Diagnostic approach to metastatic adenocarcinoma

Role of IHC in diagnosis of
metastatic adenocarcinoma of
unknown primary origin
Presenter: Sivaranjini. N
Guide: Dr. Manisha Singh

 Carcinoma of unknown primary origin
(CUP) is a heterogeneous group of
cancers defined by the presence of
metastatic disease with no identified
primary tumor at presentation.
 Identifying patients with prognostically
favorable disease is important, since
they may derive substantial benefit,
including prolonged survival, from
directed treatment.

The approach to definitive diagnosis of
the patient with CUP effectively follows
five sequential steps

I
• Determine the cell line of differentiation by using
major lineage markers
II
• Cytokeratin profile
III
• Is vimentin co-expressed?
IV
• Expression of supplemental antigens of epithelial or
germ cell derivation is present i.e. CEA, EMA or PLAP
V
• Organ specific markers/unique identifiers of cell types

Step I: Markers for major
lineages

Epithelial:
Pankerati
n,
EMA
Skeletal
muscle
Neuronal:
NeuN
Neurofilament
Desmin,
MSA, SMA,
Calponin
H-
caldesmon
Smooth
muscle
CD34,31,14
1
Factor VIII
FLI-I, D2-40
Ulex
Europaeus I
Mesenchym
al: Vimentin
S100,
HMB45,
Melan-A
MITF,
Tyrosinase
Schwann
Cells:
S100
Glial cells:
GFAP
Endothelial
Desmin
Myogenin
MyoD
MSA
Melanocytic

Hematopoeitic
CD45/LCA
CD3 (pan-T cell),
CD20, CD79a,
PAX5 (pan-B
cell),
CD138
κ/λ light chains
(plasma cell)
Histiocytic
CD68,
CD163,
HAM56,
MAC 387
Lysozyme
α1-antitrypsin
Neuroendocrin
e
Synaptophysi
n
Chromograni
n
Neuron
specific
enolase
CD56,CD57

Antigens
expressed in
cellular nuclei or on
the nuclear
membrane.
Antigens located
within the cell
membrane
Staining specific
organelles

Step I: Screening Immunohistochemistry
An abbreviated first-line panel should
be used, consisting of:-
 Epithelial markers: Pankeratin
AE1/AE3 and CAM5.2, both used
together
 Lymphoid marker: Leukocyte common
antigen/CD45
 Dendritic cell marker: CD56
 Mesenchymal marker: Vimentin
 Melanocytic markers: s-100 protein
with either Melan A or HMB45.

 Although vimentin is included in the
above panel, it is generally the least
helpful and should therefore be
interpreted with caution
 Except for vimentin, a diffuse strong
expression of any of the markers
above is generally suggestive of a
particular line of differentiation

Pankeratin
+
Carcinoma
Non-epithelial
tumors
CD45
S100
Vimentin
Lymphoma
Sarcoma
(maybe?)
HMB45
Melan A CD 68
Dendritic
cells
Melanoma

CD 45: Leukocyte Common
Antigen
 Transmembrane protein essential for
haematopoietic signal transduction
and cell activation
 Positive in large majority of
haematolymphoid cells
 Lost in maturing erythocytes,
megakaryocytes and plasma cells
 Never found in non-haematolymphoid
cells
M
E
M
B
R
A
N
O
U
S

Controls
 In tonsil all B-
and T-cells must
show strong and
distinct
membranous
staining reaction,
while Kupffer cells
in liver tissue
must show an at
least weak to
moderate but
distinct staining
reaction.
 No staining
should be seen in
the squamous
epithelial cells and
hepatocytes.

S 100
 Name is from its solubility in 100% saturated
ammonium sulfate at neutral pH
 Family of acid calcium binding proteins
 Located in nuclei, cytoplasm and cell
membranes. Hence shows nuclear and
cytoplasmic positivity.
 At least 10 α-chains and one β-chain creating
homo-and heterodimers
Polyclonal antibodies primarily detects the
β-chain
C
O
M
B
I
N
E
D

 A diffuse strong staining with S-100 in
a tumor of unknown origin that is
negative for cytokeratins is good
evidence that it may be a melanoma.
 However, this diagnosis still must be
confirmed by additional melanoma
markers such as HMB-45, melan-A or
tyrosinase.
 Additional markers are needed
because, S-100 is not a specific
marker for melanoma.

 s-100 is also expressed by some
sarcomas and carcinomas
(liposarcoma, chondrosarcoma, neural
tumors).
 An additional pitfall is that some
melanomas may show polyclonal CEA
and/or focal cytokeratin positivity
In addition to melanocytes, s-100 is positive in
Glial cells Langerhans’ cells
Fat cells Myoepithelial cells

Positive control: In the appendix, all adipocytes,
Schwann cells and dendritic cells must be stained
as strongly as possible without any staining
reaction of the smooth muscle or epithelial cells.

Vimentin
 Cytoplasmic intermediate filament
 Present in all mesenchymal cells
 Present in early stages of all cells,
replaced by other intermediate filaments
in most non-mesenchymal cells
 Strong vimentin expression in a
nonmelanocytic, nonlymphoid neoplasm
is generally an indication of a sarcoma.
 Most sarcomas, but not all, are negative
for epithelial markers.
C
y
t
o
p
l
a
s
m
i
c

The real use of vimentin (?)
 Because of its ubiquity, staining for
vimentin is also helpful as a reporter
molecule, to give a general idea as to
the adequacy of fixation and
processing of the tissue
 The intensity of staining of vimentin
provides a crude indication of
overfixation.

Controls
Colon/Appendix: Endothelial cells of large
vessels and stromal cells must display a
strong but distinct cytoplasmic staining
intensity. Epithelial cells of the colon
mucosa should be negative.

