Lecture delivered at CNCI, September, 2013

1. IMMUNO-HISTO-
CHEMISTRY
IN
GYNAECOLOGICAL
ONCOLOGY
SUJOY DASGUPTA
CNCI

3. HISTOLOGY
CHEMISTRYIMMUNOLOGY

4. IMMUNO-
HISTO-
CHEMISTRY
(IHC)

5. IMMUNOLOGY
CHEMISTRY
HISTOLOGY

6. HISTOLOGY
• Study of structural changes
by naked eye examination
as well as light/ electron
microscopy
• Rudolf Virchow
(1821-1905), Germany is
credited as the “Father of
Cellular pathology”

7. Histopathology
Surgical
Pathology
Forensic
Pathology
Cytopathology

8. CHEMICAL PATHOLOGY
Study of biochemical
constituents of
Blood, urine, semen,
body fluids
Tissues, cells
DNA

9. IMMUNOPATHOLOGY
Detection of abnormalities of immune
system of the body

10. HISTOLOGY
CHEMISTRY
HISTOCHEMISTRY

11. HISTOCHEMISTRY
• Use of special stains
to demonstrate certain
specific substances/
constituents of the
cells/ tissues
• Depends on physico-
chemical properties and
solubility of the stains Robert Feulgen
(1884-1955),
Germany

12. Amyloid Congo Red
Toluidine blue
Carbohydrate Periodic acid Schiff (PAS)
Mucicarmine
Alcian blue
Lipids Oild red O
Sudan black
Osmium tetroxide
Iron Perl’s prussian blue
Melanin Massson-Fontana
Calcium Von Kossa
Alizarin red S
Copper Rubeanic acid
DNA Feulgen
DNA, RNA Methyl green pyronin
Collagen Van Gieson’s
Micro-organisms Gram’s
Ziehl-Neelsen
Silver methanamine

13. ENZYME HISTOCHEMISTRY
• Detects enzyme-activities of cellular/ extracellular
components
Enzymes Dyes Positive results
Alkaline phosphatase Na β glycerophosphate
(pH 9.0)
Brown/ black
Acid phosphatase Na β glycerophosphate
(pH 5.0)
Black
Nonspecific esterase
(Neural tumour)
α naphthyl acetate Reddish brown
Tyrosinase
(Melanoma)
L-tyrosine, DL-DOPA Brown
Dehydrogenase Tetrazolium soln Purple
ATPase
(Myopathy)
ATP Dark
Acetyl cholineseterase
(Hirschprung’s Ds)
Acetyl choline iodide Red brown

14. ENZYME HISTOCHEMISTRY
(Contd.)
• Very limited application
• Needs special preparation of fresh tissues
• Cannot be applied to
 Paraffine-embedded sections
 Formalin-fixed tissues

15. HISTOLOGY
IMMUNOLOGY
IMMUNO-
HISTOLOGY

16. IMMUNOFLUORESCENCE
(IF)
• Coons (1941)
• To locate antigenic molecules on the cells
by microscopic examination
• Using specific antibody against that
particular antigen
• Ab is coated with a fluorochrome that
absorbs uv light (360nm) to emit light in the
visible spectrum
Fluorescein isothiocyanate (blue green)
Lissamine rhodamine (orange-red)

17. IMMUNOFLUORESCENCE
(Contd.)
• Direct IF-
• Indirect IF (Sandwich IF)-

18. IMMUNOFLUORESCENCE
(Contd.)
• Uses
Glomerular diseases
Dermatological diseases
Mononuclear cell surface markers
• Ag-Ab complex is visible under fluorescent
microscope
• Needs fresh unfixed tissue

19. IMMUNO-HISTO-CHEMISTRY
• Use of Antibody to detect specific chemical
Antigen in cells/ tissue samples evaluated
under the microscope

20. IF
-Fluorochrome
-Fluorescent microscope
-Fresh tissue
Enzyme labelling technique
Avrameas and Nakane (1967)
-HRP (Horse Radish Peroxide)
-Chromogens
-Light microscopy
-Frozen section
Immunoperoxidase technique
Taylor and Burns (1974)
-Formalin-fixed paraffin-embedded
(FFPE) tissues

21. BASIC STEPS
Tissue
processing
Diagnosis
in H/E
sections
Ag Retrieval (AR)
1. Physical
2. Chemical
Antibody
1. Polyclonal
2. Monoclonal
(Hybridoma)
Washing
-Blocking non-
specific binding
Reagents
Incubation Automation
Positive
Control

22. Chromogens-
1. Diaminobenzidine tetrahydrochloride (DAB)
2. Aminoethyl carbazole (AEC)

23. DIFFERENT TECHNIQUES
• Direct conjugate labeled Ab
• Indirect/ Sandwich Ab
• Unlabelled enzyme methods (Antibody bridging)
 Peroxidase-Antiperoxidase (PAP)
• Biotin-Avidin
• Avidin-Biotin-Conjugate (ABC)
• Biotin-Streptavidin (B-SA)
• Alkaline Phosphatase
• Tyramine signal amplification
• ‘Sausage’ tissue block techniques

