role of immunohistochemistry in head and neck pathology

1. IN Head and neck pathology
ROLE OF IHC

2. CONTENTS
• INTRODUCTION
• HISTORY
• PRINCIPLE
• APPLICATION
• MARKERS
• LESIONS
• REFERENCES

3. INTRODUCTION
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.
• Widely used for diagnosis of cancers; specific tumor antigens are expressed de novo or
up-regulated in certain cancers.

4. • Plays an important role in pathology, particularly in the subspecialties of oncologic pathology,
neuropathology, and hematopathology.
• In autopsy pathology while basic histologic examination of tissue is considered a useful and necessary
component IHC may provide a greater insight.

5. • IHC requires the availability of biopsies; these are processed into sections with a microtome and then the
sections are incubated with an appropriate antibody.
• Ordinary or fluorescent microscope by a marker such as fluorescent dye, enzyme, radioactive element, or
colloidal gold, which is directly linked to the primary antibody or to an appropriate secondary antibody.

6. PRINCIPLE..
• The principle of IHC has existed since the 1930s, but it was not until 1941 that
the first IHC study was reported.
• The aim of IHC is to perform most IHC staining by causing least damage on the
cell or tissue, and by using least amount of antibody, it finds a way in the tumor
typing and tumor markers.

7. • IHC involves specific antigen–antibody reactions, it has apparent advantage over traditionally used
special enzyme staining techniques that identify only a limited number of proteins, enzymes, and
tissue structures.
• Become a crucial technique and is widely used in many medical research laboratories as well as
clinical diagnostics.

8. Prognostic markers of cancer…
 Analysis of tumors by these methods is a significant improvement over the
conventional prognostic considerations by clinical staging and histologic
grading.
 Physicians use IHC to diagnose a cancer as benign or malignant.
 Identify the cell type and origin of a metastasis to find the site of the primary
tumor.
 IHC is also used in drug development to test drug efficacy by detecting either the
activity or the up- or down-regulation of disease targets.

9. Tumors Of Uncertain Histogenesis
• Brought about a revolution in approach to diagnosis of tumors of uncertain origin, primary as well as
metastatic from unknown primary tumor.
• The selection of antibodies being made is based on clinical history, morphological features, and results of
other relevant investigations.

10. IHC - HEAD AND NECK PATHOLOGY
• Immunohistochemistry is important in diagnosis, investigation, and determining the
behavior and pathogenesis of oral tumors.
• Application of immunohistochemistry in distinguishing undifferentiated oral
neoplasms of different origins was achieved through the detection of tumor antigens
using known antibodies.

11. MARKERS
Working Classification of Antibodies Used In Immunohistochemistry
1. Squamous epithelial
 Pancytokeratin.
Vimentin
 Carcino embryonic antigen.(CEA)
 Epithelial membrane antigen.(EMA)
4. Melanocytes
S-100
 Melan –A
 Mart -1
 HMB – 45
 Tyrosinase
2. Odontogenic epithelium
Cytokeratin (ck) -5,13,14,19
Basal layer and intermediate layer - ck 5, 14, 19
Superficial cell layer - ck4,13
 Enamelin
 Ameloginin
 Laminin
3. Glandular epithelium
 S-100
 Actin
 Calponin- myoepithelial cells
 CK 7
 Lysozyme.
 Estrogen receptors.
 Progesterone receptors.
 CK -14
 EMA- ductal cells
 P63- myoepithelial cells

12. Muscle Markers
Vascular Markers
Smooth Muscle Marker
Desmin
 Smooth muscle actin (SMA)
 Calponin
 Caldesmon
Skeletal Muscle Marker
Myogenin (Myf- 4, Myf- 5)
 Myo D1
CD 31(Cluster of differentiation)
CD 34
CD105/ Endoglin
VEGF
Von willebrand factor (VIII)
Ulex europaeus
Podoplanin
Podocalynin

13. Neural Markers Neuro-Ectodermal Markers
S-100
Neuron specific enolase (NSE)
CD57
p75 NTR
CD99
CD56
Neuro filament protein (NFP)
Synaptophysin
Chromogranin
Claudin – 1
Glut – 1.
CD99
CD56/ NCAM - neural cell adhesion molecule
NB84
Histiocyte/Dendritic Cell Markers
Lysozyme
CD68
CD21
Clusterin,