Liver: an intense staining reaction
of all Kupffer cells (arrow), whereas,
endothelial cells of the sinusoids
must display an at least weak
intensity. Liver cells should be
negative.
Pancreas: Epithelial cells
of exocrine acini should
display a weak to strong
cytoplasmic staining
reaction.

Cytokeratins
 Highly complex family of intermediate
filaments
 50 distinct types Types CK1-20
diagnostically most relevant:
Class I (type A -Acidic):
 CK9(64 kDa) -CK20(40 kDa)
Class II (type B -Basic/neutral):
 CK1(68 kDa) -CK8 (52.5 kDa)

Cytokeratins
 Cancers generally express CK
patterns that at least in part represent
the pattern of the putative cell of origin
 Metastases express CK patterns fairly
concordant with those of the primary
tumours

Micro-metastases identified by
cytokeratin

Pairing of cytokeratins
 One CK class I and one CK class II ‘always’
paired
 CK class I in a pair ~ 8 kDa smaller than
class II

Pan-cytokeratins
 AE1/AE3 is a mixture of both AE1 and
AE3, whereas AE1 reacts with type I
cytokeratins and AE3 with type II
cytokeratins. It lacks the reactivity with
cytokeratin 18. It shows crossreactivity
with GFAP
 CAM 5.2 is a cytokeratin clone that
reacts with the cytokeratins 8/18/19.
 34ßE12 is a high molecular weight
keratin which reacts to CK1, CK5, CK10
and CK14.

 Cytokeratin OSCAR is a broad-
spectrum cytokeratin that reacts with
cytokeratins 7, 8, 18, and 19.
Cytokeratin OSCAR does not show
cross-reactivity with GFAP, but it
reacts with follicular dendritic cells in
lymphatic tissue.

Complex keratins: CK 5/6
 Present in basal cell layer of stratified and
squamous epithelium.
 Good indicators of squamous and transitional cell
differentiation.
 Together with p63, identify squamous origin in
poorly differentiated metastatic carcinomas.
 Good discriminators of mesothelial differentiation
versus adenocarcinoma in lung.
 Sensitive and specific markers of basal-like
phenotype of breast carcinoma.

CK 5/6 is positive in myoepithelial cells of
breast and basal cells of prostate; used to
distinguish between insitu and invasive
lesions.

Distinguish breast usual ductal hyperplasia
(UDH) and papillary lesions (mosaic-like
pattern) from DCIS (usually negative, rarely
diffusely positive)
DCIS

Cytokeratin 19 (CK19)
 Cytokeratin with the lowest molecular
weight of the group.
 Present in the basal layer of the
squamous epithelium of mucosal
surfaces, and may be seen in
epidermal basal cells.

CK19 confirms diagnosis of papillary thyroid carcinoma in
cytology or equivocal cases
It also helps to distinguish between FVPTC (CK19+) from
follicular adenoma (CK19-),

Other uses
 Breast: presence of CK19+ peripheral
blood tumor cells or CK19+ fragments
is a poor prognostic factor for breast
cancer (predicts CNS relapse)
 Liver: distinguish HCC (CK19-) from
either hepatoid adenocarcinoma
metastatic to liver or
cholangiocarcinoma (CK19+)

Cytokeratin 7 (CK7)
 Type II simple
keratin with
restricted
distribution.
 Useful in evaluating
the origin of
metastatic
adenocarcinomas.
 Strong diffuse CK7
immunostaining
may be used as a
starting point for
further IHC study

Main diagnostic use of CK7 Other uses
Adenocarcinomas of :-
• Lung,
• Salivary glands,
• Upper GI tract,
• Pancreas,
• Biliary tract,
• Breast,
• Endometrium,
• Transitional cell carcinoma,
• Ovarian serous tumors
Differentiating between different
types of RCC:-
 Chromophobe RCC is diffuse
CK7+
 Papillary RCC is CK7+
 Clear cell RCC is CK7-
Endometrioid and clear cell
carcinoma (CK7+) from yolk sac
tumors (CK7- or focal)
CK7 expression in esophageal
squamous cell carcinoma is an
independent prognostic factor for
poor overall survival

A diagnostic pitfall in the interpretation
of CK7 is that it stains subsets of
endothelial cells of normal soft tissues
in addition to endothelial cells in
venules and lymphatics.

Cytokeratin 20 (CK20)
CK20 is LMW
limited
predominantly to
GI epithelium and
tumors, mucinous
tumors of the
ovary, and Merkel
cell neoplasms.

Characteristic dot-like perinuclear expression
of CK20 in Merkel cell carcinoma

Diagnostic utility of CK 20
When combined with the specific tissue
distribution of other keratins, such as
CK7, it is possible to :-
 Identify colon cancer metastases in
the lung
 Distinguish pulmonary small cell
carcinoma from Merkel cell carcinoma
 Distinguish transitional cell carcinoma
from other squamous cell carcinomas
and poorly differentiated carcinomas.

• HCC
• RCC
• Prostate
SCC
• Lung
• Thyroid
• FGT
• Upper GI
•Colorectal
•Merkel cell
•Mucinous
ovary
• Urothelial
• Pancreatic
• Cholangio-
carcinoma
CK 7+
CK 20+
CK7 -
CK20 +
CK7-
CK20-
CK7+
CK20 -

Supplemental Epithelial Markers
CARCINOEMBRYONIC ANTIGEN
(CD66a):
 Cell surface glycoprotein normally
expressed by colonic mucosa of fetal
colon and to a lesser degree in adult
colonic mucosa

Controls
Appendix: Epithelial cells show a
weak to moderate cytoplasmic
staining reaction.
Liver: No staining reaction is
seen in the Kupffer cells,
leucocytes and the bile
canaliculi. No background
staining is seen.

pCEA in bile
canaliculi
pCEA in HCC: canalicular
pattern for polyclonal CEA is 50-
90% sensitive