24. APPLICATIONS OF IHC
• Tumours of uncertain histogenesis
• Prognostic markers in cancer
• Prediction of response to therapy
• Unknown primary
• Infection study

25. LIMITATIONS OF IHC
• Technical problems
• Expense
• Expertise
• Cannot replace conventional surgical
pathology
• Always be interpreted along with conventional
morphological description
• Cannot be applied to d/d between
neoplasm vs non-neoplasm
benign vs malignant

26. LOWER GENITAL TRACT
(LGT)

27. p16
• Most common Ag of LGT
• Tumor suppressor protein
• Inactivates cyclin-dependent kinases that
phosphorylate Rb
• HPV oncogenes E6 and E7 can inactivate pRB and
thus lead to p16 overexpression.
• p16 overexpression is a surrogate biomarker of HPV
infection (in particular high-risk HPV types)
• The intensity and distribution of p16 as well as in
nuclear versus cytoplasmic localization are important
• HPV-independent mechanisms of p16
overexpression- ovarian serous carcinoma

28. VULVAR AND VAGINAL
NEOPLASMS

29. PAGET’S DISEASE
• Primary Paget’s ds of vulva-
 expresses CK7, GCDFP, and CEA
 rare cases express CK20.16 HER2/neu
 but not CDX2, S-100, HMB45, or ER/PR
• Questionable cells near surgical margins- evaluated by CK7
since normal epidermis is CK7 negative
• Stromal invasion- Ck7 +, p16 overexpression
• Secondary vulvar Paget disease
 Of colorectal origin- CK20, CDX2, and CEA; rarely GCDFP
 Of urothelial origin- CK20, uroplakin, and thrombomodulin
• Panel approach is advised rather than reliance on a single
marker
• Pagetoid VIN- HMWK, p16, and p63
• Pagetoid melanoma- S-100 and HMB45

30. IHC for in Paget’s disease

31. VULVAR INTRAEPITHELIAL
NEOPLASIA (VIN)
• Normal and atrophic vulvar epithelium-
minimal MIB1 expression in parabasal cells
• Common type of highgrade VIN (undifferentiated or
bowenoid type)
expresses MIB1 in the middle and upper epithelial layers.
p16 expression similar to those seen in CIN (although some
cases may be negative)
• Simplex (differentiated) VIN
typically not associated with HPV
p16 is usually absent
may harbor p53 gene mutations
staining should be interpreted in context of the
morphologic findings as benign lesions (lichen sclerosis)
may be focally p53-positive

32. MISCELLANOEOUS LESIONS
• Papillary squamous lesions-
 MIB1 is confined to parabasal cells in-
normal vulvar epithelium
fibroepithelial polyps
squamous papilloma
 MIB1 in the middle & upper 1/3 of epithelium— Condyloma
• Vulvar Mesenchymal tumours-
 Most entities (e.g. aggressive angiofibroma) express vimentin, ER, PR,
desmin, actin, and CD34
 Cellular angiofibroma lacks desmin and actin
 CD117 help distinguish vaginal GISTs that mimic smooth muscle
tumors
• Granular cell tumors-
 Express S-100 protein, inhibin, and calretinin
• Caution is advised in interpreting specimens with tangential sectioning
or sub-optimal orientatIion

33. CERVICAL NEOPLASMS

34. SQUAMOUS INTRAEPITHELIAL
LESIONS (SIL)
• Role of IHC-
 distinguish dysplasia from benign mimics
 evaluating cauterized margins
 Grading dysplasia
• The markers used most widely are MIB1 and p16
• MIB1
 helps distinguish benign squamous lesions from SIL
 less helpful in distinguishing LSILs from HSILs
• MIB1 is confined to parabasal layers in
 normal cervical squamous epithelium
 immature squamous metaplasia
 Atrophy
 transitional cell metaplasia
• Full-thickness MIB1 expression - well-developed HSIL
• Lesser degrees of dysplasia - MIB1 expression in cells above the basal
layer to varying degrees
• Proper tissue orientation is critical to interpreting MIB1

36. SIL (Contd.)
• p16 expression is seen in most high-risk HPV-associated
squamous lesions
• p16 is absent or occasional focal weak expression is seen
in
 Normal
 Inflamed
 Atrophic cervical epithelium
 Transitional cell metaplasia
• HSIL-
diffuse strong p16 staining of nuclei and cytoplasm
from the upper two thirds to entire full thickness of the
epithelium
though not all cases are positive
• LSIL-
p16 expression is variable
typically limited to the lower half of the epithelium

37. SIL (Contd.)
HPV in situ
hybridization
p16 IHC
• Excellent specificity
• may be useful to further
evaluate cases of focal p16
positivity
• p16 positivity in LSIL appears to be
a function of whether high-risk HPV
is present
• diffuse strong p16
immunoexpression appears to be a
more sensitive marker
• wider availability
• easier interpretation
• better suited as a first step in
evaluating questionable morphology

