Immuno histo chemistry in gyn oncology

1. INTRODUCTION
2. MARKERS
3. UTERUS &GTD
4. CERVIX, VAGINA, VULVA
5. OVARY & PERITONEUM
Immuno Histo Chemistry
Gynecologic Cancers
Dr Shruthi Shivdas
Moderator : Dr Champaka
Dr ShruthiShivdas

Definition
⚫Immuno Histo Chemistry is the detection of
antigens of interest in tissue sections and cells
using antibodies.
IMMUNO-HISTO-
CHEMISTRY
Antigen – Antibody
Based
Tissue
Based
Reacti
on
Dr ShruthiShivdas

Applications of IHC
⚫Poorly differentiated tumors
⚫Mixed carcinomas.
⚫Malignancy of Unknown origin
⚫Treatment based on sub-type of cancer:
Personalized Medicine
⚫Predictive of Prognosis
⚪ ER PR , BRCA + breast ca better prognosis
⚪ C_Kit + GIST – good prognosis
⚪ TCGA ; ER PR good prognosis in EC & LMS
⚪ SOC
Dr ShruthiShivdas

PRINCIPLE
DIRECT DETECTION INDIRECT DETECTION
Dr ShruthiShivdas

Colour Detection
REPORTER
• enzyme acts on substrate / chromogen to give a
coloured product – Horse Radish Peroxidase ;
Alkaline Phosphatase)
•Flouresence Tag
•Substrate : DAB (Diaminobenzidine)
Dr ShruthiShivdas

Direct vs Indirect
Dr ShruthiShivdas

Multistep Staining
Dr ShruthiShivdas

Antigen Retrieval – Why?
⚪ Heat induced (950C) epitope retreival
⚪ Proteolytic (pepsin, trypsin, etc) epitope retrieval.
Dr ShruthiShivdas

1. “CONTROLS”
2. P53
3. P16
4. HR
Defining Positivity
Dr ShruthiShivdas

Controls for quality control
▪ Positive Tissue Controls
⚪ For positive tissue controls, a tissue type known to express your protein of
interest is stained. If the results are positive, then the assay is working correctly.
⚪ Reduces false negative
⚪ If no staining occurs in the positive control, it’s time to troubleshoot the staining
protocol!
⚫ Negative tissue controls
⚪ are used to test specificity of the antibody
⚪ reduce false positive results.
⚪ Tissue known not to express the protein of interest is stained
⚪ Negative controls should not express the target antigen. If staining occurs, you
can confirm that the staining is unspecific.
⚫ Controls can be external (tissue slide known to express the antigen
stained along with the test slides) or internal (a normal cell included
in the tissue slice being subjected to staining)
Dr ShruthiShivdas

Controls
P57 expressed by extravillous trophoblast in abortus act as positive internal
control for PHM vs CM
Dr ShruthiShivdas

P53 staining patterns
Wild type
(Missense
Mutation)
variable
proportion of
tumor cell
nuclei staining
with variable
intensity
overexpression
strong staining
in virtually all
tumor cell
nuclei,(>>C)
much stronger
compared with
the internal
control of
fibroblasts in the
center
Null pattern
(Nonsense / Truncating
mutation)
complete
absence of p53
expression with
internal control
(positive)
showing
moderate to
strong but
variable staining
cytoplasmic
p53 expression
internal
NEGATIVE
control (stroma
and normal
endometrial
glands)
showing
nuclear wild-
type pattern
C+N staining of
diffuse strong nuclear positivity involving at least 80%
of the tumor cells
Ca Endometrium - >=75% cells
Dr ShruthiShivdas

P16 staining
⚫Positive staining is defined as "block" staining:
⚪ strong nuclear and cytoplasmic expression in a
continuous segment of cells (at least 10 - 20 cells); in
squamous epithelium, block positivity needs to involve
basal and parabasal layers
⚪ In addition, complete absence of staining is also
abnormal, since it has been correlated
with CDKN2A gene silencing mutations (Histopathol
2017;70:1138) – Negative staining / p16 silencing
⚫nuclear only staining, diffuse blush / weak intensity
staining and other focal / patchy patterns should
be considered negative
Dr ShruthiShivdas

P16 staining patterns
Negative
patchy
nuclear
Staining
POSITIVE
P16 Block
N+C / C
Negative
nuclear
only
staining
Negative
Patchy
cytoplasmic
staining
Dr ShruthiShivdas

ERPR Staining
⚫Optimal cut-offs for ER and PR staining have not
been defined in malignancies of FGT.
⚫Historically, many investigators had defined 10% or
greater nuclear staining as the threshold for positive
ER and PR
⚫Historically, many investigators had defined 10% or
greater nuclear staining as the threshold for positive
ER and PR
⚫Similar cut off have been used by CAP guidelines in
Ca Breast
Guan et al, 2019
Dr ShruthiShivdas

Negative Expression
⚫Negative Expression can also be a positive marker
⚪ Loss of PTEN
⚪ Loss of ARID1A
⚪ P53 null expression
⚪ Loss of SMARCA4 (BRG1)
⚪ Loss of BAP1 in mesothelioma
Dr ShruthiShivdas

LINEAGE MARKERS
IHC – Antigens of Significance
in Gyn Oncology
Dr ShruthiShivdas

Tissue Type Markers
S 100 , SOX10
MyoG1
Dr ShruthiShivdas

Epithelial lineage markers
⚪ Cytokerartins C - keratin is the intermediate filament found in
epithelial cells.
⚪ EMA M
⚪ Ber-EP4 C+M
⚪ Broad spectrum Anti CK a/bs :
⯍ AE1/3 reacts against almost all of the CK family of proteins (AE1
recognises most of the type 1 CKs / acidic (CK9-20)whereas AE3
reacts against most of the type II CKs / basic (CK1-9).
⯍ Useful in distinguishing a poorly differentiated carcinoma from
sarcoma, melanoma or lymphoma, reactivity with AE1/3,
especially if widespread, favors a diagnosis of carcinoma.
Dr ShruthiShivdas

Mesenchymal Lineage Markers
⚫Vimentin C is the most widely distributed of the
intermediate filament proteins and is expressed in
virtually all mesenchymal cells.
⚫Most mesenchymal neoplasms in the FGT are
reactive for vimentin
⚫Certain Carcinomas of FGT with frequent and
strong vimentin co expression include endometrial
endometrioid adenocarcinomas and serous ovarian
carcinomas.
Dr ShruthiShivdas

Smooth Muscle Markers
1. α smooth muscle actin (α SMA) C+M
2. desmin C
3. h-caldesmon.
⚫ Some smooth muscle neoplasms, especially epithelioid
variants, are negative or only focally reactive.
⚫ H-caldesmon is the most specific, but is less sensitive than
desmin.
⚫ Desmin is not a specific smooth muscle marker, as it also stains
skeletal muscle.
⚪ Desmin sometimes assists in the distinction between benign and malignant mesothelial
proliferations. Benign mesothelial cells are usually desmin reactive whereas the cells of
malignant mesothelioma are generally negative
Dr ShruthiShivdas

Skeletal Mu Markers
1. Myoglobin
2. myogenin
3. myoD1
4. sarcomeric actin.
⯍ confirming the presence of RMS; diffreentiating from
small blue cell tumors of childhood in vagina (MC
site)
⯍ assist in confirming rhabdomyoblastic differentiation
in a uterine or ovarian carcinosarcoma
Dr ShruthiShivdas

Endometrial Stromal Markers
⚫ CD10 C
⚪ is zinc-dependent membrane metallo-endo peptidase located on the
cell surface.
⚪ Initially, CD10 was identified as a tumor-specific antigen of leukemia
cells, (common acute lymphoblastic leukemia antigen- CALLA)
⚫ CD10 is a reliable and sensitive IHC marker of normal endometrial
stroma.
⚫ Strong and/or diffuse positivity is found in endometrial stromal nodules
and low-grade ESS.
⚫ CD10 is also of value in confirming the presence of endometrial stroma
and in establishing a diagnosis of endometriosis.
⚫ Mesonephric remnants and tumors are CD10+
⚫ CD10 differentiates metastatic renal cell carcinoma (CD10+,) from
primary clear cell carcinoma (CD10-)
⚫ CD 10 differentiates HCC (CD10+) from metastatic lesions to liver.
Dr ShruthiShivdas

Mesothelial Markers
⚫Calretinin is a cytoplasmic calcium binding protein.
⚪ In the distinction between a mesothelioma and an
adenocarcinoma, calretinin and Ber-EP4 are the two most
useful antibodies.
⯍ Most serous (epithelial) proliferations are Ber-EP4 reactive and
calretinin negative, the converse being the rule for mesothelial
lesions.
⚪ Staining is both cytoplasmic and nuclear, with nuclear
staining required for specificity for mesothelioma.
⚪ Calretinin is also found in most ovarian SCSTs
⚫WT1
Dr ShruthiShivdas

NARROW SPECTRUM DIFFERENTIATION MARKERS
Trophoblastic
Markers
HCG – syncitiotrophoblast
PLAP- IT (chorionic > implamtation
site
HPL – IT
Mel-CAM - IT
HLA – G – all IT
Melanocytic Markers HMB 45 – most specific
Melan A / MART1
S 100
Neuroendocrine
Markers
Chromogranin – Specific but poor
sensitivity
CD 56 – sensitive not specific
Synaptophysin
NSE
PGP9.5
Lymphoid Markers LCA , CD 45
These are cell
type specific
(pathognomo
nic) markers
that are often
useful to rule
in or exclude
a targeted
question.
Dr ShruthiShivdas