Diagnostic Utility of monoclonal CEA
• 97% specific for lung adenocarcinoma; negative in
mesothelioma and reactive mesothelial cells.
• As a tumor marker: monitor serum levels to detect
recurrence in colorectal cancer; elevated preoperative
serum level is also a poor prognostic factor
• Cysts (various): CEA levels over 5 ng/dl in ascites fluid
are associated with malignancy
• Pancreatic adenocarcinoma: monoclonal CEA is 92%
sensitive for metastases vs. 0% for bile duct adenoma

Epithelial membrane antigen
 Also known as
MUC-I
 Normally acts
as barrier to
apical surface
of epithelial
cells.
 Inhibits
formation of E-
cadherin / beta
catenin
complex

Positive control: Appendix, tonsil
Main diagnostic use of EMA Main diagnostic use
• Shed into the bloodstream
of adenocarcinoma patients,
used in commercial serum
tumor marker assays
(CA15-3)
• Distinguish NLPHL (EMA+)
from Classical HL (EMA-)
• Useful as a pan-epithelial
marker for detecting early
metastatic loci of carcinoma
in bone marrow or liver.
• Epithelioid sarcoma,
• Choroid plexus tumors,
• Ependymoma,
• Chordoma and
parachordoma,
• Plasmacytoma
• Marker of meningioma
& Paget disease

p63/p40
 Also called KET
or p73L is a
member of the
p53 gene family.
 It plays an
important role in
the
differentiation of
stratified
epithelia and
regulation of cell
cycle
progression.
TONSIL

Myoepithelial cell marker: Used to
discriminate between benign and
malignant salivary gland, prostatic and
breast lesions

Positive control: prostate, tonsil, placenta
(cytotrophoblasts)
Expression in normal cells Also used to
• Stratified epithelium
• Transitional epithelium
• Myoepithelial cells
• Determine squamous
differentiation (p63+) along
with CK5/6
• Differentiate renal collecting
duct carcinoma & high
grade prostatic carcinoma
(p63−) from urothelial
carcinoma (p63+)
• Used to discriminate
between FVPTC (p63
positive) and other benign
follicular lesions of the
thyroid gland (p63 negative)

Ep-CAM
 Also known as human
epithelial antigen
 It is a transmembrane
glycoprotein involved in
cell signaling, migration,
proliferation and
differentiation
 Ber-EP4 is the most
commonly used clone.
Liver

Uses of Ep-CAM
Differential diagnosis
Ep-CAM positive Ep-CAM negative
Pulmonary
Adenocarcinoma
Mesothelioma
BCC SCC
Metastasis to liver HCC (MOC 31 clone is
used)

Thyroglobulin
 Specific marker for thyroid
follicular cells.
 Expression correlates with
the differentiation grade of
thyroid tumors
 Specific and sensitive
markers of both primary
and metastatic carcinomas
of the thyroid
 Excellent marker of
papillary and follicular
carcinomas
 Poor marker of anaplastic
and poorly differentiated
carcinoma
 NOT a marker for medullary
carcinoma.

THYROID TRANSCRIPTION FACTOR-1
 Nuclear tissue
specific protein
transcription factor.
 Selectively
expressed during
embryogenesis in
the thyroid,
diencephalon and
respiratory
epithelium
CONTROLS
 Positive: Lung,
thyroid
 Negative: Colon,
uterus

TTF-I
 Even more-sensitive marker of thyroid
carcinomas than thyroglobulin
 Medullary carcinoma also shows
positivity.
 Anaplastic carcinoma  negative

Nuclear staining
with TTF-1 is
seen in the vast
majority of
carcinomas of the
lung:
 Adenocarcinom
as (~70% to
80%),
 Large cell
neuroendocrine
carcinomas,
 Small cell
carcinomas
SCC &
mesothelioma are
negative

PAIRED BOX GENE-8
 Nuclear transcriptional regulator in the
paired-box family expressed during
organogenesis of the thyroid gland,
kidney, and Müllerian tract.
 PAX8 regulates the expression of the
WT1 gene.

PAX8
Controls:-
 A moderate to strong,
distinct nuclear
staining of the
distal/collecting
tubular cells in the
kidney is seen.
 A weak to moderate,
distinct nuclear
staining of the
majority of the ciliated
epithelial cells of
fallopian tube.
KIDNEY
FT

 Follicular and papillary thyroid carcinoma are virtually
always PAX8 positive
 Anaplastic carcinoma is positive in most cases
 Medullary thyroid carcinoma is negative

PAX 8 is highly sensitive and relatively specific marker for thyroid
follicular cell tumors, RCCs, ovarian carcinomas, endometrial
carcinomas, and thymic tumors.

 PAX8 is not expressed in mammary
carcinomas, including ductal and
lobular types
 Because the ovary is a common
site of involvement for metastasis
by breast carcinoma, PAX8 can be a
useful marker in the differential
diagnosis of ovarian and breast
carcinomas

Other uses of PAX8
PAX8 positive PAX8 negative
Collecting duct carcinoma Urothelial carcinoma
Anaplastic thyroid
carcinoma
Head & neck SCC
• Marker of nephrogenic adenoma
• Determine renal tubular origin.

Trophoblastic cell surface
antigen 2 (Trop-2)
 Transmembrane glycoprotein
functioning as calcium signal
transducer.
 Expression is up-regulated during
malignant transformation
 More than 90% of papillary thyroid
carcinomas express Trop-2 while
follicular adenomas and follicular
carcinomas are negative.