14. Lymphoid Markers
Bone Markers
Germ Cell Tumor Markers
T cell markers
CD 2
CD3
CD30
CD99
CD43
CD45 RO
TDT
B cell markers
CD 20
CD75 a
CD26
Pax5
RANKL
CD99
Osteocalcin
Osteonectin
Alkaline phosphatase.
Osteoclast trap.
Osteoprotegrin Alpha feto protein(AFP)
Human chorionic gonadotropin.
(HCG) – In pregnancy.
Germ cell tumor.
Choriocarcinoma
Islet cell tumor.
CA125(Cancer Antigen)

15. Metastatic Epithelial
Markers
Spindle cell tumor
Markers
Pleomorphic cell tumor
Markers
CK 7 and 20 – Low molecular
weight cytokeratin
Presence for metastatic lung
and rectal adenocarcinoma.
Villin - Actin binding protein.
 Smooth muscle actin
 Desmin
 S100
 Cytokeratins
 CD34
 EMA
CK
S100
Actin
Desmin
CD30

16. Tumor markers can be broadly classified as…
• Oncofetal antigens (e.g. alpha-fetoprotein (AFP), Carcinoembryonic antigen (CEA), Pancreatic
oncofetal antigen, fetal sulfoglycoprotein.
• Tumor associated antigens /Cancer Antigens e.g.CA125, CA19-9, CA15-3, CA72-4 CA50 etc.
• Hormones e.g. Beta human chorionic gonadotropin, calcitonin, placental lactogen etc.
• Hormone receptors (e.g. estrogen and progesterone receptors)

17. • Enzymes and Isoenzymes (e.g. prostate specific antigen (PSA), prostatic acid phosphatase (PAP), neuron
specific enolase (NSE), glycosyl transferases, placental alkaline phosphatase (PALP), terminal deoxy
nucleotidyl transferase (TDT), lysozyme, alpha amylase
• Serum and tissue proteins (beta-2 microglobulin, monoclonal immunoglobulin/para proteins, glial fibrillary
acidic protein (GFAP), protein S-100, ferritin, fibrinogen degradation products)
• Other biomolecules e.g. polyamines

18. LESIONS
Vascular malignancies
Oral Salivary Gland Tumors
Oral Melanoma
Benign Mesenchymal Tumors of Oral Cavity
Oral Sarcomas
Rhabdomyosarcomas
Leiomyosarcomas
Osteosarcoma
Ewing’s tumor
Oral Squamous Cell Carcinoma
Lymphomas
Plasmacytoma
Peripheral Nerve Tumors
Organ Specific Antigens in the Identification
of Metastatic Carcinomas

19. OSCC
the most important form of oral cancer
markers of interest in squamous cell carcinoma are keratins.
Keratins are the cytoskeletal intermediate filament
proteins typical of all epithelial cells.
More than 20 different keratins are known to be
located in epithelial cells.
Type II (basic) keratins and. The Type I (acidic)
keratins
To differentiate adenocarcinomas from the adenoid squamous cell carcinomas,keratin 20
can be used since it is negative in squamous cell carcinoma

20. • Squamous cell carcinomas have been the main objects of immunohistochemical studies evaluating the
markers of cell proliferation and their possible prognostic correlations
• Ki-67 antigen, a nuclear protein, was originally isolated from Reed–Sternberg’s cell line.
• Expressed in all cells except for those in G0 phase (resting, noncycling cells)
• The immunoreactivity of these proliferation markers appears limited to the proliferating basal cells in
the normal and hyperplastic mucosa, but it increases and also appears in suprabasal layers in the
dysplastic mucosa.
Ki-67 cells which are more prolifertive Ki-67 cells which are less prolifertive
P53 cells which are more prolifertive i.e, in soft
tisse sarcomas
p53 cells which are less prolifertive

21. • Pleomorphic adenoma is the most common benign salivary gland tumor.
• Components - Ductal cells & Myoepithelial cells
Staining for glial fibrillary acidic protein (GFAP) has been localized to the
myoepithelial calls
This marker has utility in differentiating the pleomorphic adenomas from the polymorphous
low-grade adenocarcinomas.
Adenoid cystic carcinoma- extensive immunoreactivity for S-100 protein, muscle actins,
and epithelial membrane antigen.
Oral Salivary Gland Tumors
Marker -