NE Markers
⚫used to confirm
⚪ diagnosis of a small cell or large cell neuroendocrine
carcinoma
⚪ neuroendocrine differentiation within a neoplasm
⚫ Reactivity with NE markers is not necessary to
establish a diagnosis of a small cell NEC because
many of these are sparsely granulated and negative
with neuroendocrine markers.
⚫ In contrast, reactivity with NE markers is necessary
for a diagnosis of large cell NEC, although this
diagnosis can be strongly suspected on morphology.
Dr ShruthiShivdas

Melanotic Markers
⚫HMB45 +
⚪ Epitheloid LMS with a clear cell appearance
⚪ PEComa
⚪ Ovarian Steroid cell tumours
⚫Melan A
⚪ Ovarian SCSTs
⚫S100
⚪ ovarian SCST
⚪ cartilaginous areas within carcinosarcomas
Dr ShruthiShivdas

Tumor Suprpressor
Genes in IHC
⚫ DPC4 (Deleted in
Pancreatic Cancer, locus
4)
⚫ P53
⚫ P63
⚫ PTEN
⚫ P16
⚫ p57
⚫ WT1
⚫ BCL2
⚫ CD117 (C-Kit)
PROTO
ONCOGENES
PROLIFERATION
MARKERS
•Ki 67/MIB1
•PCNA
• A result of less than 6%
is considered low, 6-10% intermediate,
and more than 10% is considered
Dr ShruthiShivdas

Ca EM -
Pathogenesi
s
TYPE 1
TYPE II
Dr ShruthiShivdas

EH vs EIN vs EEC (Negative markers)
⚫ PTEN is a tumor suppressor gene which is mutated with
progressive loss of protein expression with progression from
EIN to EEC .
⚪ Cyclic endometrium reveals a diffuse positive staining pattern with PTEN
IHC whereas endometrial hyperplasia gives intermediate and
endometrioid carcinoma negative staining helping in differentiation of
these three closely mimicking entities
⚪ EIN vs EEC Gr1 - although both lesions show PTEN loss, additional loss
of ARID1A and increased Ki-67 proliferation index may warn for
endometrioid carcinoma.
⚪ The PTEN null rate in endometrial adenocarcinoma varies by tumor
subtype, ranging from a low of 13% of serous cancers to almost 60% of
all endometrioid cancers
⚫ bcl-2 is diffusely expressed in proliferative endometrium, in
the gland cell cytoplasm.
⚪ Activity reduced in AH and Ca.; absent in type 2.
Dr ShruthiShivdas

Subtyping of Endometrial Ca’s
⚫ Usually CK7+ Vimentin+ CA 125+; CEA & CK20 negative
⚫ In some instances, such as glandular–cribriform USC,
papillary endometrioid carcinoma and endometrioid carcinoma
with clear cells, their discrimination may be difficult and IHC
aids may be necessary.
⚫ ER PR
⚪ EEC - positive
⚪ USC and CCC - negative.
⚫ P53pattern
⚪ UPSC – mutant
⚪ EEC & CCC - TP53 wild-type.
⚫ EEC and CCC harbour ARID1A,CTNNB1 and DNA mismatch
repair gene (MMR) mutations and a mutant IHC pattern
⚫ diffuse HNF1β and NapsinA favour CCC.
Dr ShruthiShivdas

Subtyping of Endometrial Ca’s
Undifferentiated carcinoma
✔ Loss of ER/PR, CK
✔ Retains EMA
✔Aberrant expression of DNA MMR proteins
(association with HNPCC)
Dr ShruthiShivdas

HG EEC vs UPSC
Characteristics HG EEC UPSC
Morphology Solid with morules (grade1/2)
less nuclear pleomorphism
Glandular with severe
pleomorphism
ARID1A; PTEN; MMR Lost Present
P53 Wt >M (37% HG vs 3% LG) M (70-90%) >>wt
p16 Patchy / negative (20% +) Strong
Dr ShruthiShivdas

CCC EM
⚪ Express CK7, CA125, and vimentin
⚪ HNF -1 β 67-100% cases
⚪ Napsin A 56 – 93% cases
⚪ AMACR (P504S) 75 – 88% cases
⚫Unlike serous carcinomas, p53 and p16
overexpression is rare
⚫Unlike typical endometrioid carcinomas, ER and PR
expression is focal or absent.
⚫Loss of DNA MMR protein expression can be
encountered
ALMOST
ALL
CELLS
Dr ShruthiShivdas

Undifferentiated & Dedifferentiated Carcinomas
EM
⚫UC – no overt ell lineage differentiation - 2% EC
⚪ Can resemble lymphoma / plasmacytoma / HG ESS / Small
cell ca
⚫DDC – Biphasic with UC + differentiated component
(usually FIGO Grade 1 /2 EEC ; rarely G3 / USC)
⚪ Undiff component from dedifferentiation of the other
component
⚪ 40% of UC are DDC
⚫LS association in 50 -75% UC & 50% DDC
Dr ShruthiShivdas

ER
PR
P53
EM
UC
Dr ShruthiShivdas

UC
⚫ Epithelial markers are usualy focal but intense staining
⚫ As panCK can often be negative, more than one
epithelial marker to be tested for lineage confirmation
⚪ CK8/18 most likely to be positive
⚫ Vimentin +/- but ER PR negative
⚫ PAX 8 NEGATIVE
⚫ Chromogranin, synaptophysin maybe positive ;
SMARCA4(BRG1) / SMARCB1(INI1) loss may be there
⚫ Mimics :
⚪ Grade 3 EEC – PAX 8 neg in UC
USC – Vimentin neg; Bcatenin + in UC
NE Ca – NE marker positivity < 10 % in UC
Dr ShruthiShivdas

MIXED Carcinoma EM
⚫ Admixture of >=2 diff histological types of Ca EM;
atleastone of which is either serous or clear cell Ca.
⚫ ANY PERCENTAGE of confidently demonstrable high
grade carcinoma is sufficient to label as MC , as any
proprtion of USC or CC confers an adverse prognosis.
⚫ MC – EEC + USC (10% of all EC’s)
⚫ Behaviour of tumour dictated by nature of the highest
grade component ; graded as high grade
IRRESPECTIVE of the relative % of USC/CCC
component.
⚫ Should NOT be used to indicate morphological vairnats
of EEC ( clear cell / serous changes)/ CCC/ USC or DDC
or CS.
Dr ShruthiShivdas

Carcinosarcoma
⚫ Sarcoma component predominates (40 – 60% cases)
⚫ CA component is usually EEC / USC
⚫ Sarcoma is usually HG sarcoma
⚫ Metastasis is carcinomatous in 90%
⚫ The sarcoma component usually shows CK positivity.
⚫ IHC may be needed to confirm specific mesenchymal
differentition if heterologous sarcoma component ( eg:
RMS ; chondrosarcoa)
⚫ USC and RMS diferentiation associated with worse
survival
Dr ShruthiShivdas

EMC vs ECC
Vimentin - endocervical carcinomas rarely stain (weak focal staining in up to
13% of endocervical carcinomas) compared to almost all EEC
McCluggage et al - “membrane expression of mCEA in the glandular
component of EMC vs more common cytoplasmic localization in ECC.
vimentin-positive/CEA-negative phenotype remained the most constant among all
endometrial cancers, whereas ER PR expression may depend on endometroid type
& degree of differentiation.
Reed et al, 2006
Dr ShruthiShivdas

Cx Bx
Clinically, Uterus and endocervix ballooned with large polypoid tumour protruding
through os ; obese; DM; MRI s/o large EM growth with no cx stromal inv
Dr ShruthiShivdas

PD Ca on EB
p53
Dr ShruthiShivdas

EB
72 yr old with PMB ; no CM ; not obese; thyroid swelling (PTC FNA) ; EM growth
and SC adenexal mass . TM normal
EB
Final HPE
Final IHC
Dr ShruthiShivdas

Endometrial METAPLASIA
⚫ Morphological alteration of the endometrial epithelium
from one mature cell type to another.
⚫ More in postmenopausal women; esply if ERT within 3
months prior to EM sampling. ; chronic inflammation/
irritation.
⚪ Eosinophilic(oncocytic)
⚪ Tubal / ciliated
⚪ Squamos
⚪ Hobail
⚪ Clear cell
⚫ Each MAYBE associated with a precursor lesion or
carcinoma, esply if extensive or architecturally complex /
if IHC markers positive.
Dr ShruthiShivdas

Synchronous EM and Ovarian Primaries
Dr ShruthiShivdas

WHO 2020
⚫4 criteria to be met to label “Synchronous”
(endometroid types) justifying conservative
management:
1. Both tumors are low grade
2. <50% MM invasion
3. Absence of ‘extensive’ (>=5 vessels) LVSI at any site
4. No involvement at any other site
ESMO ESTRO joint Recommendations 2020
“If all WHO 2020 criteria mentioned above are met and
the ovarian carcinoma is pT1a, no adjuvant
treatment is recommended”
Dr ShruthiShivdas

