Immunohistochemistry
has become an important tool in the diagnosis of the vast range of
neoplastic and non-neoplastic diseases in dermatopathology. There are
many established markers that have become an indispensable adjunct to
diagnostic dermatopathology. These markers evaluate various
differential diagnoses and help in determining tumour subtype in small
biopsies .
I have highlighted certain aspects in the interpretation of the
results of immunohistochemical techniques when applied to
dermatopathology.
-
Immunohistological features of the various skin components.
- Role of immunohistology in non-neoplastic skin lesions
- Role of intermediate filaments in differential diagnosis of skin
tumours
- Role of immunohistochemistry in evaluation of vimentin positive
cutaneous tumours .
- Role
of immunohistochemistry in the diagnosis of small blue cell tumours of
skin.
- Role of immunohistochemistry in the diagnosis of problematic adnexal
& epidermal tumours.
- Immunohistochemistry in the differential diagnosis of
intraepithelial malignant tumours
-
Immunohistochemistry in the diagnosis of melanoma.
- Role of immunohistochemistry in the diagnosis of cutaneous lymphoid
infiltrate.
- Immunohistochemistry in the differential diagnosis of malignant
tumours of skin with clear cell differentiation.
Immunohistological
features of the various skin components:
Keratinocytes: Express
keratin (pan-epithelial marker).
Keratin is an intermediate filament. Keratin polypeptides are
classified according to their molecular weight.
Low mol.wt.- Type I
(K10-K20)- Acidic ; High mol.wt.- Type II (K1-K9)-Basic
In normal epidermis
the basal keratinocytes express K5 and K14
.
Suprabasal
keratinocytes express K1 and K10.
Stratum granulosum
cells express K2
and K11.
Hyperproliferative
keratinocytes express
K6 and K16.
Keratinocytes also express desmosomal proteins . Desmogleins1 and 3 , E-cadherins may be used in
paraffin embedded material.
Langerhans cells:
CD1a, S100 protein positive. Vimentin (not very specific)
Melanocytes: Express S100 protein,
HMB45, bcl2, tyrosinase, MART1/melan A antigen, Vimentin .
HMB45
is expressed by faetal melanocytes and not adult melanocytes. Normal
hair bulb and epidermal melanocytes over inflammatory dermatosis also
express HMB45.
Merkel cells:
These are neuroendocrine cells in which are
immunopositive to
low mol.wt. keratin 8, 18, 19, 20, neurofilament ,chromogranin A, NSE,
synaptophysin, protein gene product 9.5 . The cell may also express EMA.
EPIDERMAL APPENDAGE-
Hair
follicle: Keratinocytes express pilar keratin.
Type I (Ha1-4 and Hax) and Type II (Hb1-4 and Hbx).
Sebaceous glands: Markers include Keratin, EMA , Thomsen-Friedenreich
antigen and less frequently
Tn antigen.
Sweat glands: CEA is the most useful
marker. Stains the luminal border of cells of eccrine sweat gland.
EMA is detected on the apical pole of
secretary cells of eccrine and apocrine sweat glands.
Keratin (8,18,19),
demonstrates cytoplasmic positivity of secretary cells of eccrine and
apocrine sweat glands.
GCDFP-15
is expressed apocrine sweat glands and eccrine glands (variable).
Myoepithelial cells are smooth muscle actin positive
DERMO-EPIDERMAL JUNCTION-
Type IV collagen (used for diagnosis
of bullous dermatosis) and laminin can be detected on
routinely processed tissue.
DERMIS-
Cellular components:
Fibroblast: Vimentin positive. FibAS
is specific for fibroblasts.
Myofibroblast : Express muscle specific actin (sometimes desmin)
Dermal dendrocytes: TypeI - present in papillary dermis and around
capillary vessels. Express factor XIIIa
TypeII-
present in reticular dermis and around secretary portion of
eccrine sweat glands- Express CD34
Lymphocytes:
Express CD3, 8,15,20,30,45,68 etc
Cutaneous blood vessels: Markers include Factor VIII related
antigen (vWf) most
specific marker,CD31,
CD34 ,PAL- E, Vimentin. Lymphatic
endothelial cells express vimentin but do not stain with antibodies
to Factor VIII related
antigen and do not express
CD34 . Type IV collagen and
laminin are detected in the
basement membrane that surround the blood vessels . The pericytes
express vimentin and muscle specific actin.