22. • Malignant melanoma can present as a primary tumor in oral mucosa or as a metastasis in adjacent soft tissues and jaw
bones.
• Histologic patterns of melanoma also are variable and may include epithelioid, spindle cell sarcomatous, or round cell
appearances that must be distinguished from carcinoma, sarcoma, and lymphoma, respectively.
• Marker –
 S-100 protein is present in almost 95% cases. S-100
 Melan –A
 Mart -1
 HMB – 45
 Tyrosinase
Oral Melanoma
Clear Cell Sarcomas…!!!
Unique tumors that may produce melanin and are intimately
associated with tendons or aponeuroses.
S-100 and often HMB-45, NSE, and Leu-7

23. • Granular cell tumor,
• Granular cell epulis,
• Melanotic neuroectodermal tumor of infancy and
• Schwannoma.
Benign Mesenchymal Tumors of Oral Cavity
Granular cell tumor (formerly granular cell myoblastoma) occurs within the oral cavity,
predominantly in the tongue of the adult patient
Positive for S-100 protein and vimentin, similar to schwannoma, but negative for the muscle cell
markers desmin and muscle actins,

24. • Melanotic neuroectodermal tumor of infancy (melanotic prognoma) is a rare, usually benign tumor
that occurs in the jawbones, predominantly in infants. This tumor consists of small neural-like cells and
large, pigmented epithelial-like cells and shows a complex immunohistochemical profile reflecting its
divergent differentiation properties. The large cell (cuboidal cell) component is positive for keratins and
melanoma-specific antigen (HMB-45), but they are usually negative for S-100.

25. • Most of the sarcomas contain vimentin, the intermediate filament protein typical of
mesenchymal cells and negative for keratins
Oral Sarcomas
Rhabdomyosarcomas
•Children in the first decade,
•Histologically as a “small round blue cell neoplasm.”
Marker-desmin is the most consistently detectable in paraffin embedded specimens
•Myglobin has been a marker that has been traditionally used for the diagnosis of these tumors.

26. • The reliable recognition of osseous matrix production in
malignant lesions.
• Late 1990s, number of putatively osteoblast-specific markers
have been developed including bone morphogenetic protein, type
I collagen, COL-I-C peptide, bone sialoprotein, and bone
glycoprotein 75.
• Best markers-osteonectin and osteocalcin (OCN)
• Sometimes fibroblasts can also cross-react with the polyclonal
anti-OCN reagents therefore; monoclonal antibodies with
selective peptide recognition are preferred for diagnostic work.
Thus, it can be used with reasonable success as a single marker
to detect such neoplasms.
Osteosarcoma

27. • Ewing’s tumor and Primitive NeuroEctodermal Tumors (PNETs)
• Share similar chromosomal translocations, predominantly t(11;22) translocations.
• These tumors also express cell surface glycoprotein p30/32 (CD99) encoded by the MIC2 gene.
• Definitive diagnosis of Ewing’s tumor/ PNETs can be made with either cytogenetic, fluorescence
in situ hybridization, or reverse transcriptase polymerase chain reaction analyses to identify the
characteristic chromosomal/ molecular defects in these tumors.
Ewing’s tumor

28. • Malignant smooth muscle cell tumors are also seen in the oral cavity.
• It probably arises from smooth muscle cells, especially those found in blood vessel walls and from
undifferentiated mesenchymal cells.
• Marker -
Leiomyosarcomas
Positive for muscle cell markers such as desmin and actin but may also contain
keratin.
Another novel marker, caldesmon a protein involved in cell contraction that is widely
distributed in both smooth and nonsmooth muscle cells has been recently used for the
diagnosis of leiomyosarcoma and tumors with smooth muscle cell-like differentiation

29. • Angiosarcoma is the rare malignant tumors recapitulating the features of endothelial cells.
• Many tumors other than angiosarcomas can show slit-like structures and pools of
extravasated erythrocytes, causing differential diagnostic difficulties.
• Marker-
Vascular malignancies
CD34,CD31, the platelet-endothelium cell adhesion molecule-1.
This antigen is present in endothelial cells and platelets but is
practically absent in carcinomas and is detectable in about 90% of
angiosarcomas and also in Kaposi’s sarcoma.
A new marker FLI1, a nuclear transcription factor appears to be expressed in almost
100% of different vascular tumors, including Kaposi’s sarcoma