Synchronous EM and Ovarian Primaries
⚫ Vimentin has been suggested to differentiate ovarian and
uterine endometroid cas.
⚪ Desouki et al - 82% of uterine corpus cases were positive for Vimentin,
while 97% of ovarian endometrioid adenocarcinoma cases were
negative for vimentin.
⚪ Most dependable when both sites similar
⚪ Cannot comment when EM+/ Ovary neg
⚫ One of the ancillary techniques to differentiate double primary
from metastatic carcinoma is using molecular methods as
MSI, mutations in PTEN .
⚪ Synchronous ovarian primary lower incidence of MSI and PTEN
mutations compared to uterine primary; B catenin can be positive in
both.
⚫ UPSC & HGSOC
⚪ WT1 positive in HGSOC; neg in USC
Dr ShruthiShivdas

d/d of AdenoCa in uterus
Focal
/neg
GCDFP
Dr ShruthiShivdas

Her 2 testing in USC
⚫ HER2 protein (Membrane) overexpression and/or gene amplification
is present in approximately 25% to 30% of USC’s
⚫ A recent ph2 (n=61) clinical trial showed significant prolonged PFS
in advanced-stage (+9months) and recurrent (+3.5mo) HER2–
positive USC when trastuzumab was added to the standard
chemotherapy regimen.
Fader et al. Randomized Phase II Trial of Carboplatin-Paclitaxel Versus Carboplatin-Paclitaxel-Trastuzumab in Uterine Serous
Carcinomas That Overexpress Human Epidermal Growth Factor Receptor 2/neu. J Clin Oncol. 2018
⚫ This targeted therapeutic approach was recently endorsed by NCCN
⚫ Fader et al criteria :
⚪ IHC –
⯍ 3+score - 30% strong complete or basolateral/lateral membrane staining-⯍
”POSITIVE”
⯍ 2+ - <=30%-----⯍ Go for FISH to confirm positive / negative
⯍ 1+ - <10% cells
⚪ FISH (FFPE) –
⯍ HER2/CEP17 ratio >2.0 --⯍positive
⯍ CEP : Centromere Enumeration Probe
Dr ShruthiShivdas

TCGA (EEC+USC)
18%
40% 12% 30%
Dr ShruthiShivdas

NCCN 2021
NSMP
Dr ShruthiShivdas

ESGO ESTRO 2020
⚫ Performing one of the surrogate marker tests in isolation
is insufficient, as a combination of positive tests can
occur in approximatively 5% of al carcinomas.
⚫ Smaller studies showed that the molecular classification
is also applicable to non-endometrioid tumors including
serous, clear cell, undifferentiated carcinomas, and
uterine carcinosarcomas.
⚫ For adjuvant treatment recommendations, the molecular
classification seems to be particularly relevant in the
context of high-grade and/or high-risk endometrial
carcinomas.
⚫ In low-risk endometrioid carcinomas, the molecular
classification may not be required.
Dr ShruthiShivdas

ESGO ESTRO 2020
modified binary FIGO grading is recommended lumping
together grade 1 and grade 2 endometrioid carcinomas as
low-grade and grade 3 as high-grade.
NO adjuvant
NO adj if polyp with no MMI
VBT
May be omitted if <60 yrs
VBT +
EBRT if substantial LVSI / stage II.
Adj CT can be considered, especially for
high-grade and/or substantial LVSI
EBRT with concurrent and adjuvant
CT or
alternatively sequential CT & RT.
CT alone is an alternative optional
Tumor debulking with resection of
only enlarged LN
Primary systemic therapy should be
used if upfront surgery is not feasible
or acceptable .
In cases of a good response to
therapy, delayed surgery can be
considered .
Dr ShruthiShivdas

NCCN 2021
Recurrent / Met Ca Endo
TMB – H >= 1o mutations / Megabase
NTRK - Neurotrophic Tyrosin Kinase
Dr ShruthiShivdas

Other uses of IHC in Ca EM
⚫Staging
⚪ Myometrial invasion- CD10
⚪ LVSI- CD34, CD31, D2-40 (podoplanin)
⚪ LN mets and sentinel LN- Keratin (?)
Euscher ED, Malpica A, Deavers MT, et al., 2005 Irving JA, Catasus L, Gallardo A, et al.,
2005
Dr ShruthiShivdas

Leiomyomatous (SM) neoplasm vs Stromal
Neoplasm
⯍Leiomyomatous neoplasms are diffusely reactive with desmin and h-caldesmon.
CD10 is usually negative or focally reactive.
⯍Endometrial stromal neoplasms are usually diffusely CD10 reactive, and negative
or focally positive with desmin and h-caldesmon.
⯍SMA is of limited value because many endometrial stromal neoplasms are diffusely
positive, an indication that considerable immunophenotypic overlap exists between
uterine smooth muscle and endometrial stromal neoplasms, as these two cell types
develop from a common progenitor within the uterus
Dr ShruthiShivdas

ESS
Z3H7B-
BCOR
BCOR ITD
LG ESS and HG ESS are characterised by distinctive pathognomonic
chromosomal translocations, resulting in gene fuions
•JAZF1– SUZ12 for LG ESS
• YWHAE–NUTM2A/B or Z3H7B-BCOR or BCOR ITD (Internal Tandem Duplication) in HGESS.
Dr ShruthiShivdas

LG ESS HG ESS
Tumour with
haemorrhagic and
necrotic areas
Dr ShruthiShivdas

EM Stromal Sarcomas - Subtyping
⚫ However, molecular testing is not currently standard practice; therefore,
immunophenotype is helpful in the differential diagnosis.
Dr ShruthiShivdas

ESS
⚫ LGESS - diffuse immunopositivity for CD10, ER and PR
⚪ Pan CK Positive
⚪ SM markers + - Desmin; Caldeson
⚪ SCST mrkers + - Calretinin; WT1; Inh; CD99
⚫ HGESS – CD 10/ER/PR negative ; cyclin D1strong nuclear + (80%)
and c-Kit (M/C)
⚫ UUS include a heterogeneous group of aggressive neoplasias showing
marked pleomorphism, necrosis and mitotic activity and variable
expression of CD10, ER and PR.
⚫ UUS should be diagnosed only after exclusion of the most
common mimics: leiomyosarcoma, carcinosarcoma, undifferentiated
carcinoma, rhabdomyosarcoma .
⚪ Exclusion of gene fusions associates with HG ESS
⚪ Often + for p53 & p16 ; ER PR & CD 10 vairable
⚪ Several authors advocate removal of this group as a lot of overlap with HG ESS
Dr ShruthiShivdas

LM vs Sarcoma
Kuhn E, Ayhan A. J Clin Pathol 2018;71:98–109. doi:10.1136/jclinpath-2017-204787
LMS vs LM & STUMP
Diffuse p53 and p16 and a high Ki-67 proliferation index favour a LMS;
however, some smooth muscle tumours with uncertain malignant potential and
some leiomyomas rarely show overlapping patterns.
LGESS vs LM (particularly cellular LM)
LM , as opposed to LG ESS, usually express desmin and h-caldesmon but not
CD10.
Dr ShruthiShivdas

Uterine leiomyosarcoma
⚫Criteria : Severe nuclear atypia + Tumor cell
necrosis / mitosis > 4 / 10 HPF
⚫typically express smooth muscle actin and desmin ,
caldesmon(most specific) and SMA
SM marker expression patchy in myxoid variety / poorly
differentiated.
⚫It may express CD10, cytokeratins (patchy)
⚫Novel markers- histone deacetylase 8 (HDAC8),
oxytocin receptors, anti-phosphohistone H3 (PHH3)
⚫p16 positivity- higher risk of relapse
Dr ShruthiShivdas

uLMS variants
frequently express cytokeratins
and lack desmin expression
Loss of INI1/SMARCB1
differentiate epithelioid sarcoma
from epithelioid
hemangioendothelioma
Df PEComa
Dr ShruthiShivdas

ER/PR in Sarcomas
✔ESSs frequently express ER in 40% to 100% of tumors and PR in 60% to 100% of
tumors
✔LMS : ER (7–71%) and PR (17–60%)
✔ hormonal therapy has been confirmed effective for recurrent, metastatic or
unresectable LGESS and ER+/PR+ uLMS .
✔Athough ER PR expression in HGESS & UUS vary from 20 – 40%, both have
rapidly deteriorating course & poor response to HT.
Dr ShruthiShivdas

Case 1
60 year old lady with post menopausal Bleeding
16 weeks size uterus with a fundal EM polypoid lesion on
imaging with >1/2 MM invasion
EB (scanty)was repoted as carcinosarcoma with
predominant sarcomatous elements(No IHC required)
Underwent TAH BSO RPLND ICO
C/S: fibroid like lesion
Final HPE : Tumor cells have an epithelioid appearance
but are less cohesive. Intercellular bridges are absent.
IHC : Positive for SMA, H caldesmon ; Pan CK, ER/PR ;
p16; p53 mutant
Negative for EMA, Cyclin D1 and CD10
Imp : Epitheloid LMS
Dr ShruthiShivdas

