Immunohistochemistry of breast lesions(IHC)

DR. NOOPUR S. PATIL
IMMUNOHISTOCHEMISTRY IN
BREAST PATHOLOGY

Immunohistochemistry (IHC) BASICS:
•Technique used to detect & localize antigens by means of labeled antibodies
through Ag – Ab interaction that are visualized by enzyme - substrate –
chromogen reaction
•It takes its name from the roots "immuno," in reference to antibodies used in
the procedure, "histo," meaning tissue & “chemistry” meaning enzymatic
reaction.
•Visualizing an antibody-antigen interaction can be accomplished in a number
of ways.
•In the most common instance, an antibody is conjugated to an enzyme, such
as peroxidase, that can catalyse a colour-producing reaction.
PRINCIPLES:
•Direct labeling method - enzymes are directly labeled with the primary
antibody, less sensitive
••Indirect labeling method – enzymes are labeled with the secondary Ab
which is produced against the primary Ab, more sensitive.
••ABC method – the sec. Ab & the avidin are labeled with biotin & enzyme
respectively. The avidin has high affinity to biotin . IHC makes use of this high
affinity by using biotinylated Abs to amplify tissue Ags

INTRODUCTION:
•Immunohistochemistry (IHC) is used to characterize
intracellular proteins or various cell surfaces in all tissues.
•Individual markers or more often panels of various
marker proteins can be used to characterize various
tumour subtypes, confirm tissue of origin, distinguish
metastatic from primary tumour and provide additional
information which may be important for prognosis, predicting
response to therapy or evaluating residual tumour
post-treatment.
•There is a growing list of available products
(antibodies) or antigen retrieval techniques, which all
contribute to the broader utility of immunohistochemistry
for solving diagnostic problems or for determining
prognosis and response to therapy in breast pathology.

It is well known that normal glandular breast tissue is composed of three cell
types which express different subsets of proteins:
The most important diagnostic problems that occur in mammary gland
tumor pathology are: the differential diagnosis of various types of benign
lesions and carcinoma; differentiating between carcinoma in situ and
invasive carcinoma, diagnosis and differentiation of microinvasion and its
imitating lesions and confirming the breast as the primary site in
metastatic carcinoma.
In the absence of advanced molecular biological techniques, IHC can be
use to identify histologic subtype or molecular phenotype.

basal cell
types express
different
cytokeratins
(5/6, 14, 17)

1. Analysis of prognostic markers e.g. Prognostic
or Predictive Factors in Breast Carcinoma:
Hormone Receptors
2. Solving common diagnostic dilemmas-
Differential Diagnosis of Epithelial Lesions:
Myoepithelial Cells and papillary lesions of
breast
3. Tumour typing and confirming diagnoses- e.g.
Lobular v Ductal; Luminal v Basal/Myoepithelial;
Distinction DIN (DCIS) and LIN (LCIS)
4. Cell Population in Intraductal Proliferative
Lesions: Homogeneous Versus Heterogeneous
Cell Population (Neoplasia Versus Hyperplasia)
INDICATIONS OF IHC IN BREAST LESIONS:

Subtle foci of invasion/Microinvasive
Carcinoma and status of margins
Lymph node metastases- Micrometastatic Disease
in Axillary Lymph Nodes (Including Sentinel
Nodes)
Demonstrating epithelial cells in necrotic
material
Paget’s Disease
Systemic Metastasis of Breast Carcinoma
Metastases to breast
Spindle cell lesions

Endocrine treatment for breast cancer- late 1890s by
Beatson, when he observed what would now be called
responses in a few young women with apparent locally
advanced breast cancer after he performed surgical
oophorectomy.
Beatson presumed that he was interrupting neural
connections between the ovaries and the breast,
although he was actually removing the ligand
estrogen from the patient’s circulation, which
diminished estrogen availability to its protein receptor
(ER) in the breast
HISTORICAL IMPORTANCE:

MARKERS OF PROGNOSIS AND RESPONSE TO THERAPY
The most common immunohistochemical breast
cancer prognostic and therapeutic markers used
include: ER, HER2, Ki-67, PR, and p53. In addition
markers of angiogenesis and apoptosis are used.
Nowadays, immunohistochemical detection of ER
and PR is part of the routine work-up of breast
cancer, and in some cases of DCIS the presence of
ERs is an indication for tamoxifen therapy.