1) Papillary thyroid carcinoma shows strong membranous staining
pattern.
2) Thyroid follicular carcinoma: No TROP-2 membranous staining
pattern was identified in follicular neoplasms.
PTC
Follicula
r

Calcitonin
 Polypeptide hormone
synthesized by the
parafollicular (C)
thyroid cells involved in
the regulation of
calcium and
phosphorous
metabolism
 Specific marker for the
parafollicular cells.
 Tumors originating
from the thyroid
follicular cells are
constantly negative for

Immunoprofile of thyroid
tumors
Cytokeratin profile
• CK7+/CK20-
• CK8/18/19+
• PAX8+
• Well differentiated carcinomas are positive for
TTF-1, thyroglobulin, thyroid peroxidase

Tumour Commonly expressed
markers
Negative markers
Follicular thyroid
adenoma
• Pan-CK Calcitonin, CD44V6,
Trop-2, galectin-3
CK-19
Follicular thyroid
carcinoma
• Galectin-3,
• Vimentin ,HBME1,
• E-cadherin, Bcl-2,
• Calcitonin,
• Trop-2, CEA,
• HER-2
Papillary thyroid
carcinoma
• Trop-2,
• p63
• Galectin-3
• CD15, vimentin
• HBME-1
• CK19
• CEA, calcitonin,
• synaptophysin,
• chromogranin,
• CD56

markers
Negative markers
Anaplastic thyroid
carcinoma
• Pan-CK,
• CEA,
• Vimentin
• Thyroglobulin,
• Calcitonin
Medullary thyroid
carcinoma
• Calcitonin,
• chromogranin,
• synaptophysin,
• TTF-1, BCL-2
• CD56, Leu7, S100,
• NSE, CEA,
• PAX-8,
• Thyroglobulin

NAPSIN A
 Aspartic protease that
is crucial to the
maturation of
surfactant B and
present in the
cytoplasm of type 2
pneumocytes and
alveolar macrophages
Controls:
 Positive: Kidney
 Negative: Colon
Kidney: All epithelial cells of the PCT must
show an at least moderate, distinct
granular cytoplasmic staining reaction.

Uses of Napsin A
 To distinguish lung adenocarcinoma
(positive) from SCC (negative)
 As part of panel to classify poorly
differentiated non small cell lung
carcinoma .
 Superior to TTF1 in distinguishing
primary lung adenocarcinoma from
other carcinomas (except kidney),
particularly primary lung small cell
carcinoma and primary thyroid
carcinoma.

Immunoprofile of lung tumors
Cytokeratin profile:
• CK7+/CK20-
• CK8/18/19+

markers
Negative markers
Squamous cell
carcinoma
• CK5/6,
• p63
• TTF-1
Pulmonary
adenocarcinoma
• TTF-1,
• CEA,
• Napsin A
• Calretinin
• p63
• CDX-2
Small cell carcinoma • CD56, NSE, s100
• Synaptophysin
• Chromogranin
Proliferation index (Ki-67):
>90%
• TTF-1
• CK5/6/14
Large cell carcinoma EMA • TTF-I
• Napsin-A

Pleural neoplasms
The differential diagnosis of a malignant
epithelial neoplasm in the pleura
includes
 metastatic lung carcinoma,
 metastatic non pulmonary carcinoma,
 malignant mesothelioma.

 A panel approach is generally used to
arrive at the correct diagnosis.
 IMIG (International Mesothelioma
Interest Group) recommends 2
mesothelial markers and 2 carcinoma
markers be included in the panel

Marker Mesothelioma Adenocarcinoma
CK5/6 Positive Negative
Calretinin Positive Negative
D2-40 (podoplanin) Positive Negative
WT1 Positive Negative
B72.3 Negative Positive
MOC31 Negative Positive
TTF1/Napsin-A Negative Positive
Claudin4 Negative Positive
Adeno-
Lung

CDX2
 CDX2 is a homeobox gene that
encodes a transcription protein factor
that guides development of intestinal
epithelial cells from the region of the
duodenum to the rectum
 CDX-2 is highly sensitive and specific
for intestinal differentiation.

Intestine
- All cell types incl.
endocrine
-Intestinal metaplasia
-Chronic gastritis
-Barrett’s esophagus
Pancreas/biliary tract
(heterogenous)
colon
pancrea
s

Uses of CDX-2
CDX-2 positive CDX-2 negative
Colorectal
Adenocarcinoma
Primary bladder
adenocarcinoma
Metastatic mucinous
colorectal
adenocarcinoma
Mucinous
bronchioloalveolar
adenocarcinoma
Used to determine origin of metastatic
adenocarcinoma as part of panel

 The specificity of CDX-2 for metastatic
colorectal carcinoma in the liver is
enhanced by the concomitant use of a
CK20-positive/CK7-negative profile,
because CDX-2 may be positive in upper
GI carcinomas.
 Endometrioid carcinomas of the uterus
or ovary may mimic colorectal
carcinomas, and they may demonstrate
nuclear CDX-2, in which case a panel of
antibodies that includes CK7, CK20,
Pax-8, villin, vimentin, and estrogen
receptor would be needed to
discriminate from colorectal carcinomas.

SATB2
 Special Adenine Thymine-rich
sequence-Binding protein 2
 In the GI tract, SATB-2 is selectively
expressed in colorectal epithelium,
while gastric and small intestinal
mucosa and pancreatic epithelium
lack the expression of SATB-2.

Nuclear SATB-2 expression in lung
metastasis from rectal adenocarcinoma

Diagnostic uses
 SATB-2 is a specific marker for
colorectal adenocarcinomas including
medullary carcinoma
 SATB2 in combination with cytokeratin
20 identifies over 95% of all colorectal
carcinomas
 Expression of SATB2 is associated with
better prognosis in colorectal carcinoma.
 SATB-2 is also an important diagnostic
marker for osteosarcoma