Case 2
⚫ 42-year-old female patient presented with complaints of
irregular menstrual cycles for 2 years with excessive
bleeding; TAH done with a preop/ intraop diagnosis of 18
weeks fibroid
⚫ Gross :multiple gray-yellowish ropy nodular masses
ranging in size from 0.1 to 1.5 cm seen over the entire
endomyometrium
⚫ Microscopy :
Endometrial stromal cells were seen without any
evidence of nuclear atypia, showing extensive
myometrial permeation (tongue-like growth) by sharply
defined tumor islands with pointed edge
⚫ IHC : CD10+; ER PR +; Cyclin D1 neg; SMA+, desmin-
⚫ LG ESS
Dr ShruthiShivdas

Case 3
⚫ 21-year-old, G0P0 woman presented with a four-
month history of excessive vaginal bleeding with a
14 weeks uterus & 3 cm exophytic mass at the
posterior lip of the cervix.
⚫ Cervical Bx : Small round cells with signficant
necrosis; negative staining for multi-cytokeratin
(AE1/AE3), S-100 protein, CD 10, cyclin D1,
caldesmon, myogenin, and desmin; Ki67 >70%.
The preoperative differential diagnoses were HG-
ESS and UUS.
⚫ Final HPE :uniformly high-grade round cells with
brisk mitotic activity arranged in tight nests, with
extensive LVSI.
⚫ Additional IHC studies revealed positive diffuse
staining for vimentin, CD 10, and cyclin D1. The
tumor stained negative for desmin, ER, and PR.
⚫ Diagnosis: HG-ESS
Dr ShruthiShivdas

Case 4
⚫ A 69-year-old female, postmenopausal with abdominal discomfort and
enlarged uterus.
⚫ MRI s/o degenerated fibroid
⚫ (a) Uterine tumoral mass with areas of hemorrhage and necrosis. (b)
Atypical nuclear were seen in myxoid stroma
⚫ The mitotic index accounts three to four mitotic figures per 10 high-power
fields (HPFs). The presence of tumor cell necrosis is noted.
⚫ IHC : SMA& caldesmon + CD117+ ; Pan CK neg; ER PR neg; Ki67 – 20%
⚫ Imp : Myxoid LMS
Dr ShruthiShivdas

Case 5
⚫ 58 yr old with PMB
⚫ 14 weeks uterus with a 11 cm EM lesion extending into cervix
with < ½ MM invasion
⚫ EB – scanty pleomorphic neoplastic cells in a background of
extensive necrosis
⚫ IHC : CD10+; ER diffuse +. PR patchy; Cyclin D1neg.
Negative for SMA, desmin, CD 117. Ki67 – 15%
Possibility of HGESS
⚫ Final HPE :Tr cells arranged in sheetlike pattern with
extensive tumour necrosis and high mitotic activity p53+; Pan
CK negative
Imp : UUS
Dr ShruthiShivdas

TestingforLS
Dr ShruthiShivdas
TUMOUR TISSUE
(FFPE)
MSI Testing
(PCR)
Direct IHC
for MMR
proteins
MMR Gene
Testing

⯍Cytotrophoblast cells are the stem cells which give rise to SCT and IT.
⯍SCT --🡪 HCG
⯍ The IT cells that infiltrate the decidua, myometrium, and spiral arteries
of the placental site are termed implantation site IT, and those in the
chorion laeve in the fetal membranes are termed chorionic-type IT.
Villous
Tropho
blast
Extravill
ous
& ETT
PSTT & EPS
Dr ShruthiShivdas

Pathology Gross
Dr ShruthiShivdas

Why IHC in GTN
1. CCs , especially those with a monomorphic appearance, can be
confused with a poorly differentiated neoplasm, PSTT, or ETT.
Choriocarcinoma is highly metastatic & sensitive to chemotherapy,
whereas PSTT and ETT are relatively indolent neoplasms for which
surgical resection or total hysterectomy is considered the treatment of
choice because they tend to be refractory to conventional
chemotherapy.
1. ETTs may be confused with hyalinizing SCC of the cervix
morphologically (epithelioid growth and eosinophilic fibrinoid deposition
in ETT that may resemble keratin in SCC) and also because many ETTs
develop at the cervix or lower uterine segment from reproductive ages of
women.
1. ETT vs PSN can be at times very difficult because they are composed of
chorionic-type IT cells. ETTs have malignant potential that requires
surgical intervention and close follow-up. In contrast, PSNs are benign
Dr ShruthiShivdas

Cytotrophoblasts
express β-catenin, p63, PLAP
Pantrophoblastic markers negative
CC may be positive for any of the markers ; HCG(ST)/ HPL (ST)/ HLAG(IT)/
Inhibinα (ST)/ MCAM(IIT)/ p63(CIT& CT))/ SALL4(ST), as it can have all cell types
Ki 67 very high (>90%)
(SALL4
)
Pan Trophoblastic markers
Pan Ck, CK 18, Inhibin; and the
following
hydroxyl-d-5-steroid dehydrogenase
(HSD3B1
CC – Cyto-🡪+SCT+IT
PSTT- Imp IT
ETT – Chor- IT
Dr ShruthiShivdas

TOPHOGRAM - GTN
The stepwise immunohistochemical assessment of a lesion suspected of a
trophoblastic tumor or a tumorlike lesion
3-tiered sequential staining procedure
1. The first tier is to discriminate a trophoblastic vs a nontrophoblastic lesion.
2. The second tier is to determine if the lesion is a choriocarcinoma, a lesion
related to implantation site IT cells, or a lesion related to chorionic-type IT
cells.
3. The third tier is to distinguish a benign tumorlike lesion vs a true
trophoblastic neoplasm.
PAN
Trophobl
astic
markers
CYCLI
N E +
CYCLI
N E +
++
Dr ShruthiShivdas

IHC for PDL1
⚫Strong and constant overexpression of PDL1 has
been found in all subtypes and settings of GTN
tumors from French reference gestational
trophoblastic center
⚫IHC detection may give scope for treatment in
multiagent resistant GTN
Dr ShruthiShivdas

Gestational CC Non gestational CC
Dr ShruthiShivdas

Gestational vs Non gestational CC
⚫ Distinction of non-gestational choriocarcinoma from
gestational choriocarcinoma is impossible on the
histomorphology unless an evidence of pregnancy or other
germ cell neoplasm is encountered.
⚫ Non gestational is less chemo-sensitive ; poor prognosis; in
assocation with mixed GCT.
⚫ To differentiate gestational from nongestational tumors, it is
necessary to determine whether a paternal contribution is
present in the genome of the tumor.
⚫ Genotyping including short tandem repeats (STR) analysis
from the tumor, to identify paternal alleles and comparison
with those found in the patient and her partner should define
the presence or absence of paternal DNA and establish
whether or not a tumor is gestational.
Dr ShruthiShivdas

P57 / Kip 2
⚫ A particular use is in the distinction of CHM from PHM ,
hydropic abortion and normal placenta.
⚫ p57 is expressed in the nuclei of cytotrophoblast and
villous mesenchyme in the normal placenta, hydropic villi
and villi of partial mole because all have a maternal
component.
⚫ In contrast, p57 is negative in the villi of complete mole,
because these villi are paternally derived and lack
maternal DNA.
⚪ Positive reactivity in extravillous trophoblast acts as an internal
positive control.
⚫ p57 cannot determine whether a trophoblastic neoplasm
has arisen from a preexisting partial or complete mole or
nonmolar pregnancy (as no villi)
Dr ShruthiShivdas

Trophogram - Moles
5%
persistenc
e
0.5% CC
Hydropic
Abortion
Monogynic
diandric
triploid
15%
persist
5% CC
Differentiating molar pregnancy from mimics like hydropic abortion and
trisomies in early gestation period is often difficult
Differentiating between CHM amd PHM also important for prognostication.
P57 Kip2 is a maternally expressed paternally imprinted gene.
Dr ShruthiShivdas

Malignant potential of Moles
⚫greater expression of Mcl-1(Myeloid Cell
Leukaemia), which is an antiapoptotic gene
⚫Overexpression of p21
⚫Loss of p27 (kip1)
⚫Overexpression of Cyclin E.
⚫Serum hCG-H activity in HM-
⯍ Early diagnosis of GTN
⯍ start of chemotherapy
Dr ShruthiShivdas

Case
⚫ A 28-year-old gravida 4 para 2, underwent a C-section 10 months ago, followed
by breast-feeding for 10 months thereafter with postpartum irregular
menstruation and anasarca
⚫ HCG – 1000 to 1500
⚫ MRI indicated SOLs in the left posterior wall of the uterus, with undetermined
nature and multiple cystic lesions in the right posterior wall of the uterus.
⚫ Underwent TAH BSO as per pt preference.
⚫ Uters C/S: Placenta-like tissue mass of approximately 5 cm in diameter, which
was blue-violet in color, as well as a rough and fragile surface.
⚫ HPE : strongly suggested PSTT, which invaded the walls of the uterus, and
affected the cervical canals and the cervix .
⚫ IHC : results revealed CK (+), HCG (weak+), p63 negative, HPL++ and Ki-67
(+30%).
⚫ Imp : PSTT
Dr ShruthiShivdas

Cervix & Vagina
Dr ShruthiShivdas

WHO 2020
✔Hpv /Non HPV differentiation also to be done for EC adenocarcinomas.
✔Also for AIS.
✔True endometroid adenoca extremely rare ; other mimics to be excluded.
✔Serous Ca removed as no true SC of cervix.
✔Adenoca NOS removed as all adenoca to be alloted either HPV asso/ HPV independent
Dr ShruthiShivdas