ER and PR in Carcinoma breast
Normal breast epithelial cells-ER and PR receptors ,
proliferate under their influence.
75% to 85% Ca breast stimulated to grow when these
hormones are present.
ER
Clinical rationale: identify patients who may benefit from
hormonal therapy. –Strong predictive factor
It is also a minor prognostic factor.
PR
PR status is determined to identify a small number of
carcinomas (in most series <5%) that are PgR positive and ER
negative but which may respond to hormonal therapy. –
Predictive factor
Progesterone receptor is also a minor prognostic
factor

 Prognostic factor - any measurement available at the time
of diagnosis or surgery that is associated with clinical
outcome in the absence of systemic adjuvant therapy.
 Predictive factor - associated with response or lack of
response to a particular therapy.
 ER- Strong predictive factor for response to
endocrine therapy.
 Weak prognostic factor
 PR expression is induced by ER - surrogate marker
for ER activity(Additional predictive factor for hormonal
therapy in breast cancer)
 Oneof the effects of estrogen is to induce the PR(ERand PR+tumors
form 99%of receptor positive tumors) and ER-negative, PR-positive
tumors account for fewer than 1%of all breastcancers
 Nuclear immunostaining for both receptor proteins canbe
demonstrated in normal breast acini (15-20%), which serve
as internal controls for the testingprocedure

Estrogen receptors (ER)
●ER-alpha: “classic”
functions of ER;
susceptible to
proliferative stimulation
of estrogen; breast and
endometrium;
● ER-
beta:“ housekeeping”
functions; normal
ovary and granulosa
cells, carcinoma of
breast, colon, prostate;

ER status is strongly influenced by tumor grade
and histology.
Virtually all grade I tumors are ER positive, bcl2+
tumors, as are pure tubular, colloid, and classic
lobular carcinoma.
PR is an estrogen-regulated gene, and its
synthesis in normal and cancer cells requires
estrogen and ER.
 Some studies have shown that ER and PR status can
change over the natural history of the disease or
during treatment

Response rate
ER(+) PR(+) 78%
ER(+) PR(-) 34%
ER(-) PR(+) 45%
ER(-) PR(-) 10%

Assay methods
Cytosol ligand binding assay
Enzyme immunoassay
Immunohistochemical assay
Antibody used :
* H 222
* 1D5
ER is detected in the nucleus by
immunohistochemical studies.

Tissue fixation
ER are thermolabile and short half life
Incised immediately after resection to ensure rapid penetration of fixative
Most tissue fixatives can be used to preserve receptor reactivity if microwave predigest is used
Control : To avoid risk of false negative classification

FALSE NEGATIVE
 Exposure of the carcinoma to heat ( cautery during
surgery).
 Prolonged cold ischemic time (the time between
tissue removal at surgery and initiation of fixation).
 Type of fixative: ER is degraded in acidic fixatives
such as Bouin’s and B-5.
 Prolonged fixation in formalin: Optimally at least 8
hours in buffered formalin.
 Fixation for more than 3 weeks can diminish
immunoreactivity
 Decalcification: results in loss of immunoreactivity.
 Incorrect antigen retrieval method.
 Non-optimized antigen retrieval.

Quantification systems
Quantification systems may use only the proportion of
positive cells or may include the intensity of
immunoreactivity.
Number of positive cells: As a percentage orwithin
discrete categories.
Intensity: Refers to degree of nuclear positivity (ie, pale
to dark). The intensity can be affected by the amount
of protein present, as well as the antibody used and
the antigen retrieval system.
Most cancers - Heterogeneous immunoreactivity with
pale to darkly positive cells present

Scoring for ER Immunostains
Conventional scoring: Semi quantitative fashion
incorporating both the intensity and the distribution of
specific staining as described by Mc Carthy, Jr et al.
Allred scoring: Semi quantitative system that takes into
consideration the proportion of positive cells(scored
on a scale of 0-5) and staining intensity (scored on a
scale of 0-3).
The proportion and intensity were then summed to
produce total scores of 0 or 2 through 8.