Immunoprofile of GI tumors
Cytokeratin profile
• CK8/18/19+
• Upper GI: CK7+, CK20 variable
• Lower GI: CK7-, CK20+

markers
Negative markers
Adenocarcinoma,
oesphagus
• E-Cadherin,
• CDX-2,
• Cyclin D1,
• Villin
• CK 5/6
• p40
Adenocarcinoma,
stomach
• CDX-2,
• CEA,
• Cyclin D1,
• Villin
• CK5/6,
• CA125,
• SATB-2
Adenocarcinoma,
duodenum and small
bowel
• CDX-2
• Villin
• PDX-1
• AMACR
• SATB-2
Adenocarcinoma of the
ampullary region
PDX-1

markers
Negative markers
Colorectal
adenocarcinoma
• CDX-2,
• SATB-2, CEA,
• Villin,
• MUC-2
• β-Catenin,
• CD10
• CA125, CK5/6,
• AMACR,
• GATA-3,
• Thrombomodulin
GI neuroendocrine
tumors/ carcinoma
• Synaptophysin,
• Chromogranin,
• NSE, S100, CD56
• CDX-2, villin
CK20

Alpha fetoprotein
 Fetal counterpart of
albumin
(transporter)
 Fetal yolk sac
 Fetal liver and GI
tract
Neoplasms:
 Yolk sac tumour
 HCC
 Hepatoblastoma
 Hepatoid carcinoma
 Pancreatoblastoma

Diagnostic pitfalls
 About 5% of hepatocellular carcinoma
is negative for AFP.
 Frequently results in high-background
serum staining, making interpretation
difficult.

Glutamine synthetase
 Catalyzes the synthesis of glutamine,
which is the major energy source of
tumor cells.
 When a panel of GS, Heat Shock
Protein 70 and Glypican 3 is used, if
any 2 of the 3 are positive, the
sensitivity and specificity for the
detection of early and HCC-G1 were
72% and 100% respectively.
 GS activity is a marker for astrocytes
and can be used to distinguish

Other uses
 Strongly expressed
in the cytoplasm of
hepatocytes in
Focal Nodular
Hyperplasia and
forms large
hepatocytic areas,
anastomosed in a
‘map-like’ pattern.
 In normal liver, it is
restricted to 1 or 2
centrilobular
plates.

Glypican 3
 Cell surface
heparan sulphate
proteoglycan
regulating cell
growth and
differentiation.
 In normal tissues
largely restricted to
embryonic and
foetal tissue and
placental
syncytiotrophoblast

Uses
 Distinguish
hepatocellular
carcinoma (usually
GPC3+) from
nonmalignant
liver(GPC3-) as part of
a panel but cirrhotic
nodules may show focal
strong staining
 Distinguish
hepatocellular
carcinoma from other
primary and metastatic
hepatic lesions.
 Distinguish ovarian yolk
sac (GPC3+) from
embryonal carcinoma
(GCP3-)

Heat-Shock Protein-70
 It is an anti-apoptotic
regulator.
 Can be used as a
marker to discriminate
between HCC positive
for HSP70
(nuclear/cytoplasmic
staining pattern) and
dysplastic nodules or
hepatocellular adenoma
negative for HSP70.
 Since HSP70 is
expressed in different
malignant tumors, it
cannot be used to
discriminate between
HCC and metastatic
carcinoma.

Hepatocyte paraffin-I/
HepPar I
 Sensitive and
highly specific
marker of
hepatocytic
differentiation.
 Directed against a
mitochondrial
antigen present
within hepatocytes
and shows a
characteristic
granular
cytoplasmic
staining.

Uses
 Determine
hepatocellular origin,
particularly in panel
with alpha-
fetoprotein and CEA
or CD10.
 Differentiate HCC
from
cholangiocarcinoma
or metastases to
liver, as part of panel.

Arginase-1
 Enzyme involved in the
urea cycle
 The most-sensitive
and most-specific
marker of HCC.
 Shows Cytoplasmic,
granular pattern
 High level of sensitivity
even in the context of
high-grade HCC

Immunoprofile of liver tumors
Cytokeratin profile
• CK7/CK20-
• CK8/18/19+

markers
Negative markers
Hepatocellular adenoma • Arginase-1, Hep Par-
1, CD34
• ER, PR
• Glypican-3,
• AFP, HSP70
HCC • AFP, Arginase, Hep
Par-1,
• Glypican-3
• CD34
• SATB-2, pCEA,
HSP70,
• EMA, inhibin,
• Melan A
• EPCAM
Hepatoblastoma • Hep Par-1, pan-CK,
• Glypican-3, AFP,
• CEA, CD34, EMA,
Chromogranin,
• Vimentin, ß-catenin
• CA125, SATB-2
Cholangiocarcinoma • CEA, EMA
• S100, PDX-1
• CDH17, CD5
• CK7+/CK20-
AFP, CK5/6,
CD56
Adenocarcinoma, gall-
bladder
EMA, CEA, S100 Arginase-1,
CK5/6

Pancreatic and duodenal
homeobox1(PDX-I)
 Also known as insulin promoter factor 1
 It is a transcription factor involved in the
pancreatic development and maturation
of the endocrine β-cells in addition to
Brunner’s glands, duodenal papilla, and
bile ducts.
 PDX-1 strongly labels pancreatic
endocrine tumors and pancreatobiliary
adenocarcinomas including
adenocarcinoma of the gallbladder and
cholangiocarcinoma.