HPV DNA – Episome (LR) vs
Integrated(HR)
⚫ A hallmark of HPV associated carcinogenesis is the integration of the viral DNA
into the cellular genome, usually accompanied by the loss of expression of
the viral E2 gene. E2 regulates the expression of the E6 and E7 oncogenes.
Loss of
regulatory
E2
Dr ShruthiShivdas

P16 overexpresion in HPV related
cancers
✔Cyclin D-cyclin dependent kinase (CDK) 4/6 complex initiates phosphorylation of the
tumor suppressor protein, pRb.
✔ The hyperphosphorylation of pRb leads to release of the transcription factor E2F
into its active state, which drives the expression of downstream gene products
allowing the cell to transition from the G1 to S phase.
P16 = CDK2A inhibitor (CDKN2A)
Dr ShruthiShivdas

Surrogate Markers of HR HPV infection
&
PCR
1 2 3
or
E6E7 mRNA by PCR
Dr ShruthiShivdas

⚫ HR HPV DNA PCR on FFPE
⚫ DNA in situ hybridization (ISH) on FFPE
⚫ The Food and Drug Administration (FDA) approved the PCR to detect
E6/E7 mRNA as the “gold standard” for the detection and typing of
HPV.
⚪ Unfortunately, this assay can be performed exclusively in fresh-frozen tumor tissue,
leading its limited use in the clinical practice.
⚫ RNA in situ hybridization for detecting the HPV E6/E7 mRNA
transcripts can detect the virus in its transcriptionally-active status also
in FFPE.
⚪ RNA ISH is incredibly sensitive, being near to 100%, with a specificity approximately
of 90%
⚫ p16INK4a overexpression on IHC
⚪ relatively inexpensive & highly sensitive for transcriptionally active HR HPV.
⚪ good concordance between RNA ISH and p16 IHC has been reported
Dr ShruthiShivdas

HPV RNA ISH / p16 (FFPE)
P16 IHC HPV E6/E7 mRNA Insitu Hybridisation
⯍more sensitive marker
⯍wider availability
⯍cheaper
⯍easier interpretation
⯍better suited as a first step in
evaluating questionable morphology
⯍+ in HSIL
⯍Neg in LSIL
✔Excellent specificity
✔ may be useful to further evaluate
cases of focal p16 positivity
✔ LSIL vs mimics (reactive /
inflammatory changes)
+ ve in ASC/LSIL(cytology) lesions
harbouring HG changes, even when p16
negative
Most imp application for E6E7 mRNA ISH are in
1. To reduce overdiagnosis of LSIL in reactive changes in morphologically
ambiguous cases. LSIL overdiagnoses on biopsy may result in the
erroneous clinical impression that a cervical lesion has been sampled
appropriately. So, if LR/HR HPV mRNA negative, resample.
2. ASC/LSIL lesions on cytology, which would generally go for F/U as per
ASCCP, but may harbour HG change (These lesions would generally be
p16 negative)
Dr ShruthiShivdas

SILs versus benign mimics
⚫ MIMICS of SILs
⚪ reactive and metaplastic squamous changes, atrophy and cytological atypia due to
cautery artefact.
⚪ p16 helps differentiate ; positive in SILs
⚫ The maturation level of the metaplastic epithelium at the time of HR
HPV infection determines the type of HSIL that develops there
⚪ Thick HSIL ( >10 cells thick) from
mature metaplasia.
⚪ Thin HSIL (<=9 cells thick) develops from
immature metaplastic cells.
⚪ Both are p16 +
H&
E
P16
IHC
Dr ShruthiShivdas

LSIL vs HSIL
⚫ LAST recommendation include the use of p16 IHC in lesions morphologically
doubtful for CIN 2 to stratify the management of CIN2 into immediate treatment
or a period of observation.
1. CIN2 are histologically heterogeneous, may behave like CIN3 / CIN1.
2. High inter-observer variation & poor reproducibility
⚫ p16 block positivity (basal & lower 1/3 epithelium) is found in the majority of
HSILs
⚪ Negative in 1/3 of HSILs
⚪ Positive in 10% of LSILs (cases driven by HR HPV; likely to progress to HSIL).
⚫ But, this practice could jeopardise diagnosis of the p16-negative HSILs,
resulting in underdiagnosis as LSIL. WHO 2020
⚫ Kuhn et al, 2017 – “whether p16-negative CIN-2 behave like p16-positive
ones?
⚪ p16-positive L-SIL progression rates 36%- 62.2%
⚪ p16-negative L-SIL progression rates 4%-28.6%
P16
MIB-1
SCJ markers
clinical ‘correctness’ of LAST
recommendations
Dr ShruthiShivdas

WHO 2020 –When to perform p16
HSIL vs mimics
HSIL (CIN2) vs LSIL –
LAST
To r/o missed high grade disease in
biopsy specimes morphologcally
interpreted as <=LSIL, but with Pap
>=ASC-H
Dr ShruthiShivdas

LSIL vs HSIL – MIB1
⚫ Along with p16 positivity, Ki-67 may also reliably assist the differential
diagnosis between HSIL and LSIL, whereby full-thickness / >lower1/3
proliferation favours HSIL.
⚪ MIB1 is confined to parabasal layers in
⯍ normal cervical squamous epithelium
⯍ immature squamous metaplasia
⯍ Atrophy
⚪ Full-thickness MIB1 expression (>90%)- well-developed HSIL
⚪ LSIL- MIB1 expression in cells above the basal layer to varying degrees
⚫ MIB1 may also be used to evaluate cauterized cervical resection
margins, i.e., cauterized CIN 3 from cauterized nondysplastic
squamous epithelium
⚫ “Clearly, appropriate orientation of the epithelium is
necessary in order to prevent misinterpretation.”
⚫ Avoid tangential sectioning
Dr ShruthiShivdas

SCJ markers in SIL
⚫ SCJ cells are the cells of origin of SILs
⚫ SCJ proteins
⚪ CK7
⚪ CK1
⚪ MMP7
⚪ p63
⚫ SCJ markers are strongly and diffusely expressed in HSILs,
but either negative or patchy in LSILs, so that they may be
used as adjunct IHC markers in distinguishing between HSIL
and LSIL.
⚫ Of these, CK7 is widely used.
⚫ CK7 expression has been correlated with an increased risk of
LSIL progression and accordingly proposed as a risk stratifier.
Kuhn E, Ayhan A. J Clin Pathol 2018;71:98–109.
Dr ShruthiShivdas

SCC Cervix
⚫ 80-90% cervical carcinomas are SCCs.
⚫ >95% SCCs are HPV associated
⚫ Macroscopic/ morphological appearance are similar
⚫ If p16 IHC / molecular HPV typing of SCC not available, to be
designated as “SCC, NOS of the uterine cervix”
Type HPVA (Associated) HPVI (Independent)
Frequency >95% of SCCs 5 – 7% of SCCs
Median Age 51 yrs 60 yrs
P16 Strong diffuse block Negative in most
P53 Negative Positive in many
Morphological pattern (MC) Non keratinizing Keratinizing
Prognosis ⯍Advanced stage
⯍More LN involvement
⯍Worse DFI &OS
Dr ShruthiShivdas

HPVA Adenocarcinoma Cervix
⚫ Adenoca accounts for 5% of all cervical ca’s in non-screened
populations, and 10-15% in screened populations
⚫ Mean age 40-42 yrs ; ER PR NEGATIVE
⚫ It is important to note that p16 in a cervival adenoca may also be
positive in a drop metastasis from any of these non HPV Cas :
1. Serous Ca of EM / adenexa – will also resemble the usual type due to
papillary pattern
2. HG EEC (EC Adenoca ER PR neg, vimentin+)
3. CCC EM /( Endocx also)
All are p16 positive(block)
HR HPV mRNA ISH helps to differentiate (+ve in Endocx Usual
adenoca)
Usual Type Mucinous Type
75% of all EC adenoca 10% of all EC adenoca
Cells with mucinous
cytoplasm <50% of tumour
Cells with mucinous
cytoplasm =>50% tumour
Dr ShruthiShivdas

Gastric Type Adenocarcinoma Cervix
⚫ HPV independent ; 10-15% of all cervical AC
⚫ Previously called (NOT recommended) : Minimal Deviation / Mucinous
Adenocarcinoma / Adenoma Malignum
⚪ Morphology varies from extremely well differentiated (MDA) to poorly differentiated.
⚪ Median age 50 – 55y : significantly older than usual
⚫ PJ association ; higher incidence in Japan
⚫ Precursors : Atypical lobular endocervical hyperplasia & gAIS
⚫ Typically upper cervix with extension into LUS
⚫ Barrel cervix with induration of walls; There may be no visible lesion esply if early
⚫ MRI – multilocular lesion with solid components from endocervical glands into
deep strom or a stromal solid lesion
⚫ IHC
⚪ gastric pyloric markers HK1083 + ; MUC6 + (non specific)
⚪ ER/PR/p16 negative
⚪ PAX 8/CEA/ CK7 +
⚪ P53+ (50% cases)
⚫ Aggressive with invasive destruction, extrauterine and advnaced disease,
independent of the degree of differentiation.
Dr ShruthiShivdas