Cutaneous nerves:
Axons- Neurofilament and
neuron specific enolase. Schwann cells- S100, GFAP, myelin basic
protein , Schwann cell associated antigen (AHMY1). Perineural cells express - EMA and
Vimentin.
Cutaneous muscle:
Smooth muscle cells are present in Arrector pili muscles, muscle
fibres in vessel wall , dartos muscle and areola. Skeletal
muscle (myoglobin positive) may be present in the deep dermis. Desmin and muscle specific
actin are expressed by both
smooth and striated muscle cells.
Extracellular
matrix: Antibody to Collagen, elastin
and fibronectin may be detected in
dermal connective tissue.
Role of
immunohistology in non-neoplastic skin lesions:
Infectious diseases:
Viral
Infection:
Cytomegalovirus ; Herpes Simplex I & II ; Varicella Zoster virus ;
Human Papilloma virus ; Epstein Barr virus:
Fungal Infection
- Candida,
Histoplasma, Aspergillus, Pnuemocystiis Carinii , Sporothrix,
Coccidioides ;
Bacterial Infection-
Mycobacteria
(M. tuberculosis ; M Leprae; Atypical
Mycobacteria; M paratuberculosis)
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Bullous lesion: Bullous pemphigoid ;
Pemphigus and variants (p vulgaris ; p foliaceus ; p
vegetans; p erythematosus); Epidermolysis
Bullosa Acquisita ; Dermatitis Herpetiformis; Linear IgA disease;
Herpes Gestationis ; Bullous Lupus Erythematosus
Other cutaneous lesions:
Lichen Planus; Cutaneous Vasculitides (selected lesions- Henoch
Schonlein Vasculitis of skin).
The following
intermediate filaments play an important role in the differential
diagnosis of skin tumours:
1.
Cytokeratin 2. Vimentin 3. Desmin 4. Neurofilament
CYTOKERATIN:
Cytokeratins are divided into two broad groups Simple (K7,8,18,19)
and Complex (K1,4,5,6,10,13,14,16,19)
according to the epithelia in which they are expressed.
( Simple epithelium is characterized by single row of epithelial cells
lying on their basement membrane. Complex epithelium are stratified
epithelium eg.stratified squamous epithelium, ductal epithelium. )
Tumours arising from complex epithelium eg. well- differentiated
squamous cell carcinoma may be negative for simple epithelial keratin, CAM 5.2
.Tumours arising from simple epithelium may be negative for the
complex epithelial keratins, AE1
. In less differentiated squamous cell carcinomas cytokeratins from
simple and non-squamous epithelium are expressed.
Broad spectrum antibody is selected- Eg.
MNF116 (K5,6,17 and 19) or a combination AE1(K10,13,14,19 ) ;
AE3 (K1-8,15,16).
Cytokeratin 20 (
low mol. wt.) shows
perinuclear and dot like positivity in merkel cell carcinoma.
VIMENTIN:
Vimentin is present in mesenchymal cells and their tumours. This
intermediate filament is detected in endothelial
cells, fibroblasts,smooth muscle cells and in lymphoid cells,
melanocytes and Schwann cells.
DESMIN:
Desmin is helpful in the identification of smooth muscle tumours and
is particularly used in the diagnosis of cutaneous
leiomyosarcoma.
Vimentin is expressed in a wide spectrum
of cutaneous tumours. Hence further immunological differentiation of
vimentin positive tumours is necessary to establish the correct
diagnosis.
Immunohistochemical staining of Vimentin
positive tumours:
3. CD34 negative-
stromal cells 3. CD34 positive spindle shaped
cells surrounding the
cellular islands
4. Stronger and
more diffuse expression 4. Less prominent
of Ki67 and PCNA.