In adult tissue, PDX-1 is intensely expressed in endocrine
cells of the upper gastrointestinal tract and pancreas

SMAD4/DPC4
 Similar to Mothers Against Drosophila
4
 Deleted in pancreatic cancer-4
(DPC4)
 Nuclear transcription activator in all
normal cells.
 Deleted in ~ 50% of pancreatic
carcinomas

SMAD 4 expression lost in
pancreatic carcinoma

CK7+
CK20 NEG
ER
ER+
GYNECOLOGICAL
GATA3 &
PAX8
PAX8+
UTERUS/
OVARY
WT1+
OVARY
GATA3+
BREAST
ER NEGATIVE
NON-GYNEC

Estrogen receptor (ER)
 Member of the steroid family and
includes two types encoded by two
different genes on different
chromosomes, ER-α and ER-β
 ER-alpha: Expressed in breast and
endometrium
 ER-beta: Expressed in normal ovary and
granulosa cells
 The expression of ER-α type is an
important predictor for the response to
the antihormone therapy

Significance
 Diffuse and strong staining for ER in
the right clinical context, along with the
appropriate cytokeratin expression
profile (CK7+/CK20−), is highly
indicative of a breast or gynecologic
primary tumor
 Distinguishes endocervical (ER-) from
endometrial (ER+) adenocarcinomas

Progesterone Receptor (PR)
Main diagnostic
use
Also expressed in
• In breast cancer, predicts
response to anti-estrogens,
only weakly associated with
prognosis
• For metastatic tumors with
unknown primary, relatively
specific for breast origin
• Endometriosis (glands and
stroma)
• Hepatic adenoma
• Lymphangiomyomatosis
• Ovarian tumors: endometrioid,
fibroma, granulosa cell (juvenile)
• Cellular angiofibroma
• Solitary fibrous tumor
• Meningioma
• SPPT
• Uterus: endometrial carcinoma,
endometrial stromal tumors,
leiomyoma, STUMP
Positive control: normal breast tissue

 Adequate and rapid tissue fixation with
buffered neutral formalin is required
for optimal stain results.
 For all steroid receptors, any stain
pattern other than nuclear must be
interpreted as negative.
 More than 80% of breast carcinomas
are ER+. An ER positivity of less than
30% points to a problem with the
assay.

GCDFP 15(Gross Cystic
Disease Fluid Protein)
 Expressed by
apocrine cells or
cells with apocrine
metaplasia and
regulated by the
androgen receptor
 In the identification
of CUP, it is a
highly specific and
fairly sensitive
marker for breast
carcinoma along
with Mammaglobin
and ER alpha.
Positive control, SKIN: cytoplasmic
staining of epithelial cells in the sweat
glands.

Significance
 GCDFP-15 has
greater than 90%
specificity but much
lower sensitivity for
breast carcinoma
 Greatest in lobular
(particularly those
with signet ring cells)
and those with
apocrine features
 Triple negative breast
carcinoma is usually
negative for GCDFP-
15.

MAMMAGLOBIN A
 Highly specific for
most breast
cancers; useful to
detect circulating
or metastatic
breast cancer .
 Superior to
GCDFP15
 Positive in
endometrial and
endocervical
lesions (benign &
malignant) Cytoplasmic staining

Endothelial transcription factor 3,
(GATA-3)
 1 of 6 members of zinc finger transcription
factor family
 Very sensitive for breast and urothelial
carcinoma
 Sensitivity :Ductal 91%, lobular 100%
diffuse and strong nuclear staining
 Maintained in metastatic breast cancer
(>90%)
 In breast carcinomas, the expression of
GATA-3 strongly correlates with the
expression of the estrogen receptors but
lacks the therapeutic and prognostic value.

Bone metastasis from CA breast showing GATA-3
positivity

Other uses of GATA 3
GATA3 positive GATA3 negative
Metastatic lobular breast
carcinomas
Gastric signet ring cell carcinoma
Urothelial carcinoma Prostatic carcinoma
SCC skin SCC lung
Mesothelioma Pulmonary adenocarcinoma
T-ALL Other acute leukemias
Diffuse GATA3 staining is seen in 100% of mammary analogue
secretory carcinoma and salivary duct carcinomas

Immunoprofiles of breast
tumours
Cytokeratin profile of breast tumours:-
• CK7+/CK20-
• CK8/18/19+

DCIS
• ER, PR,
• Myoepithelial markers
• E-cadherin, BCL-2
• Cyclin D1, HER-2, p53 (in
high grade DCIS)
LCIS
• ER
• GATA3
• Negative markers:-
• Her2,
• E-cadherin
IDC-NOS
• GATA-3, E-cadherin,
• β-catenin, EGFR
• ER and PR expression in 70–
80%
• Negative markers
Lobular carcinoma
• GATA-3,
• GCDFP15,
• CyclinD1
• ER and PR expression in 70–80%
• Negative markers:-
• E-cadherin

markers
Negative markers
Medullary carcinoma • p53, EGFR
• ER and PR
expression in 70–
80%
• CK7/CK20
(exception)
• CK19, Her2,
• GCDFP15
Carcinoma with
apocrine differentiation
• Androgen receptors,
• GCDFP-15, CEA
• ER-ß
• HER-2, ER-α, PR,
• S100
Basal like carcinoma Vimentin, Pan-CK ER/PR, Her2,
GCDFP15
Metaplastic carcinoma Vimentin, Pan-CK ER, PR
Paget’s disease of
nipple
EMA, CEA
GCDFP15, Her2
CK5/6

Hepatocyte Nuclear Factor-1β
(HNF-1β)
 It is a transcription factor regulating
the growth and differentiation of
hepatocytes and cells of the biliary
system.
 Used to differentiate between different
types of ovarian and endometrial
carcinomas.
 HNF1-ß is associated with clear cell
tumours.

Normal
kidney
Clear
cell
Endom
etrial
Clear
cell
ovary
Mucinou
s
endometr
ial-weak

CA125: Oncofetal glycoprotein. In fetal life
associated with amnion, coelomic and
Müllerian epithelium
 Mesothelial
cells(visceral)
 Fallopian tube
(normal ovary
is negative)
 Breast
 Pancreas /
biliary tract
 Apocrine sweat
gland

Cancer antigen 125 in
carcinomas
 Used in ovarian
carcinomas as a
tumor marker to
follow response
to treatment and
predict prognosis
 Useful in IHC to
confirm ovarian
origin of tumor.