38. SIL (Contd.)
• Recent studies suggest that
(Guimaraes MC, Goncalves MA, Soares CP, et al. 2005 Hariri J, Oster A. 2007 Negri G, Vittadello
F, Romano F, et al. 2004)
 Cervical LSIL that express p16 diffusely may be more likely to
progress to HSIL than those that are p16-negative
 p16-positive L-SIL progression rates 36%- 62.2%
 p16-negative L-SIL progression rates 4%-28.6%
• p16 is not a perfect predictor in this setting
 some p16-positive L-SIL may regress
 some p16-negative L-SIL may progress
• The prognostic value of p16 may be enhanced by use of an
additional marker, the HPV capsidic protein HPVL1(L1)
(Negri G, Bellisano G, Zannoni GF, et al 2008)
 L1 is expressed in the early productive phase of cervical carcinogenesis
and diminishes in the later phase of proliferation, correlating with onset
of p16 overexpression
 L1 may enhance the value of p16 in stratifying behavior of LSIL

39. SIL (Contd.)
• Recent studies suggest potential value for novel markers
 involved in early steps of DNA replication
 overexpressed in HPV infection
minichromosome maintenance protein 2 (MCM2)
DNA topoisomerase II alpha (TOP 2A)
• Antibodies to a cocktail of the two, ProEx C, show promise
• In a study (Shi J, Liu H, Wilkerson M, et al. 2007)
 Detection rate in L-SIL- p16- 76.5%, ProEx C -94.1%, both - 100%
 Stain intensity and distribution for ProEx C greater than that for p16
 Detection rate in H-SIL- ProEx C 78.6% and p16 alone 100%
• Normal ectocervix expresses ProEx C in one or two basal
layers
• Another study- better specificity for ProEx C combined with p16 than
for p16 or MIB1 alone (Pinto AP, Schlecht NF, Woo TY, et al. 2008)

40. ENDOCERVICAL
ADENOCARCINOMA in SITU
• The constellation of nuclear enlargement, hyperchromasia,
crowding, atypia, mitoses, and cribriform growth
• Endocervical AIS expresses
 increased MIB1, p16 (diffuse, strong), monoclonal CEA
 not ER, PR, vimentin, or BCL-2
• Tubal metaplasia and endometriosis
 show cytoplasmic BCL-2
 no increase in MIB1 or CEA
 p16 is either negative or focal and weak.
• Microglandular hyperplasia-
 lacks increased MIB1, p16 and CEA
• But IHC CANNOT detect stromal invasion

41. INVASIVE ENDOCERVICAL
ADENOCARCINOMA
• Must be d/d from endometrial adenoCa
• Generally endocervical adenocarcinoma expresses
 monoclonal CEA (cytoplasmic and at luminal borders)
 p16 diffuse strong nuclear and cytoplasmic pattern
 but not vimentin, ER or PR
• Low-grade endometrial endometrioid adenocarcinoma
 shows the converse profile
 CEA can be positive, though usually in a weak, luminal pattern
 p16 can also be expressed, usually weak patchy but occasionally
strong diffuse pattern
• No single (even a panel) immunostain is accurate
• Correlate clinico-radiologically
• p16 is also positive in uterine serous carcinoma
 the morphology is strikingly different

42. INVASIVE ENDOCERVICAL
ADENOCA (Contd.)
• Rarely, endocervical microglandular hyperplasia may
mimic endometrial mucinous adenocarcinoma
 Increased MIB1 and positive vimentin occurs in the latter
 Both entities lack CEA and have variable ER and PR expression
• Intestinal-type endocervical adenoCa and AIS
 May be mistaken for spread of primary intestinal adenoCa
 Primary endocervical adenocarcinoma expresses CK7 and p16 but not
CK20
 Primary colorectal adenoCa mets to Cx- the converse is true
• Minimal deviation endocervical adenocarcinoma
 Diagnosis can be challenging
 Immunostains are of limited value
 CEA, MIB1 and p53can be positive in some but not all
 p16 is not helpful because most cases are not HPV associated

43. MISCELLANEOUS
• Neuro-endocrine carcinoma (NEC)
 Small cell carcinoma- mimics small cell squamous carcinoma and
lymphoma
 Morphologic features are often sufficient
 IHC can help if the sample is small or questionable
 Most will express at least one neuroendocrine marker such as
synaptophysin, chromogranin, or CD56
 p16 tends to be present as well, creating diagnostic problems
 In such cases, the useful marker is p63, which is negative in small cell
carcinoma.
 TTF-1 can be positive in primary cervical neuroendocrine tumors
• Adenoid cystic carcinoma
 The major cell type is modified myoepithelial cell, positive for p63 and
smooth muscle actin
 The minor cell type is an epithelial cell, positive for keratin and CD117
 Distinction from basaloid squamous carcinoma is based on the distinct
morphology because overlap exists in immunostains
• Adenoid basal carcinoma
 Express keratin, p63, and p16

44. UTERUS AND PLACENTA

45. IHC MARKERS
Epithelial markers
• CK7, CK20, EMA (epithelial membrane Ag)
• Found in epithelia, stroma
• Sarcomatous elements of carcinosarcoma (MMMT)
stains +ve for CK
Vimentin
• Intermediate filament
• In mesenchymal cells, proliferating normal
endometrium, endometrial Ca
• d/d between endometrial and endocervical adenoCa

46. IHC MARKERS (Contd.)
p53
• Tumour suppressor gene
• Mutated gene is overexpressed in endometrial intraepithelial Ca (EIC) and
carcinoma
• Complex atypical hyperplasia and FIGO grade 1 endometrioid Ca focally positive
or negative
• FIGO grade 3 Ca strongly +ve (in 75% of cell nuclei)
• Negative prognostic marker in endometrial Ca
β-Catenin
• Normally membranous location
• Nuclear location in endometrioid Ca (50%)
PTEN
• Tx suppressor gene
• Lost in endometriod Ca
Estrogen and progesterone receptors (ER,PR)
• Nuclear localisation
• Strong +ve in differentiated endometrioid Ca
• -ve or focal +ve in poorly differentiated endometrioid ca and clear cell Ca
• Favourable prognostic marker in endometrial Ca and leiomyosarcoma

47. IHC MARKERS (Contd.)
Muscle markers
• Normal stromal cells +ve for vimentin and muscle actin and –ve for CK
and EMA
• h-caldesomn, desmin, SMA, SMSA- d/d between stromal neoplasms
and Tx showing smooth muscle differentiation
• Antidesmin Ab, myogenin, myo-D +ve in rhabdomyoblastic
differentiation
• Inhibin +ve in sex-cord stromal differentiation
CD10 (ALLAg)
• Distinguishing adenomyosis from invasive endometrial cancer
• distinguishing endometrial stromal neoplasms from smooth muscle
neoplasms
• But also +ve in
myometrium surrounding invading endometrial cancer cells,
occasional smooth muscle neoplasms
loss of expression in endometrial stromal neoplasms with
divergent differentiation

48. ENDOMETRIAL CARCINOMA
Endometrioid Adenocarcinoma
• Typically express CK7, CA125, EMA, ER, PR,
and vimentin
• Usually negative for CEA and CK20
• p53 and p16 are only occasionally overexpressed
(especially FIGO grade 1, 2 with morular
metaplasia)
• In significant minorities (33%- FIGO grade 2, 3)
Nuclear β-catenin expression
Loss of PTEN
Loss of DNA MMR proteins (mismatch repair
proteins- MLH1, MHS2, MHS6, PMS2)

50. ENDOMETRIAL CA (Contd.)
Endometrial serous carcinoma
• Express CK7, CA125, EMA and vimentin
• Usually negative for CEA and CK20, just like
endometrioid Ca
• Unlike endometrioid Ca, typically overexpress
p53 and p16 (not due to HPV)
• Extremely high proliferative indices with Ki-67
• Most cases have low-level expression of ER and
low or absent PR
• β-catenin overexpression, loss of PTEN and DNA
MMR are extremely rare
• HER2/neu expression- poor prognosis

51. ENDOMETRIAL CA (Contd.)
Clear cell carcinoma
• express CK7, CA125, and vimentin
• usually negative for CEA and CK20 (like
endometrioid and serous Ca)
• Unlike serous carcinomas, p53 and p16
overexpression is rare
• Unlike typical endometrioid carcinomas, ER and
PR expression is low or absent.
• Loss of DNA MMR protein expression can be
encountered
• Nuclear expression HNF-1β (hepatocyte nuclear
factor) may emerge as a specific marker

52. ENDOMETRIAL CA (Contd.)
Undifferentiated carcinoma
• Loss of ER/PR, CK
• Retains EMA
• Aberrant expression of DNA MMR proteins (association
with HNPCC)
ER/PR P53, p16
overexpresseion
% of Ki67
positivity
Endometrioid +++ (except
FIGO grade 3)
+ Rare
Serous + +++ Very high
Clear cell - + Intermediate

53. ENDOMETRIAL CA (Contd.)
d/d of AdenoCa in uterus
ER/PR WT1 CK20 CEA TTF1
Endometrioid
endometrum
+++
(except
grade 3)
_ _ _ _
Serous
endometrium
+ + _ _ _
Serous ovary/
tubes etc
++ +++ _ _ _
Endocervix _ _ _ +++ _
Breast ++ _ _ + _
Colorectal _ _ +++ +++ _
Pulmonary _ _ _ +++ +++

54. ENDOMETRIAL CA (Contd.)
Staging
• Myometrial invasion- CD10 can d/d from
adenomyosis (?)
• LVSI- CD34, CD31, D2-40 (podoplanin)
• LN mets and sentinel LN- Keratin (?)
Synchronous and metastatic ovarian Tx
• WT1 -ve, β-catenin +ve in synchronous Tx
(Euscher ED, Malpica A, Deavers MT, et al., 2005
Irving JA, Catasus L, Gallardo A, et al., 2005)