Scoring system forER/PR
Score forproportion Score forintensity
0=No staining 0=No staining
1<1% staining 1=Weak staining
2=(1-10)% staining 2=Moderate staining
3=(11-33)% staining 3=Strong staining
4=(34-66)% staining
5=(67-100)% staining
Totalscore rangesfrom 0to8.
Tumorsscoring ≤2are regarded asERnegative and have anegligible chanceof response.

Preanalytical
optimum formalin exposure time for ER
determination is 8 hours
antigen can be retrieved with increasing retrieval
times for over-exposed tissue, but an under-fixed
tissue is completely useless for biomarker study.
Analytical
All commercially available antibodies for ER
assessment in breast carcinoma target only ER-
alpha isoform.
Post analytical
NIH Consensus Conference of December 2000
states, “Any nuclear expression of HRs should be
regarded as a positive result and render a patient
eligible for hormonal therapy

HER 2/neu (c- erb B2)
Proto oncogene
Homologous to human epidermal growth factors
Encodes 185 KD transmembrane protein having intra and extra cellular domain
Intra cellular domain with tyrosine kinase activity and extracellular domain with binding site for growth factor.

Over expression of c-erb B2 in 1/3 rd of breast
cancer
Over expression is associated with
High histologic grade
Reduced survival
Lower response to methotrexate,
tamoxifen
Higher responsiveness to doxorubicin
Monoclonal antibody trantuzumab (Herceptin)
have role in treatment of tumor cell lines over
expressing the HER – 2 protein

HER2 Testing by Immunohistochemistry
•It is a marker for sensitivity to Herceptin (trastuzumab),
and resistance to tamoxifen.
•Although Her-2/neu can be detected using many methods,
only two are currently approved and recommended for its
detection: IHC and fluorescence in situ hybridization (FISH).
Scientific rationale: A subset of breast carcinomas
(approximately 15% to 25%) overexpress the epidermal
growth factor receptor HER2.
The mechanism of overexpression - amplificationof the gene
resulting in increased amounts of protein
Clinical rationale: To determine if a carcinoma will
respond to treatment directed against the protein (eg,
treatment with trastuzumab or lapatinib

Clinical
Implication
- Theavailability of
trastuzumab,a
humanized monoclonal
antibody against HER2
hasimproved
progression-free survival
and
- overall survival, aspart of
adjuvant treatment for
both early high-risk
disease and metastatic
disease

BRCA 1 mutation(17q21)
IHC - new and powerful predictor of BRCA1
mutation status
ER-negative(usually triple negative) will
roughly double the probability that the
individual is a BRCA1 carrier.
Er positive - reduce the probability by
approximately fivefold.(a/w BRCA2-
13q12)

Markers of apoptosis and cell proliferation:
Ki-67 proliferation index, BCl-2, p53
Ki-67 (MIB-1):
•Ki-67, a non-histone protein, involved in the early steps
of polymerase I-dependent ribosomal RNA synthesis is a
predictive and prognostic marker in cancers and has been
extensively studied. When Ki-67 level is above 10%-14%,
breast cancer patients are deﬁned as high-risk
Proliferation marker, stains cells in all cell cycle
phases except the resting phase (G0); the
percentage of stained nuclei is the proliferation
index.
A low proliferation index is associated with slower tumor
growth,better prognosis, whereas the converse is true
for a high proliferation index.

•According to the St. Gallen Consensus (2009), the Ki-67 index is useful for
selecting patients with hormone receptor-positive breast cancers for the
addition of chemotherapy to endocrine therapy.
•Thus, breast tumours are classiﬁed as low, intermediate, and highly
proliferating according to a Ki-67 labelling index of under 15%, 16%-30%, and
over 30%, respectively.
•Data from the Clinical Cancer Registry Regensburg showed that Ki-67 expression
was associated with common histopathological parameters, especially grading
and survival, but is an additional independent prognostic parameter for
diseasefree survival and overall survival in breast cancer patients.
•Ki-67 expression has been used to determine the effects of different doses of
tamoxifen on breast cancer proliferation[42,46]. The change in Ki-67 expression
induced by lower doses of tamoxifen was comparable to that achieved with the
standard dose, indicating that tamoxifen retains antiproliferative activity at low
doses

With regard to the molecular breast cancers, high Ki-67
proliferation index can be used to classify triple negative breast
cancer into subtypes with different prognosis or responses to
treatment.
For this purpose, the number of Ki-67 positive cells among the
total number of counted tumour cells was determined and the
high expression of Ki-67 was defined as ≥ 10%.
It is known that patients with triple negative breast cancer have a
poor survival, despite their high response rate to neoadjuvant
chemotherapy, and those with high Ki-67 have more aggressive
clinical features.