Other positive lesions
 Carcinomas of biliary tree, liver,
pancreas
 Carcinomas of breast, cervix,
endometrium, fallopian tubes, ovary
(serous, usually not mucinous), ovarian
sex cord tumors
 Sarcomas: Alveolar rhabdomyosarcoma
of uterus, desmoplastic small round cell
tumor of pelvic peritoneum, epithelioid
sarcoma
 Lung: carcinomas, intralobar pulmonary
sequestration, mesothelioma,
 Seminal vessel adenocarcinoma

WT1
 It is a transcriptional regulator encoded by the
WT-1 gene on chromosome 11p13 with four
isoforms.
 Regulates growth factors involved in
development of tissues from the inner layer of
intermediate mesoderm including the
genitourinary system, mesothelial cells, and
spleen.
 WT1 influences cell proliferation by
suppressing bcl-2 and regulating cadherin
and p53.
 In routine immunohistochemistry, WT-1
shows two different expression patterns

In normal epithelia, nuclear WT1 expression is largely restricted
to ovary (surface epithelium and inclusion cysts) and fallopian
tube, while WT1 is not found in endometrial or cervical epithelium.
True nuclear expression pattern characteristic for different tumors
such as serous carcinomas of ovary

Cytoplasmic staining pattern found in
endothelium, semineferous tubules (above)
and vascular tumors in addition to some
carcinoma types such as pulmonary
adenocarcinoma. It is probably due to Ab cross
reaction with an unrelated epitope.

Uses
 WT1 is mainly used to determine
ovarian origin of carcinoma
 In a poorly differentiated ovarian
carcinoma, nuclear WT1 reactivity
favors a serous neoplasm because
endometrioid, clear cell and mucinous
carcinomas are negative.

Marker HGSC LGSC ENDOMETR
IOID
CLEAR
CELL
MUCINOUS
WT1 Diffuse Diffuse - - -
p53 Abnormal - - - -
p16 Diffuse Focal Focal Variable Variable
ER Diffuse Diffuse Diffuse - -
HNF1-ß - - - Diffuse Variable
NAPSIN-A - - - Diffuse -

Immunoprofile of tumors of
FGT
Cytokeratin profile
• CK7+/CK20 variable
• CK8/18/19 +

markers
Negative markers
Endocervical
adenocarcinoma
• CEA, EMA,
• p16
(overexpressed in
>90%)
• PAX-8
• ER, PR, CK5/6,
• WT-1, PAX-2
• GFAP
Endometrial
adenocarcinoma
• PAX-8, EMA, CA125
• PR, ER
• Vimentin, GFAP
• CEA, WT-1,
• CDX-2
Brenner tumor
(benign/malignant)
Epithelial components:
• CK7, CK5/6/14,
• EMA, p63, CEA,
CA125, Uroplakin III
Fibrous stroma:
Vimentin
• CK19/20,
• Thrombomodulin

Prostate specific antigen
(PSA)
 Also known as
kallikrein-3
 Single chain
glycoprotein
synthesized by the
epithelium of
prostatic gland and
secreted into
prostatic ducts.
 Normally, protease
inhibitors rapidly
inactivate PSA that
enter the blood
circulation.

Diagnostic uses
 PSA is one of the most specific
markers for prostatic parenchyma and
prostatic carcinoma.
 Metastatic carcinoma positive for pan-
cytokeratin but negative for
cytokeratins suggests a primary
prostatic carcinoma,
 The expression of PSA and/or NKX3.1
will confirm the prostatic origin.

Prostatic acid phosphatase
 An enzyme secreted by
prostatic epithelium and
a major component of
prostatic fluid.
 PAP is more sensitive
but less specific than
PSA for prostatic glands
and prostatic carcinoma.
 Can be successfully
used in a panel with PSA
to classify metastases of
unknown primary tumor.

Prostate specific antigen and
Prostatic Acid Phosphatase
 Antibodies to prostate-specific antigen
(PSA) and prostatic acid phosphatase
(PAP) will stain more than 95% of
prostate carcinomas.
 The immunostaining of tumor cells
falls off with increasing Gleason grade
with both antibodies
 PSA / PAP may become negative after
hormonal treatment

Prostein
 A transmembrane
transporter protein
found in the Golgi
apparatus of prostatic
secretory epithelia.
 Prostein is more
specific to determine a
prostatic origin than
PSA and slightly more
sensitive.
 The loss of prostein
expression is
associated with
unfavorable clinical
course.

Prostate-Specific Membrane
Antigen, Protein (P501S), NKX3.1
 Prostate-specific
membrane antigen
(PSMA) is highly
specific for prostate
cells.
 Upregulated in
prostate carcinoma,
so that stronger
staining is seen in
higher-grade
carcinomas.
 Highly specific for
prostate carcinoma.

Alpha-methylacyl-CoA racemase
(AMACR)
 Also known as p504S
 A member of the isomerases enzyme
family involved in the metabolism of
branched-chain fatty acids and
synthesis of bile acids.
 It is expressed in the mitochondria and
peroxisomes of various normal and
neoplastic cells.

Diagnostic uses
 p504S is
overexpressed in
prostatic carcinoma
compared to benign
prostatic glands
 In combination with
p63, AMACR is used
for the diagnosis of
prostatic carcinoma
(so-called PIN
cocktail).
 CK5/6/14 can be used
as alternatives to p63
in a separate reaction

The immunohistochemical double stain
with the PIN cocktail can show one of
the following three results:
• AMACR-positive
prostatic glands
• p63-negative
myoepithelial cells
ABSENT
• AMACR-positive
glands
surrounded by
• p63-positive
myoepithelial
cells;
• AMACR-negative
prostatic glands
surrounded
• by p63-positive
myoepithelial
cells;
Normal
prostate
High
grade
PIN
Neoplasti
c glands

Immunoprofile of prostatic
tumors
Cytokeratin profile
• CK8/18/19+
• CK7/20-

markers
Negative markers
Adenocarcinoma,
prostate
• PSA, PAP, NKX3.1,
• Prostein, p504S
• androgen receptors,
ERG
• Uroplakin
Loss of basal
myoepithelial cell
layer is diagnostic :
• high molecular
weight cytokeratins
(CK5/6/14, CK-
34E12),
• p63, p40
Basal cell carcinoma.
prostate
• CK5/6, p63, p40,
• HER-2, androgen
receptors,
• Bcl-2
• CK7 in luminal
cells
• P504S (AMACR),
• PSA
Adenocarcinoma,
seminal vesicle
• CK 7, CEA, CA125,
• PAX-8,
• PSA
• PAP

Transitional cell markers
• Cytokeratin profile (CK5/6/7/20)
• GATA-3 is positive.