30. • Often diagnosed as undifferentiated malignant neoplasms.
• Marker -LCA is an excellent screening marker for lymphoid cells, including the non-Hodgkin’s lymphomas.
Lymphomas
T-lymphocytes (UCHL-1, CD3, L60, and MT1) B-lymphocytes (L26, LN1, LN2, LMB1, and MB2)
The most important B-cell marker that can be identified in paraffin sections is CD20.
•Markers for Reed–Sternberg cells include CD15, CD30, and BLA36.
•In lymphocyte predominant Hodgkin’s disease and the Reed–Sternberg cells differ in phenotype
and are CD15, CD30 negative but CD45, CD20 and BLA36 positive

31. • Malignant tumors arising from nerves or showing nerve sheath differentiation are better
designated as “malignant peripheral nerve sheath tumors”
• Markers-
Peripheral Nerve Tumors
S-100
Neuron specific enolase (NSE)
CD57
p75 NTR
CD99
CD56
Neuro filament protein (NFP)
Synaptophysin
Chromogranin
Claudin – 1
Glut – 1.

32. INFECTIONS
• Also being applied to confirm infectious agent in tissues by use of specific antibodies against
microbial DNA or RNA.
• e.g. in Cytomegalo virus, Hepatitis B virus, Hepatitis C virus, etc
• Concept was introduced as early as the 1940s when fluorescein dye (visible under ultraviolet
light) was tagged to antibodies directed against pneumococci for identification of this organism
with specific anti-serum.

33. • IHC can also be used to detect organisms in cytological preparations such as
fluids, sputum samples, and material obtained from fine needle aspiration procedures
• This can be very helpful in certain situations such as detection of pneumocystis from the sputum of an
immunocompromised patient who needs rapid and precise confirmation of infection in order to begin immediate
and appropriate therapy.

34. GENETICS
• IHC is used to determine the function of specific gene products in fundamental biological processes such
as development and apoptosis.
• Using a custom made monoclonal antibody against p53 homologue of the pro-apoptotic pathways of p53
was identified

35. NEURODEGENERATIVE DISORDERS
• Degenerative disorders of the nervous system include a wide range of diseases characterized by the
dysfunction and death of specific, selectively vulnerable populations of nerve cells.
• It has played an increasingly important role in the sub-classification of neurodegenerative disorders and
the development of consensus criteria for their diagnosis.

36. Brain Trauma
• In the last few years, immuno histochemical staining for beta amyloid precursor protein has been validated as
a method to detect axonal injury within as little as 2–3 hrs of head injury…!!
• Detection of axonal injury can be useful in establishing timing of a traumatic insult in medico-legal settings

37. IHC in Muscle Diseases
• Specific diagnosis of muscular dystrophy is important because of the genetic counseling implications of
inherited disease and accurate prognostication.
• Skeletal muscle biopsy can play a main role in differentiating vascular dystrophy from non-dystrophic
disorders and IHC can assist in establishing a specific diagnosis of the dystrophies for which specific
protein abnormalities are known.

38. Research Application
• Much of the current research into the causes of neurodegenerative diseases is directed at identifying the factors
that result in the formation of paired helical filaments, the deposition of beta amyloid, cytoplasmic
accumulations of alpha synuclein, etc
• To localize and quantify the abnormal proteins that constitute reasons of neurodegenerative diseases are of
central importance.
• IHC using antibodies to beta amyloid, alpha synuclein, ubiquitin, huntingt in, polyglutamine, and others has
become a routine tool for a sensitive detection and quantification of these abnormal proteins in both human
tissues and in experimental animals that are used to model some of the features of these diseases

39. • Despite the routine use of IHC and its ample availability for clinical and surgical personnel, it is worthwhile
discussing its use and precise indications in diagnostics, prognostics, the possible impacts it may have upon
therapy, and the technique’s limitations.
• IHC has played a significant role in primary tumor site identification in metastatic disease. Panels labeling
SCC, adenocarcinoma, thyroid carcinoma, sarcoma, lymphoma, neuroendocrine carcinoma, and melanoma
can improve diagnostic accuracy and thus provide valuable input to the determination of the most effective
course of therapy.
Pitfalls of immunohistochemistry in head and neck cancer

40. References
1. Bancroft Histological Techniques
2. Sheeyans Textbook
3. Review Article -Applications Of Immunohistochemistry-jeyapradha Duraiyan
4. Emerging Applications Of Immunohistochemistry In Head And Neck Pathology-review Article.
Dipak Baliram Patil
5. Review Article The Role Of Immunohistochemistry In Diagnosis Of Various Oral Lesions - A
Review Bhavana Gupta1 And Vivek Gupta
6. Indications and pitfalls of immunohistochemistry in head and neck cancer-Décio de
Natale Caly