MDA vs Benign glandular lesions
⚫The presence of many α SMA positive stromal cells
in the cervix suggests a desmoplastic response to
tumor.
⚫loss of estrogen receptor (ER) expression in the
stromal cells.
⚫P53 positive
Dr ShruthiShivdas

HPV Independent Adenoca Cx
Marker CC Mesonephric
Iteology DES related Remnants of Wolffian sys
Gross Exophytic growth Cervical deep stromal tumor
P16 Diffuse Negative/patch
HNF1B & Napsin A + (less specific) Neg
AMACR ++ Neg
PAX8 Neg +
GATA3 Neg +
CD10 (marker of
mesonephric diff)
Neg + (luminal pattern)
ER Neg Neg
HPV ISH Neg Neg
Prognosis Good prognosis Fair prognosis with late
recurrences (upto 11 yrs)
These 2 resemble one
another
Dr ShruthiShivdas

Subtyping Cervical Adenocarcinoma
HPV+, human papilloma virus infection in situ
hybridisation
EMC Cervix accounts for only 1 % of all adenoca of cervix, while 10% EEC can
extend into cervix.; It can resembles usual type ECC ; Diagnosis after exclusion of
EEC and HPV infection.
In the FGT, most adenocarcinomas of the ovary, FT, endometrium and cervix are
positive for PAX 8 (mullerian marker). However, cervical adenocarcinomas are less
likely to be positive than endometrial adenocarcinomas.
In cervical
endometriosis
Dr ShruthiShivdas

SCC Cervix vs EM (PESCC)
⚫ The majority of SCC in EM represent an extension from the cervix,
where SCC spreads superficially to the inner surface of the uterus .
⚫ To diagnose PESCC, it is important to exclude cervical SCC extension
into the endometrium and squamous differentiation of an endometrioid
adenocarcinoma.
⚫ Fluhmann’s pathological criteria:
⚪ (1) no evidence of a coexisting endometrial adenocarcinoma or primary cervical SCC;
⚪ (2) no connection between the endometrial tumour and squamous epithelium of the
cervix;
⚪ (3) no connection between the endometrial neoplasm and any existing CIN3.
⚫ PESCC - positive immunoreactivity for the CK7,5/6, p63, p53 and p16
proteins
⚪ HPV DNA/RNA ISH negative in PESCC
⚪ P53 + rare in SCC Cx
Dr ShruthiShivdas

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
⚪ NE marker such as synaptophysin, chromogranin, or
CD56
⚪ p63, is negative in small cell NEC; basal parabasal in
SCC
⚪ p16 tends to be present in both.
Dr ShruthiShivdas

Miscellaneous
⚫ 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
⚫ p53 positivity in a cervical carcinoma strongly suggests
against common cervical carcinomas, that is, squamous
carcinoma or endocervical adenocarcinoma of gastric type
⚪ diagnosis of a secondary serous carcinoma should be considered
and ruled out.
⚪ 50% endocervical adenocarcinoma of gastric type are p53+
Kuhn et al
Dr ShruthiShivdas

Case 1
⚫ 52 yr old parous lady with history of lost Copper T,
now presented with PMB
⚫ 12 weeks uterus with no cervical growth or
ballooning.
⚫ MRI : LUS EM tumour, ?loss of JZ; no cervical
extension
⚫ EB : CK5/6 +, p63,p40, p16+; Vimentin patchy, ER
PR negative; CEA negative. HPV ISH NA
⚫ Considering history and MRI preop diagnosis was
PESCC
⚫ Final HPE : C/S cut surface of uterus with uterine
cavity lining being replaced by an irregular shaggy
growth along the whole extent along with a
cervical growth. Morphologically SCC
⚫ Final diagnosis : SCC cervix with EM involvememt
? Superficial Spreading SCC of Cervix
Dr ShruthiShivdas

Case2
⚫ 35 years old, married for 8 years presented with
copious mucoid discharge from the vagina for
6 months
⚫ examination revealed profuse mucoid discharge
with a ballooned appearance of the cervix with no
obvious mass or ulceration. LBC – NILM
⚫ Imaging : a well-defined complex cystic lesion
measuring within the stroma of the cervix with
internal solid areas and inner vascularity
⚫ RH3 PLND done
⚫ Patho : barrel-shaped cervix on gross
examination with an endophytic growth pattern
and deeply invasive glands with intracytoplasmic
mucin.
⚫ Pan CK +, CK5/6-neg; CEA +; p16 neg;p53 +;
ER PR negative
⚫ Imp : MDA (not recommended anymore)
Dr ShruthiShivdas

DD of MDA
⚫ Nabothian Cysts
⚫ Tunnel clusters - Tunnel cluster (TC) is a type of retention
cyst, characterised by complex multicystic dilatation of the
endocervical glands, occasionally with mucoid discharge. TC
is found almost exclusively during pregnancy and can persist
for a variable period of time.
⚫ The presence of multiple cystic masses extending deep into
the cervical stroma as shown on transvaginal sonography or
MRI may appear like a “honeycomb.” This finding is worthy of
attention evein if negative cytological findings and HPV status,
and perhaps biopsy or cervical conization, may assist with
early detection of adenocarcinoma
Dr ShruthiShivdas

VULVA
25%
1/3
Dr ShruthiShivdas

VIN
⚫ Normal and atrophic vulvar epithelium-
⚪ minimal MIB1 expression in parabasal cells
⚪ No difference in mgt between the 2
⚪ Flat LSIL rare in vulva; LSILS restricted to condyloma
accuminatum
HSIL /VIN2&3
(UNDIFFERENTIATED OR
BOWENOID TYPE)
SIMPLEX (DIFFERENTIATED) VIN
2/3 vulval SCC
⯍HPV associated
⯍p16 expression
similar to those seen
in CIN (although
some cases may be
negative)
⯍P53 wt
✔not associated with HPV
✔C/C inflammatory vulval discorders (L. planus
& Scleorsis)
✔p16 is usually absent
✔may harbor p53 gene mutations
✔staining should be interpreted in context
of the morphologic findings as benign
lesions (lichen sclerosisdue to ischemia)
may be focally p53-positive
Dr ShruthiShivdas

Paget’s Disease (Extramammary)
⚫ Primary Paget’s ds of vulva (insitu ca of vulval skin with or without
invasion, from pleuriopotent stem cells in epidermis or skin adenxal
structures.)
⚪ expresses CK7 (not seen in normal epidermis)
⯍ Questionable cells near surgical margins- evaluated by CK7 since normal
epidermis is CK7 negative
⯍ Stromal invasion- Ck7 +in dermal cells
⚪ Express Breast Ca markers; ER/PR/ GCDFP, and CEA / Her 2 less
common
⚪ p53 mutant
⚫ Secondary vulvar Paget disease
⚪ Of colorectal origin- CK20, CDX2, and CEA
⚪ Of urothelial origin- CK20, uroplakin, and thrombomodulin
⚫ MIMICS
⚪ Pagetoid VIN- p63 +
⚪ Pagetoid melanoma- S-100 and HMB45+
Dr ShruthiShivdas

Vulvovaginal Mesenchymal Trs
⚫ Most of the vulvovaginal mesenchymal lesions are reactive with ER and
PR, including
⚪ cellular angiofibroma ----------------------------⯍ fibroblastic differentiation
⚪ aggressive angiomyxoma
⚪ Angiomyofibroblastoma
⚪ superficial cervicovaginal myofibroblastoma
⚪ smooth muscle neoplasms.
⚫ Most of these are thought to arise from the zone of hormone receptor
positive subepithelial cells that extends from the cervix to the vulva.
⚫ Most express vimentin, ER, PR, desmin, actin, and CD34
⚫ Cellular angiofibroma may lack desmin and actin ; negative staining with
smooth muscle antibodies is of value in diagnosing cellular angiofibroma
⚫ these neoplasms are often treated with GnRH agonists, especially
recurrent neoplasms and those tumors not amenable to surgical resection
Myofibroblastic
differentiation
Dr ShruthiShivdas

Malignant Melanoma
⚫Vulvovaginal (mucosal melanoma ) are the MC in
FGT
⚫IHC markers : S100; Melan A (MART1); SOX 10;
HMB 45
⚫Multiple markers to be put.
⚫Generally lower mutational burden than cutaneous
melanoma
Dr ShruthiShivdas

Epithelial Ovarian Ca’s(EOC)
▪ PAX8 (Paired Box 8 ) is the most specific marker to distinguish a primary
ovarian carcinoma from a metastasis, but also expressed in metastasis from the
endocervix, kidney and thyroid
⚪ usually expressed by the secretory cells of the fallopian tube eoithelium,
but neighboring ciliated cells do not, and neither do any cells on the
surface of healthy ovaries
⚫ WT1
⚪ sensitive and specific marker for serous histotype and can be used to
discriminate serous tumours from all other histotypes.
⚪ generally positive in SEROUS and negative in all other EOCs; approximately
10% of HGSOCs can be negative
⚫ P53 mutation (over / null) occurs in 97-10% HGSOC.
⚪ it is diffusely and intensely nuclear positive, in 60%–70% of HGSCs because of
missense mutation
⚪ entirely negative in the remaining cases due to truncating/nonsense mutation
⚫ Approximately 60%–80% of HGSOCs show diffuse p16
Dr ShruthiShivdas