Uroplakin
highlights the
cell membrane
of transitional
cell carcinoma
Normal
urothelium
Highly specific
for transitional
epithelium
Uroplakin III

Thrombomodulin
 Endothelial
anticoagulant protein
clustered as CD141.
 Transmembrane
glycoprotein
expressed on the
surface of endothelial
cells, mesothelial
cells, stratified
squamous
epithelium, and
transitional
epithelium of the
urinary tract.

Uses of Thrombomodulin
Thrombomodulin
positive
Thrombomodulin
negative
Mesothelioma Pulmonary
Adenocarcinoma
Urothelial carcinoma Other pelvic carcinomas
Squamous cell carcinoma

Immunoprofile of tumors of
urinary tract
Cytokeratin profile
• CK7/20+
• CK8/18/19+

markers
Negative markers
Transitional cell
(urothelial) carcinoma
CK5, GATA-3,
thrombomodulin
Uroplakin III
CEA, S100, fascin
PAX-8,
PAX-2,
WT-1,
vimentin
Adenocarcinoma of
urinary bladder
β-catenin, CD15
Thrombomodulin,
CEA
CK5,
PAX-8
PAP, PSA, NKX3.1
Urachal carcinoma β-catenin, CDX-2,
CEA, CD15
p63
Squamous cell
carcinoma of urinary
bladder
CK5/6, p40, CK7, CK20

Common renal clear cell carcinoma
profile
 Negative for CK7 and CK20;
 Positive for EMA, CAM5.2, CD10,
RCC marker, Pax-2,Pax-8 and
vimentin.
 Of the positive markers, none are
specific, therefore a panel approach is
preferred.

 PAX8: typical reactivity is nuclear.
 Cytoplasmic/membranous reactivity should be ignored for diagnostic
purposes.

Diagnostic uses
Clear cell, papillary, and chromophobe
RCCs besides nephroblastoma are
positive for PAX-8 in addition to the
majority of collecting duct carcinoma and
oncocytomas and about 50% of
sarcomatoid RCC.

RCC antibody
 Glycoprotein expressed
on the brush border of
proximal renal tubules but
absent in other renal
areas.
 Detected in about 90% of
primary with the highest
expression intensity noted
in clear cell carcinoma,
 Collecting duct
carcinoma, sarcomatoid
(spindle cell) carcinoma,
oncocytoma, mesoblastic
nephroma,
nephroblastoma, and
transitional cell carcinoma
are negative for RCC.

CD10: Positive in the majority of clear cell and papillary
RCCs in addition to collecting duct carcinoma
demonstrating a typical apical expression but negative in
chromophobe RCC

Carbonic anhydrase IX
 Its expression is
activated during
the malignant
transformation, and
CA IX is markedly
expressed in clear
cell and a part of
papillary RCCs.
 It is used to
discriminate
between benign
renal cysts
generally negative
for CA IX and
cystic renal cell
neoplasm

Normally, the expression of CA IX is
suppressed by the wild type of von Hippel-
Lindau protein and is negative in normal renal
tissue.

Differentiating between types of
RCC
RCC
type
CK
7
CK
20
CD
10
CD
117
E-
cadh
AMA
CR
RCC PAX
8
KIM I CA
IX
DOG
-I
Clear
cell
- - + - - - + + + + -
Chromo
-phobe
+ - - + + - - + - - +
Papillar
y
+/- +/- + - + + + + + -/+ -

Adrenal cortical tumour markers

Inhibin A
 Strongly expressed in the zona
fasciculata and zona reticularis of
adrenal cortex
 Not expressed in adrenal medulla
 α-inhibin is useful to differentiate
adrenal cortical adenomas and
carcinomas from RCCs and
metastases to the adrenal with high
specificity and sensitivity

Adrenocortical adenoma exhibiting
cytoplasmic expression of inhibin

Melan-A
 Melan –A is also known as MART-1, is
an antigen recognized by antibody
A103
 Tumors other than adrenal cortical
neoplasms and malignant melanoma
are rarely positive , rendering the
antibody useful for the discrimination
of adrenal cortical neoplasm from
renal neoplasm and metastatic

Differentiating between clear cell
tumorsTumor Pan-
CK
PAX-8 CD10 p16 Inhibin Argina
se
HMB45
SOX10
TEF-3
RCC + + + - - - - -
Adreno-
cortical
-/+ - - - + - - -
Ovary
and
endom
etrium
+ + - +/- - - - -
HCC + - + - - + - -
Clear
cell
sarcom
a
- - - - - - + -
ES + - - - - - - -
Alv.
Soft
-/+ - - - - - - +

Primary Site of Origin Immunostaining Profile
Breast ER+/PgR+, GATA3+, GCDFP15 -/+,
MGB+/-, TFF1-
Endometrium ER+, PAX8+, Vimentin+
Uterine cervix p16+, HPV+, CEA+, PR-, PAX2-,
PAX8+/-
Lung TTF1+, Napsin A+, GATA3-
Thyroid (papillary/follicular) TTF1+, Thyroglobulin+, PAX8+
Thyroid (medullary) TTF1+, Calcitonin+, CEA+
Urinary bladder GATA3+, p63+, CK5/6+, p40+, S100P+,
CK903+, UPII+/-

References
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