55. UTERINE SARCOMA
Uterine leiomyosarcoma
• composed of spindle cells that typically express smooth
muscle actin and desmin
• It may express CD10, cytokeratins (patchy)
• Novel markers- histone deacetylase 8 (HDAC8),
oxytocin receptors, anti-phosphohistone H3 (PHH3)
• Uterine epithelioid leiomyosarcomas frequently express
cytokeratins and lack desmin expression
• Uterine myxoid leiomyosarcomas frequently fail to
express desmin
• p16 positivity- higher risk of relapse
• Assays for p53, MIB1, BCL-2, ER/PR not currently
used

56. UTERINE SARCOMA (Contd.)
d/d of epithelioid Tx of uterus
Cytokeratin Desmin Inhibin S-100
Carcinoma +++ _ _ ++
Epithelioid
smooth
muscle Tx
++ ++ _ +
Trophoblastic +++ _ ++
Melanoma _ + + +++

57. UTERINE SARCOMA (Contd.)
Endometrial stromal Tumours (EST)
• Typically CD10, ER and PR positive
• lack staining for desmin, h-caldesmon and CD34
• If confronted with a possible endometrial stromal
tumor variant, concentrate the examination on
components that resemble proliferative phase
endometrial stroma
• Expression of CD10 without h-caldesmon and/or
desmin in these areas supports an endometrial
stromal component
• Absent CD34 d/d from metastatic sarcoma

58. UTERINE SARCOMA (Contd.)
CD10 Desmin/
h-caldesmon
Cytokeratin
Smooth
muscle Tx,
spindle
+ +++ +
Smooth
muscle Tx,
epithelioid
+ ++ ++
EST +++ _ ++

59. UTERINE SARCOMA (Contd.)
Carcinosarcoma (MMMT)
• Should be considered if 2 different geographical zones of CK and
mesenchymal markers
• Heterologous elements should be identified
• Myogenin, Myo-D1 for skeletal muscles
• Inhibin for sex-cord stromal components
Mullerian adenosarcoma
• Stromal components, smooth muscle components
Undifferentiated sarcoma
• Loss of CD10, ER/PR
Perivascular epithelioid cell tumors (PEComas)
• May show melanocytic components (HMB-45, Melan A)
Uterine Tumor Resembling Ovarian Sex Cord Tumor
(UTROSCT)
• Coexpress CK, muscle markers, inhibin
Inflammatory myofibroblastic Tx
• Alkaline phosphatase +ve

60. GESTATIONAL
TROPHOBLASTIC DISEASES
(GTD)
IHC Markers
• Pan –trophoblastic markers -
 strongly and diffusely with cytokeratins (AE1/AE3)
 Inhibin, CD10, CK18 and hydroxyl-d-5-steroid dehydrogenase
(HSD3B1)
• Syncytiotrophoblasts –
 Strongly hCG (human chorionic gonadotrophin)
 hPL (Human placental lactogen), PLAP (Placental alkaline
phosphatase)
• Intermediate trophoblasts-
 Mel-CAM (CD146), HLA-G, PLAP
 Implantation site intermediate trophoblasts express hPL, hCG
 Chorionic type intermediate trophoblasts express p63
• Cytotrophoblasts express β-catenin

61. GTD (Contd.)
Possible trophoblastic disease
CK18+++
HLA-G+++
P63 -ve
hPL+++
Lesion at
implantation site
intermediate
trophoblasts
Ki67<1%
Exaggerated
placental site
Ki67>1%
PSTT
P63+++
hPL+/-
Lesion at chorionic
site intermediate
trophoblasts
Ki67
<10%
Placental
site
nodules
Ki67
>10%
ETT
hCG+++
P63+/-
ChorioCa

62. GTD (Contd.)
Placental site nodules
• PLAP+ve, p63+ve
Exaggerated placental site
• hPL+ve, HLA-G+ve
Placental site trophoblastic Tx (PSTT)
• hPL +++, HLA-G +++, scattered hCG +
Epithelioid trophoblastic Tx (ETT)
• hPL and HLA-G negative
• inhibin, CD10, CK18 and HSD3B1 +ve
• ETTs lack the p16 expression that is typical of HPV-associated
squamous neoplasms
Choriocarcinoma
• hCG +ve for syncytiotrophoblasts
• Β-catenin +ve for few cytotrophoblasts
• If syncytiotrphoblasts are few, MIB1 may be helpful
• -ve desmin d/d from uterine mesenchymal Tx

63. GTD (Contd.)
Hydatidihorm mole (HM)
• p57kip2
Paternally imprinted gene, maternally expressed
Complete H mole (CHM) lacks maternal genes, so lacks p57
p57 +ve in Partial H mole (PHM), normal placenta and
hydropic abortion
Early diagnosis of CHM, Well validated, easy to perform,
inexpensive
• Malignant potential
H mole develoing chorioCa- Increased Mcl-1 gene product,
telomerase and hCG-H
Hyperglycosylated hCG
hCG-H activity in HM- needs chemotherapy
Early diagnosis of GTN
Change of chemotherapy