Sweat gland carcinoma: Squamous
cell carcinoma:
1. CEA positivity is noted in sweat gland carcinoma 1. CEA
negative
highlighting intracytoplasmic lumen formation.
2. S100 protein positivity is an indicator of sweat
gland 2. S100 protein negative
differentiation in some cases. PUBMED
Immunohistochemistry in the
diagnosis of melanoma:
Immunohistological
staining is relevant in the following areas:
1. In the diagnosis
of
desmoplastic melanomas(D/D-
dermatofibroma, scar tissue), spindle
cell melanomas (D/D- spindle cell squamous carcinoma,
atypical fibroxanthoma), superficial spreading melanomas (D/D- Bowen's disease,
extramammary Paget's disease).
2. Identification of undifferentiated metastatic melanomas where the
primary is unknown.
3. In the measurement of the thickness of primary melanomas (Eg.
melanoma over preexisting melanocytic naevi or when the tumour is
accompanied by a dense inflammatory infiltrate.)
4. To estimate prognosis of the tumour.
Melanoma markers include:
1. S100 protein
2. HMB45
3. Melan-A (MART-1)
4. NK1/C3
5. p53
6. Ki 67
7. Micropthalmia Transcription factor
8. Vimentin, cytokeratin, EMA, actin, NSE (positive in some cases)
HMB45
is a monoclonal antibody with specificity for melanoma cells. It
reacts with fetal melanocytes but not with resting adult melanocytes.
HMB45 positive
lesions :
1. Junctional melanocytes in naevi
2. Dermal naevi in HIV- positive patients
3. Deep penetrating naevi
4. Some cells in papillary dermis in dysplastic naevi
5. Melanocyes in blue naevi
6. Some cells in Spitz naevi
7. Reactive or proliferating melanocytes in inflamed adult skin.
HMB45 negative
lesions -1. Desmoplastic melanomas.
2. Some cases of nevoid melanomas.
Melan-A (MART1)
is expressed in both benign & malignant melanocytic lesion.
It is negative in Desmoplastic melanomas.
Melan A is more sensitive than HMB45 and more specific than stains for
S100 protein.
Cytokeratin
is postive in some cases of metastatic melanomas. These cases may be
negative for HMB45 although the primary lesion is HMB45 positive and
Cytokeratin negative.
Ki67
a marker of cellular proliferation and is variably expressed in
melanomas. It is identified in paraffin sections using monoclonal
antibody
MIB1.
[Note: Percentage of proliferating cells within the tumour correlate
with malignancy hence proliferating rate of melanocytic tumour is
examined. This is possible by detecting nuclear antigens which appear
during cell proliferation. Ki67(frozen material) and MIB1
antibodies(paraffin embedded material) react with cell cycle
specific nuclear antigen which is expressed in late G1,G2 and M phase
but not in G0 phase.]
More than 10% dermal melanocytic positivity of Ki67 is indicative of
melanoma.
Ki67 is of diagnostic value in borderline lesions (in distinguishing
melanomas from Spitz naevus).
Ki67 positivity is noted mainly in vertical growth phase melanomas. It
is useful in separating vertical and radial growth phase melanomas.
Thick melanomas (>4mm) with high MIB1 reactivity have a poor
prognosis.
Role of immunohistochemistry in the diagnosis of cutaneous
lymphoid infiltrate:
Immunohistochemistry plays
a crucial role in the diagnosis of primary cutaneous lymphoma. With
advances in the clinical management it is increasingly important to
subclassify lymphomas into recognized pathological groups.
It is now possible to identify the characteristic antigen profile of
each type of lymphoma in routinely formalin fixed and paraffin wax
embedded material. The basic panel of antibodies depends on the local
interest, referral practice and budget . Further antibodies are
necessary to refine the diagnosis of the lymphoma and to
differentiate difficult cases.
EORTC CLASSIFICATION OF PRIMARY CUTANEOUS
LYMPHOMA:
