Immunohistochemistry (IHC) combines histological and immunological techniques to identify specific tissue components using antigen-antibody reactions tagged with visible labels. IHC allows visualization of the distribution and localization of cellular components. Antibodies bind specifically to antigens, providing spatial location of particular cells and proteins. Important considerations for IHC include antibody selection, fixation, antigen retrieval, controls, and detection methods such as direct, indirect, and enzyme-linked methods. IHC is useful for tumor diagnosis, classification, predictive markers, and distinguishing between benign and malignant lesions.

1. Immunohistochemistry in
Oncology
Dr D. Ramu

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. • 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. 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. What cellular antigens can we
target?
• Cytoplasmic
• Nuclear
• Cell membrane
• Lipids
• Proteins

6. Important considerations for IHC
• Antibody selection
• Fixation
• Sectioning
• Antigen Retrieval
• Blocking
• Controls
• Direct method
• Indirect method
• Immunoenzyme
• Fluorescence
• Multiple labeling

7. General antibody structure

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. 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. 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. 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. 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. 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. 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. Direct method-
primary antibody only
Goat anti-actin labeled with
594

16. Indirect method – primary and
secondary antibodies
Goat anti-actin
Donkey anti-goat
labeled with 488

17. Enzyme linkage indirect method
Goat anti-actin
Flourochrome (488)
conjugated
streptavidin
Biotinylated
donkey anti-
goat

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. 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

21. 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

23. 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%)

24. 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

25. 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

26. 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

27. 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

28. 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

29. 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

30. 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

31. Primary and Additional Markers
For teaching purpose only
For teaching purpose only

32. Tumors from Unknown Primary Site
Metastatic AdenoCarcinoma from Unknown Primary
For teaching purpose only

33. CUP Diagnosis
(Cancer of Unknown Primary)
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2631214/figure/f1-gcr1_6p0229/
For teaching purpose only

34. 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

35. 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

36. 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

37. 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