64. OVARIAN
NEOPLASMS

65. IHC MARKERS
Cytokeratin
• Intermediate filament proteins
• 20 different types
• Screening for epithelial tumours
• Cocktail of AE1/AE3 and CAM5.2
Cytokeratin 7 (CK7)
• Found in all epithelia in the female genital tract
• Epithelial tumors of the ovary and fallopian tube all exhibit cytoplasmic
and/or membrane staining for CK7
• Used to differentiate primary female genital tract adenocarcinomas from
adenocarcinomas arising in other organs.
• A panel of immunostains needs to be evaluated because some primary
ovarian neoplasms fail to stain for CK7 and a proportion of metastatic
carcinomasin the ovary are CK7-positive

66. IHC MARKERS (Contd.)
Cytokeratin 20 (CK20)
• Found in ovary, GIT, urothelium
• Most primary non-mucinous Ca are CK20 –ve
Anti-Adenocarcinoma Antibodies
• d/d between adenoCa and mesothelioma
• CD15 (LeuM1)- haematological malignancies, 2/3 of serous Ca and
endometrioid and clear cell Ca- membranous/ cytoplasmic pattern
• Ber-EP4- diffuse staining in all serous ca and peritoneal Ca
• Monoclonal CEA (mCEA)- negative in most Ca ovary except mucinous Ca
and some squamous differentiation in endometrioid Ca
• TAG-72 (B-72.3)- Tumour related glycoprotein- Monoclonal Ab, positive
in almost all Ca ovary but difficult to interprete
• MOC31- In almost all ca ovary
• Ber-EP4, MOC31, TAG-72 are more sensitive than others

67. IHC MARKERS (Contd.)
CA 125
• Using monoclonal Ab
(OC125)
• In most non-mucinous
ovarian Ca,
mesothelioma, extra-
ovarian genital
malignancy, GI
malignancy
• Nonspecific, limited use

68. IHC MARKERS (Contd.)
Inhibin
• Inhibin A (α-βA) in sex-cord stromal tumour
(uncommon but very strong pattern)
• Some cases of luteinised stromal cells around
carcinoma
• Adreno-cortical neoplasm
Calretinin
• Neural tissue
• Mesothelioma (nuclear pattern)
• Sex-cord stromal Tx (more sensitive but less specific
than inhibin)

69. IHC MARKERS (Contd.)
WT1
• Serous Tx of ovary, peritoneum (nuclear pattern- 90% sensitive)
• Rarely in serous endometrial Tx
• In transitional cell Ca, sex-cord Tx, hypercalcaemic small cell Tx of
ovary
Placental Alkaline Phosphatase (PLAP)
• Germ cell Tx (Dysgerminoma, Embryonal Ca)
• Sensitive but non-specific for dysgerminoma
• Some serous Tx
CD117 (c-kit)
• Dysgerminoma but not in embryonal Ca (nuclear pattern)
• Occasional melanoma (cytoplasmic pattern)
• Helpful to evaluate round cell Tx of ovaries

70. IHC MARKERS (Contd.)
Oct4 (POUSF1)
• In pleuripotent germ cell
• Found in germ cell Tx
• Nonspecific
• Also in dysgenetic nonneoplastic gonads
Alpha-fetoprotein (AFP)
• Yolk sac Tx of ovary, some embryonal Ca
• Hepatoma, metastatic hepatic Ca
• Hepatoid ovarian ca
• Yolk sac type endometrioid Ca
• Sertoli-Leydig cell Tx with heterologous elements
• Teratoma with yolk sac differentiation
Human chorionic gonadotrophin (hCG)
• Syncytiotrophoblast (chorioCa)
• Some dysgerminoma, embryonal Ca
• Rare poorly differentiated Ca with choriocarcinomatous elements

71. IHC MARKERS (Contd.)
S100
• Melanoma
• Neural Tx
• Some sex-cord stromal Tx
HMB45, Melan A
• More specific for melanoma
CD45 (LCA-Leucocyte common Ag)
• Lymphoma of ovary (primary/ secondary)
Neuro-endocrine markers
• Carcinoid, NEC
• Neuron-specific enolase (NSE), CD56, Synaptophysin,
Chromagranin

72. EPITHELIAL TUMOURS
CK7 CK20 CDX2 DCP4 CK17 mCEA
Primary
mucinous
+ + S + N +
Primary
endometrioid
+ N N + N N
Metastatic
colorectal
N + + + N +
Metastatic
pancreatic
S S N N
(50%)
S +
+ Always Positive N Negative S Sometimes Positive
•Almost 100% CK7 +ve
•CK7 +ve, CK20-ve – primary ovarian tx
•CK7-ve, CK20+ve – GI Tx mets to ovaries

73. EPITHELIAL TUMOURS
Serous Tx-
• CK7 +ve, WT1 +ve
• P53 +ve in 30-50% of serous carcinoma but negative in
benign/ borderline serous Tx
Mucinous Tx-
• Intestinal type- CK7 +ve, CK20 patchy +ve, CDX2 –
ve, CEA +ve
• Metastatic colorectal- CK7-ve, CK20 strongly +ve,
CDX2 +ve, CEA +ve
• Endocervical type- CK7 +ve, CK20 –ve
• Helpful to evaluate pseudomyxoma peritonei

