibitors, such as pembrolizumab and nivolumab, target PD-1/PD-L1 and are used in various cancers with PD-L1 expression.
The diagnostic algorithm for workup of undifferentiated neoplasms. Abbreviations: CK, cytokeratin; ER, estrogen receptor; GATA3, GATA binding protein 3; LCA, leukocyte common antigen; TTF1, thyroid transcription factor 1.
Intermediate- Vimentin , desmin
Epithelial- EMA, AE1/AE3
Germ cell tumor markers-oct 4, plap, sall4 glypican 3
Neuroend-synp,chromo
Basement memb- ck 903, p63
Mesenchymal= myogenin, desmin
Cytokeratins are a complex family composed of more than 20 isotypes and divided into 2 types
• Type I (acidic group) including cytokeratins 9–20
• Type II (basic group) including cytokeratins 1–8
EMA- Epithelial membrane antigen
MC panel used for primary of unknown origin
In particular, it is helpful in identifying RCC, which is EMA+, but is variably reactive for CKs. Other uses include:
e clear cells are strongly positive with EMA immunostaining.
P63 {squamous and urothelial}
BerEP4 {T diff lung adenocarcinoma from mesothelioma}
Myogenic is specific for rhabdomyosarcoma
S-100 protein (Langerhans and Schwan cells)
Claudin-1 (positive in perineural cells and perinuroma)
GLUT-1 (Perineural cells)
CD57 (NK cells, oligodendroglial cells and schwann cells)
INSM-Insulinoma associated protein 1
OLIG2- Oligodendrocyte transcription factor 2
MITF- Micropthalmic transcription factor
Synaptophysin immunomarker shows positive cytoplasmic staining in the Merkel cells
SYNAPTOPHYSIN and CHROMOGRIN A
NSE
CK
), and CD56 (NCAM or neural cell adhesion molecule).
Abbreviations: CHR chromogranin A, CK cytokeratin, SYN synaptophysin, NE neuroendocrine, NET neuroendocrine tumor (arcinoid equivalent in pancreas and GI tract), NSEQHXURQVSHFL¿FHQRODVHINSM1
Angiosarcoma has a wide morphologic appearance, ranging from lesions that are cytologically bland and vasoformative, to solid sheets of highly pleomorphic cells without definitive vasoformation
Numerous irregularly shaped anastomosing vascular channels lined by atypical endothelial cells with a highly infiltrative architecture and poor demarcation
Angiosarcoma expresses endothelial cell markers, including CD31, CD34, ERG, FLI1, VEGF and factor VIII
Cytokeratin, EMA and CD30 may be expressed in a subset of epithelioid angiosarcoma
Lymphatic marker podoplanin as determined by D2-40 immunostain is variably expressed and suggests focal lymphatic differentiation in the tumo
SMMHC-Smooth muscle myosin heavy chain
AMACR- Alpha methylacyl CoA racemase
GCCDFP- Gross cyctic disease fluid protein-marker of apocrine eoithelium
HER2- Human epidermal growth factior
ACTH- Adrenocortico hormone
DOG1- Discovered on GIST
Prostate- 34BE12_(D/D benign lesion), P504S+
CEA- chorioembryonic carcinoma
Ovarian carcinoma-, CA 125+