PAX8 +ve in metastasis from
the endocervix, kidney and
thyroid
FT / EOC/
PPC vs
metastasis
SOC vs non
serous
SEROUS
Subtyping
NON - SEROUS
Subtyping
Dr ShruthiShivdas

PAX 8
POSITIVE in
ALL
SUBTYPES
OF EOC
except in
mEOC
PAX8 in
mEOC is
often focal
and weak.
SM ca – IHC
paterns are
variation of EM
type ; so
considered s
subtype of
EMOC (with
mucinous
differentiation)
V
I
M
E
N
T
I
N
+
Dr ShruthiShivdas

Aberrant p53 (positive / null) and block positivity for p16 (CDKN2A) in
>=50% HGSOCs
Help in distinguishing HGSC from LGSC when morphology is
indeterminate WHO FGT
2020
Focal&weak
Dr ShruthiShivdas

HGEOC vs HGSOC
⚫HGSC with glandular and cribriform growth may
closely resemble endometrioid carcinoma
⚫p53 may also be aberrant in almost 15 – 20% of
endometrioid carcinomas, particularly in those with
high nuclear grade
IHC marker HGSOC HGEOC
p53 + +
WT1 80 – 90 % + Negative
Vimentin Negative Positive©
p16 Diffuse strong nuclear / blank mosaic
cyclin E1
expression
Present Absent
Dr ShruthiShivdas

Serous Ca in uterus + Ovary
⚫ Disseminated serous carcinoma that involve ovaries,
uterus and peritoneum represent a challenge for
delineating the site of origin.
⚫WT1 is the most reliable marker in this setting ;
diffusely + in HGSOC ; negative in USC (30% variable
positivity).
⚫ Specifically, in cases of synchronous involvement of
both the endometrium and ovaries, WT1 is mainly worthy
if it is negative at both locations, supporting an
endometrial primary, or if the staining patterns are
different at the two sites (+in ovary, negative in EM),
suggesting two independent tumour.
Dr ShruthiShivdas

Malignant Brenners
BRENNER HGSC Urothelial Mets to Ovary
CK7 CK 7+ CK7+ +
Urothelial Markers
(Uoplakin III ,
thrombomodulin)
+ - +
P63 + - +
GATA 3 + +
WT1 - + -
P53 wt M
P16 Focal/ patchy Diffuse
ER PR negative / weak +
PAX8 Negative + neg
CK 20 - - +
The absence of a benign / borderline Brenner component should raise suspicion of
HGSC or HGEOC with transitional cell features.
WHO 2020
Bilateral Malignant Brenners without benign or borderline elements should raise
suspicion of urothelial mets to ovary
Dr ShruthiShivdas

Mucinous Carcinomas in the ovary
⚫ mEOC -typically CK7+; CK20- (10% +) ; CDX2- ( only 35% + ; mostly focal);
PAX8 is expressed in 50% of cases.
⚫ Colorectal Ca- CK20 +/ CK7- ; CDX2+ (90% - 100% ; strong); PAX8neg,
⚪ special AT-rich sequence-binding protein 2 (SATB2) staining may further distinguish appendiceal
neoplasm from MOCs, as glandular cells of lower GI (colorectal & Ax) malignancies retain expression
in metastatic foci, and SATB2 staining is negative in primary MOCs.
⚫ Gastric and pancreatobiliary malignancies may express CK7+ ; Ck20 +/_,
similar to mEOC.; both express CEA +++> mEOC
⚪ Pancreatic ductal carcinomas (mets can have IC mucin in > 50% cells like primary mEOC) lack
SMAD4/Dpc4 (Deleted in Pacnreatic Cancer) expression, while this is preserved in MOCs.
⚪ In gastric primary vs MEOC, morphology most important.
⚫ dual CK7+/CK20+ raises the possibility of a primary neoplasm in the
stomach, pancreas, biliary tree or urinary bladder
⚫ Diffuse p16 staining may help differentiate between primary MOCs and
metastatic endocervical adenocarcinomas (CK7+/ CK20 -).
Blaustein’s Pathology of Female Genital
Tract, 2019
Dr ShruthiShivdas

Non GI Mets to Ovary
⚫Metastatic renal Ca-
⚪ CK7 –ve, CD10+ve, RCC +ve (d/d clear cell Ca ovary)
⚫Metastatic urothelial Ca-
⚪ +ve for CK20, CK13 (d/d- malignant brenner)
⚫Metastatic breast Ca (micropapillary type)-
⚪ CK7, WT1, CA125, ER PR are overlaping . CK 20 negative.
⚪ Mammaglobin+, GCDFP15 +,GATA3+, PAX8 (neg) are helpful
⚫Metastatic thyroid Ca-
⚪ TTF1 (thyroid transcription factor1), thyroglobulin
Dr ShruthiShivdas

SM ca – IHC
paterns are
variation of EM
type ; so
considered s
subtype of
EMOC (with
mucinous
differentiation)
ARID1A mut is loss; ARIDIA nuclear staining is wt. (ERON)
B Catenin nuclear staining is +
Endo stroma
Dr ShruthiShivdas

UC
DDC
Undifferentiated
component
Differentiated
component
SMARCA4 staining
Dr ShruthiShivdas

PPSC vs Epitheloid mesothelioma
PPC Mesothelioma
PAX8 + Calretinin +
ER + CK 5 / 6 +
MOC31 + D2-40 + (podoplanin)
Ber-EP4 + BAP1 Loss
Although PAX8 positivity has been reported in a relevant proportion (6%–18%) of
peritoneal malignant mesotheliomas, usually the staining is weak and focal.
The most reliable recently discovered markers for the
diagnosis of mesothelioma are loss of BRCA-associated
protein 1 (BAP1) by IHC and deletion of p16 gene by
fluorescence in situ hybridisation.
Dr ShruthiShivdas

Sex Cord
Stromal
Tumours
(SCST)
Dr ShruthiShivdas

SEX-CORD STROMAL TUMOURS (SCST)
⚫ >90% SCSTs are benign (fibromas/thecomas).
⚫ The most commonly encountered malignant sex cord–
stromal tumor is granulosa cell tumor.
⚫ Juvenile granulosa cell tumor (JGCT) is the most
common SCST in premenarchal girls.
⚫ JGCT and Sertoli-Leydig cell tumor (SLCT) are the most
common malignant SCSTs in women < 30 years old.
⚫ Genetics
⚪ FOXL2 mutations in >90% AGCT and some SLCT in older pts
⚪ DICER 1 mutation in subset of SLCT (moderately & poorly
differentiated) & gynandroblastomas
⚪ Grolins Syndrome - Thecoma
Dr ShruthiShivdas

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 (eg:
sertoliform Endometroid Ca)
⚫ Inhibin is a relatively specific marker
⚪ α inhibin may also help to demonstrate luteinized stromal cells in association
with ovarian neoplasms of non SCST that have resulted in androgenic or
estrogenic manifestations
⚪ β inhibin is less useful diagnostically than α inhibin because many ovarian and
extraovarian carcinomas are reactive to the latter.
⚫ Calretinin is more sensitive, but less specific than inhibin
⚫ Other markers- CD56 (cytoplasmic and membranous
pattern), WT1 (nuclear pattern), CD 99 and SF-1
(steroidogenic factor-1)
Dr ShruthiShivdas

SCST
⯍ Granulosa cell Tx
⯍ Pan CK may be +; Negative for EMA . JGCT may be EMA +ve
⯍ +ve for inhibin, calretinin, CD99,CD56, SF1, WT1
⯍ FOXL2 expression & mutation (97%)
⚫ Thecoma-fibroma group
⯍ Thecoma- more strongly positive for inhibin and SMA
⚫ Sertoli-Leydig cell tumours
⚪ Strong positivity for Melan A
⯍ ~98% of SLCTs have a DICER1 mutation. Familial SLCTs have been associated with thyroid
disease and pleuaropulmonary blastoma; a combination of these lesions should suggest a DICER1
syndrome.
⚫ Steroid cell Tumours
⚪ Mostly –ve for WT1, CD99
⚪ Strongly +ve for SF1
⚫ All types of tumors except steroid cell tumor express WT1.
Fibroma/fibrothecoma generally donot express CD99.
Dr ShruthiShivdas

Endometroid Ov Ca vs SCST
WT1 _ +
One caveat about the use of either inhibin or calretinin immunostaining is that both
of these markers may occasionally be expressed in tumors that may be included in
the differential diagnosis of ovarian SCSTs.
in endometrioid adenocarcinoma, clear cell carcinoma, undifferentiated
carcinoma, FATWO, breast carcinoma, and melanoma
Therefore, in problematic cases, calretinin and inhibin are best used as part of a
larger panel of antibodies like EMA & WT1.
Dr ShruthiShivdas

Germ Cell
Tumours (GCT)
Dr ShruthiShivdas

GCT
B HCG may be positive in Dygerminoma , EC or IT , if syncitiotrophoblasts
present.
Gonadoblastoma
⯍Germ cells +ve for PLAP, CD117, and Oct-4
⯍ Sex cord cells stain for vimentin, cytokeratin, and inhibin
CT +SCT
Pan GCT markers
CD11
7
Dr ShruthiShivdas