74. EPITHELIAL TUMOURS
Endometrioid Tx
• CK7+ve, CK20 focal +ve
• 33% vimentin +ve
• β-catenin +ve (most likely of all epithelial Tx)-
better prognosis
• May mimic sex-cord stromal Tx but +ve for
EMA, ER/ PR, -ve for inhibin, calretinin
• d/d from metastatic colorectal Ca- CD20 –ve,
CDX2 –ve, CEA -ve
• d/d from metastatic endocervical adenoca- -ve
for p16 and HPV DNA

75. EPITHELIAL TUMOURS
Clear cell carcinoma
• CK7+ve, CK20-ve
• ER/PR –ve
• Hepatocyte nuclear factor-β (HNF-β) strongly +ve
nuclear pattern- highly sensitive (80%)
• WT1 –ve (d/d from serous Ca)
• CD 15+ve, EMA +ve, AFP –ve help to d/d from
yolk sac Tx (previously together called
“mesonephroma”)
• d/d from primary renal clear cell Ca- CK7+ve,
CK10-ve, RCC-ve

76. EPITHELIAL TUMOURS
Brenner and transitional cell Ca
• Brenner Tx-
express urothelial markers like CK7, uroplakin III,
thrombomodulin
p63 strong reactivity- d/d from other ovarian Tx
• Transitional cell Ca-
+ve for CK7, WT1
Mesothelin +ve (d/d from urothelial Tx mets to
ovary)
negative (or weakly positive) for uroplakin III,
thrombomodulin, p63

77. EPITHELIAL TUMOURS
Small cell Tx of ovaries
• Must be d/d from sex-cord stromal Tx
• Neuroendocrine type-
 +ve for NSE, CD56, chromagranin
 Thyroid transcription factor 1 (TTF1) +ve in secondary small cell Tx
• Hypercalcaemic type-
 +ve for p53, EMA, CD10, -ve for inhibin
Other rare Tx-
• CD45- Lymphoma
• S100, HMB45, Melan A- Melanoma
• Desmin, cytokeratin, FLI1- dysplastic small cell Tx
• Desmin, myogenin- rhabdomyosarcoma
• CD99- Ewing’s sarcoma/ primitive neuroendocrine Tx

78. SEX-CORD STROMAL
TUMOURS (SCST)
• Can be either positive or negative for CK
• Almost always EMA negative
• Positive staining for EMA suggests an epithelial
tumor, either primary or metastatic, that is
mimicking a SCST
• Inhibin is a relatively specific marker
• Calretinin is a more sensitive, but less specific
than inhibin
• Other markers- CD56 (cytoplasmic and
membranous pattern), WT1 (nuclear pattern), and
SF-1 (steroidogenic factor-1)

79. SCST (Contd.)
Granulosa cell Tx
• Negative for CK, EMA
• +ve for inhibin,
calretinin, CD56, SF-1,
WT1
• Juvenile Tx may be
EMA +ve
• Most wrongly-
diagnosed Tx of ovary

80. SCST (Contd.)
Inhibin Cal-
retinin
CK EMA CD45 CD99 Synapto-physin
Granulosa
cell Tx
+ + S N N + N
Carcinoma N N + + N N N
Carcinoid N N + + N N +
Lymphoma N N N N + S N
Small cell
carcinoma
N S S S N S S
+ Always Positive N Negative S Sometimes positive

81. SCST (Contd.)
Thecoma-fibroma group
• Inhibin, calretinin, CD56
• Thecoma- more strongly positive for inhibin and smooth
muscle actin
Sertoli-Leydig cell tumours
• IHC is important to determine cell types
• CK, Inhibin, Calretinin, EMA
• Sertiliform endometrioid Ca +ve for EMA, CK
SCTAT (Sex cord with stromal tumour annular tubules)
• May be additionally positive for laminin, type IV collagen
Steroid cell Tumours-
• Mostly –ve for WT1, CD99
• Strongly +ve for SF1
• Secretes testosterone

82. GERM CELL TUMOURS
• PLAP can be positive in most types of malignant germ
cell tumors, but it also can stain epithelial neoplasms
• Oct-4, CD117, and D2-40 are the most useful markers
for dysgerminoma
• Oct-4 and CD30 are the most useful markers for
embryonal carcinoma
• AFP and glypican-3 are the most useful markers for
yolk sac tumor
• Cytokeratin AE1/AE3 stains many malignant germ cell
tumors, and the pattern of staining can help with
classification
• EMA is negative in most malignant germ cell tumors.

