IHC in cancer diagnosis and managment in brief

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

26. MOST COMMON PANEL FOR METASTASIS OF UNKNWON ORIGIN

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

30. MARKERS FOR NERVE SHEATH DIFF

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

34. Surgery Department

35. HER-
PANEL FOR BREAST
Primary GCCDFP, Mammoglobin,
GATA3
Lobular Carcinoma E Cadherin, P120
Spindle cell tumor Bcl2, CD34, P63, CK, B catenin
Prognosis ER, PR, Her2, EGFR, P53, PDL1
Proliferation Ki67

36. Colorectal carcinoma CK7-, CK20+
Gastric carcinoma Keratin, EMA, CEA+,
CK7+,CK20+
PANEL FOR GIT
GIST CD117+ (100%),
DOG1
Carcinoid tumor ACTH, NSE,
Chromogranin+,
Synaptophysin +, keratin +

37. PANEL FOR MGT
Prosate carcinoma PAP snd PSA+,
Seminoma PLAP, CD117,LDH,
Vimentin+, EMA, CD30,
HMWK+
Embroyonal carcinoma EMA, CD30, HMWK+
Choriocarcinoma hCG+

38. PANEL FOR FGT
Adenocarcinoma Keratin, EMA, CEA,
HPV+
Endometrial
carcinoma
ER,PR+, Her2neu,
GLUT 1 +
Ovarian carcinoma CK7+, CK20-
Mesothelioma Calretinin,
thrombomodulin-,
CK 5/6+
Cervix P63, p40, p16

39. PANEL FOR HEPATOBILIARY
Hepatocellular carcinoma AFP, Keratin, EMA+,
Hep-par1+, CEA -
Cholangiocarcinoma EMA, CEA, Keratin +
GB carcinoma CK7+, CK20+(d/d for
cholangiocarcinoma)
Pancreatic carcinoma Keratin, EMA, CEA,
CD19-9+

40. PANEL FOR THYROID
Papillary carcinoma CK7+, CK20-. CK19+,
HMWK+
Follicular carcinoma Thyroglobulin , TTF1+,
LMWK, EMA+
Medullary carcinoma Calcitonin, CEA+,
Chromoganin+

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

43. THANK YOU 