IHC in Teratoma
⚫Glial fibrillatory acidic protein (GFAP) stain glial
tissue
⚫ GFRα-1 and MIB1 d/d between immature and
mature teratoma
⚫IHC in monodermal teratoma
⯍ TTF1 and thyroglobulin +ve for thyroid tissue
⯍ Chromogranin and synaptophysin +ve for carcinoid
⯍ CDX2 d/d from colorectal carcinoid
Dr ShruthiShivdas

SCCOHT versus other mimics
⚫can simulate mainly HGSC ,adult granulosa cell
tumour and SCC of lung.
⚫Similarly to HGSC, SCC-HT also demonstrates IHC
positivity for p53 and WT1.
⚫Characteristically, SCC-HT shows calretinin
positivity, similar to granulosa cell tumours, although
focal and weak.
⚫SMARCA4 is specifically mutated in over 90% of
SCC-HT, and this genetic aberration produces a loss
of SMARCA4 (BRG1) protein expression on IHC.
Dr ShruthiShivdas

Case1
⚫63 year old lady with bilateral Solid Cystic adenexal
masses <10 cm (predominantly solid ) with no
ascites; omental and peritoneal mets +
⚫CA125 : 157; CEA normal ; CA 199 – 35
⚫Omental Biopsy : Adenoca
⚫D/D: GI primary vs Ca Ovary
⚫IHC – PAX8+; CK7+, CK20 patchy ; WT1+; p53+;
p16+.
⚫Imp : Met from HGSOC
Dr ShruthiShivdas

Case 2
⚫62 yr old unmarried lady k/c/o Ca ovary post
treatment outside.
⚫Now presents with a vault growth bx outside stating
SCC
⚫IHC on blocks
⚪ PAX8+; p53 null type ; p16 diffuse +; WT1 negative (10%
HGSOC)
⚫Imp : HGSOC metastatic to vault
Dr ShruthiShivdas

Case 3
⚫ 48 year old lady; k/c/o HGSOC IIIC post first line treatment in 2016
⚫ Presents 3 yrs later with left breast lump; Trucut ⯍ Invasive Ca;
⚫ CT abdomen- subcapsular liver lesion in segment 7. FNAC - adenoca ; CA
125 – 201
⚫ Dilemmas :
⚪ Breast lesion is it primary/metastatic?
⚪ Liver lesion from Breast / ovarian primary?
⚫ IHC breast :
⚪ Triple negative ; GATA3 patchy GCDFP+ ; PAX8 negative; WT1 negative
⚫ IHC Liver lesion :
⚪ PAX8+; WT1+; p53mutant; immunonegative for GATA3 & GCDFP15.
⚫ Imp : Metachronous Ca Breast + Recurrence og Ovarian Ca
⚫ BRCA advised.
⚫ Went on to SCRS with MRM
Dr ShruthiShivdas

Case 4
⚫ 42 yr old with large Cystic mass with solid areas
⚫ CA125 – 56; Ca 199 – 300; CEA – 10.8
⚫ No GI symptoms, except occasional constipation
⚫ OGD / Colonoscopy – normal
⚫ Frozen – Insestinal Type Mucinous Tumour ? BOT ? Invasion. Await
paraffin sections
⚫ HPE : Mucinous Cystadenoca ; ? Primary ? Metastatic. Ax – free
⚫ IHC
⚪ CK7+; CEA + ; CK20 patchy; CDX2 negative
⚪ PAX8; ER/PR; Napsin negative
⚪ P53 mutant
⚫ Imp : Expansile MEOC
Dr ShruthiShivdas

Case 5
⚫ 50 yr old lady with large multiseptated cyst with solid enhancing areas.
⚫ CA125 – 45; CAE -3.5; CA 199- 50.
⚫ No GI symptoms.
⚫ Preop diagnosis : Mucinous BOT / Ca
⚫ Intraop : PMP like picture with 16 cm Solid cystic mass with rupture.
⚫ HPE : Mucinous Ca Ovary; FL, omentum, peritoneal deposits – acellular
mucin pools. Ax –unremarkable.
⚫ IHC
⚪ CK20; CDX2 +
⚪ CK7 negative; WT1 negative (epithelial lining of ovary CK7 and WT1 +)
⚫ Imp : Metastatic Mucinous Adenoca. Search for primary in GIT
⚫ OGD/ Colonoscopy – WNL
⚫ Final Diagnosis : Primary mEOC ( Intestinal Type).
RARE CASES of MEOC arising in a dermoid can have the same
immunoprofile as CRCa
Dr ShruthiShivdas

Case 6
⚫ 42 yr old with abdominal pain and icterus
⚫ Bil adenexal lesions <10 cm; Multiple liver and lung mets.
⚫ CA125 – 79; CEA -6; CA199- 1825
⚫ Clinical suspicion of HCC ; AFP – 1.46
⚫ D/D : HCC/ Lung primary / CA ovary
⚫ FNAC liver lesion with CB : metastatic Ca
⚫ IHC
⚪ CK7+; CK20,CDX2 neg;p40 negative;
⚪ HepPar1 negative;
⚪ WT1,PAX8,p53 negative;
⚪ Napsin & TTF1 negative;
⚪ GATA 3 & GCDFP 15 negative ;
⚪ Synaptophysin & chromogranin negative
⚪ CA199- NA
⚫ Imp : Poorly diff adeoca with CK7+
⚫ In v/o elevated CA 199 , to consider Intrahepatic CHOLANGIOCA.
Dr ShruthiShivdas

Case 7
⚫ 44 yr old lady; k/c/o Adenoca Cervix IB1 post RH3 PLND with Ov
transposition at 40 yrs of age.
⚫ Now with bilateral cystic masses with enhancing solid areas.
⚫ CA 125 – 70; CEA – 36; CA 199- 20
⚫ No GI symptoms
⚫ D/D: Primary mEOC / Late Mets from adenoca Cx
⚫ HPE – Mucinous Ca Bil ovaries . ? Primary Ovary ? Metastatic from
Cx / GI
⚫ IHC
⚪ CK 7 +; CK20 neg; CDX2 neg
⚪ PAX 8+ WT1 neg
⚪ P16 + p53 wt
⚪ ER negative
⚫ Imp : Metastatic Mucinous Ca bilateral Ovaries . Primary Adenoca
Cervix ( Usual Type)
Dr ShruthiShivdas

Case 8
⚫ 48 yr old lady h/o VH for AUB 7 months back, now with
vault growth and solid cystic adenexal mass with no
ascites. CA125 –250
⚫ Vault Bx – SCC;
⚫ D/D : Ca Vault with ovarian mets / Synchronous Trs
⚫ Planned for CCRT after BSO + TO
⚫ HPE : PD Carcinoma in ovarian mass; Omentum free
⚫ IHC
⚪ P40+
⚪ CK5/6+
⚪ WT1 neg; PR focal
⚪ p16+
⚫ Imp : Metastatic SCC to ovary
Dr ShruthiShivdas

Case 9
⚫ 41 yr old lady. K/C/O HGSOC III post Laparoscopic IDS with
CC0 and adj CT
⚫ presented 7 months later with ulceroproliferative lesion 3x3
cm at the vulva, submucosal lesion in the lower1/3 vagina with
left pelvic mass involving PWS.
⚫ CA 125 – 72
⚫ Vulval Bx – PD Ca
⚫ D/D : Ovarian met / Primary Vulvovaginal Ca
⚫ IHC – CK7+; CK5/6 negative; PAX8, WT1+; ER +; p40
negative
⚫ Imp : HGSOC metastatic to vulva
Dr ShruthiShivdas

Case 10
⚫ 54 yr old lady operated outside for bilateral ovarian
masses with elevated CA 125
⚫ R/S here : PD Ca bilateral TO masses. One of the
ovarian masses shows transitional like features. No
benign or borderline Brenner elements
⚫ D/D : To rule out urothelial mets
⚫ IHC : CK7+; CK 20 negative; p53 strong diffuse ; WT + ;
PAX 8 + ; P63 and GATA 3 neg
⚫ Imp : HGSC
Dr ShruthiShivdas

Case 11
⚫ 65 yr old with PMB
⚫ Clinically friable cervical mass
⚫ CT scan – bilateral solid-cystic masses with ascites,
omental mets and endometrial collection with
hetergenous cervical mass
⚫ CA125 – 1580
⚫ Clinical : Ca Ocary with Cx mets / Ca Cx with Abd mets
⚫ Cx Bx – high grade adenoca
⚫ IHC –
⚪ PAX8 not done ; ER PR +; Vimentin+ WT1 nega; p53 wt; p16
negative.
⚫ Imp : G3 EEC !!
Dr ShruthiShivdas

Case 12
⚫ 33 yr old lady with AUB; EB – Grade 1 EEC MMRd
⚫ MRI – 6.9cm EM lesion with >1/2 MMI; left ovary bulky with
solid area. CA 125 – 92
⚫ EFHE BSO RPLND ICO done
⚫ HPE : EEC grade 2 with <1/2 MMI ; EM Ca grade1 Ovary;
LVSI negative in both sites.
⚫ Imp : Synchronous Trs
⚫ More bits from FTs revealed atypical glands within the lumen ,
s/o transtubal spread
⚫ Imp : Stage IIIA EEC
Dr ShruthiShivdas

“ The Diagnostic Power of any
Immunohistochemical Procedure is
NO GREATER than the wisdom of
the Pathologist interpreting it.”
Dr.Allen M.Gown
Dr ShruthiShivdas

Immuno histo chemistry in gyn oncology

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