83. GERM CELL TUMOURS (Contd.)
Dysgerminoma
PLAP CD117 Oct4 CK CD30 S100 LCA MPO
Dysgerminoma +
+ + N N N N N
D4-20 (podoplanin) strongly +ve (d/d – clear cell Ca)
5% +ve for hCG (syncytiotrophoblastic giant cells)
Embryonal Ca + N + + + N N N
Yolk cell Tx + N N + N N N N
Lymphoma N N N N N N + N
Granulocytic
sarcoma
N + N N N N + +
Melanoma N N N N N + N N
PLAP= Placental Alk phosphatase LCA= Leucocyte common Ag (CD45)
MPO= Myeloperoxidase
+ Always Positive N Negative S Sometimes positive

85. GERM CELL TUMOURS (Contd.)
Yolk sac Tx (Endodermal sinus Tx)
• AFP- +ve in 75% of Schillar-Duval bodies
• Specially helpful to diagnose rare varieties like
endometrioid and glandular yolk sac Tx
• Glypican3- most useful- 95% sensitive and
specific
• +ve for broad spectrum CK (AE1/AE3) but –
ve for CK7, EMA

86. GERM CELL TUMOURS (Contd.)
Embryonal carcinoma
• CK (AE1/AE3) positive
• Most striking- Oct4 +ve and CD30 +ve
• PLAP often positive
• Rarely AFP and hCG +ve
Chriocarcinoma
• Syncytiotrophoblasts stain with hCG
• All trophoblasts +ve for CK, -ve for EMA
Gonadoblastoma
• Germ cells +ve for PLAP, CD117, and Oct-4
• Sex cord cells stain for vimentin, cytokeratin, and inhibin

87. GERM CELL TUMOURS (Contd.)
Teratoma
• Little role in mature and imature teratoma
• Glial fibrillatory acidic protein (GFAP) stain glial
tissue
• GFRα-1 and MLB1 d/d between immature and
mature teratoma
• Great role of IHC in monodermal teratoma
• TTF1 and thyroglobulin +ve for thyroid tissue
• Chromogranin and synaptophysin +ve for
carcinoid
• CDX2 d/d from colorectal carcinoid

88. METASTATIC TUMOURS
• Colorectal adenoCa is the most frequent metastasis
• Can mimic endometrioid and mucinous adenoca of the
ovary
• The most useful stains for identifying metastases are
CK7, CK20, and CDX2
• Primary ovarian Ca are usually CK7 +ve, CK20-ve, and
CDX2 –ve
But primary mucinous Ca is CK7-positive, and variably
positive for CK20 and CDX2
• Metastatic tumors are usually CK7-negative
• Metastases from colorectal adenoCa are usually, but not
always, CK20-positive
• Metastatic biliary and pancreatic Ca- CDX2 –ve, DCP4
–ve (50%), mCEA +ve

89. METASTATIC TUMOURS (Contd.)
• Metastatic stomach Ca- morphology is more important than
IHC
• Metastatic renal Ca- CK7 –ve, CK10+ve, RCC +ve (d/d-
clear cell Ca ovary)
• Metastatic urothelial Ca- +ve for CD20, uroplakin,
thrombomodulin (d/d- transitional cell Ca)
• Metastatic breast Ca (micropapillary type)- CK7, WT1,
CA125 are overlaping
Mammaglobin, gross cystic disease fluid protein-15
(GCDFP15), PAX8 are helpful
• Metastatic thyroid Ca- TTF1 (thyroid transcription factor1),
thyroglobolin, calcitonin
• Metastatic endometrial Ca- WT1 –ve (d/d serous ovarian
Tx)
• Metastatic endocervical adenoCa- p16 +ve

90. MESOTHELIOMA
• Should be d/d from serous ovarian Ca
• Traditional markers used to evaluate pleural
mesothelioma are less helpful (CD15, CEA,
WT1)
CK CK5/6 D2-40 Ber-EP4 MOC31 ER WT1
Mesothelioma
+ + + - - - +
Serous Ca
- - + + + +

91. TUBAL AND
BROAD LIGAMENT
NEOPLASMS

92. TUBAL CARCINOMA
• Epithelial tumors of the tube and peritubal region
resemble ovarian tumors and have similar
immunophenotypic features
• No stains differentiate between primary tumors of
the ovary and those of the fallopian tube
• p53 and MIB1 can detect occult tubal neoplasms
(specially in BRCA carriers)
• p53 signature lesions- precursor of tubal Ca
• Intraepithelial tubal Ca can be d/d from p53
signature lesions by MIB1

93. TUBAL CARCINOMA (Contd.)
Adenomatoid tumours-
• Immunohistochemical studies indicate that they are of
mesothelial origin
• +ve for cytokeratin, calretinin, and CK5/6 and WT1
Female adnexal tumours of Wolffian origin (FATWO)
• The immunophenotype of the FATWO overlaps with
that of epithelial and sex cord–stromal tumors, but
stains for CD10
• EMA –ve, inhibin +ve, calretinin +ve d/d FATWO from
an endometrioid carcinoma variant

94. METASTATIC
CARCINOMA OF
UNKNOWN PRIMARY

95. CARCINOMA OF UNKNOWN
PRIMARY (CUP)
1. Screening IHC
2. Line of differentiation- Lymphoid, melanocytic,
mesenchymal, epithelial
3. Analysis of cytokeratin, Vimentin, EMA, CEA
4. Analysis of cancer cell products- hormones and enzymes
5. Panel of markers
6. Anatomical correlation

96. CONCLUSIONS
• IHC is a very useful
diagnostic tool
• Should always be used along
with clinical and
routine H/P correlation
• Needs expert evaluation
• Panel of markers often necessary