P53
•The other marker, p53 is well studied in cancers, but its value in predicting
clinical outcome in breast cancer is debatable.
•The p53 gene is located on the short arm of chromosome 17 and encodes a 375
amino acid nuclear phosphoprotein that prevents propagation of genetically
modified cells.
• Wild-type p53 is a tumour suppressor protein and plays an essential role in
regulating genomic stability by controlling the cell cycle and inducing apoptosis
when cell damage cannot be repaired.
• In normal cells, p53 has a very short half-life due to ubiquitylation and
proteasome degradation.
•IHC can be used, as wild-type p53 protein is rapidly degraded, while TP53
mutations (18%-25% of primary breast carcinomas) are often associated with the
production of a stable protein. In addition, sequencing of the p53 gene in all
breast cancers would be expensive and time-consuming for routine practice.
•A higher tumour grade, negative ER and PR status, and the more aggressive
basal subtype were associated with abnormal p53 immunohistochemical
expression or p53-positive status. With regard to early breast cancers, some
scientists have reported that a p53 mutation has no influence on the outcome
and therefore, the value of p53 status is too weak to be recommended as a
routine marker in clinical practice.

Bcl 2: BCL2 belongs to a group of protein key regulators
of apoptosis or programmed cell death
Zhang et al. - bcl-2 expression - better response to
hormone therapy, and the expression of bcl-2 is a
favorable prognostic factor regardless of nodal status.
Berardo et al.- high bcl-2 expression was associated
with a significantly improved disease-free survival and
overall survival,
Gee et al. found that patients with estrogen-receptor
and bcl-2-positive tumors were particular responsive
to endocrine therapies that included an anti-estrogen.
van Slooten et al. found no association between bcl-
2 expression and response to perioperative
chemotherapy in node-negative patients.
Bonetti et al. reported a higher response rate to
chemotherapy among tumors classified as bcl-2
positive with immunostaining in ≥40% of tumor cells.

Angiogenesis markers:
•Tumor growth and metastasis are dependent on tumour
angiogenesis and this complex process involves a delicate balance
between angiogenic and antiangiogenic factors.
•Numerous studies have investigated the relationship between
tumour angiogenesis, prognosis and response to antiangiogenic
drugs.
•Analysis of these factors in tumour or serum of breast cancer
patients by IHC or multiplex protein assay (FASTQuant® Microspot
Assays) can improve diagnosis and prognosis of the disease.
•There is a large list regarding angiogenesis markers: Angiogenin,
Ang2, keratinocyte growth factor (KGF), fibroblast growth factor
basic, intercellular adhesion molecule (ICAM)-1, platelet-derived
growth factor-BB and the vascular endothelial growth factor family.

•The commonly used method to determine angiogenesis is counting
intratumoral blood vessels (MVD) stained with factor Ⅷ related
antigen or anti CD31 or CD34 using light microscopy.
•Counting newly formed stained microvessels is a useful tool in the
early detection of metastatic potential and in the selection of
patients for whom anti angiogenesis drugs might be beneficial.
•The reactivity level of CD34 antigen was assessed by IHC in all types
of invasive ductal breast cancer and its level seems to be a useful
predictor for the development of local lymph node metastasis and
can indicate the benefit of antiangiogenic treatment

Blood vessel invasion stained for CD31
positive endothelium
CD31 positive endothelium D2-40 positive endothelium.
D2-40 negative endothelium

VEGF:
•Most of the biological and clinical activity of the antiangiogenic
drugs currently approved for cancer therapy is against the VEGF-
related pathways.
• The VEGF system is part of the platelet-derived growth factor gene
family, and interacts with its specific receptors; VEGFR-1 (flt-1) and
VEGFR-2 (flt-2) for VEGF-A, a very potent angiogenic growth factor.
• VEGF-B, interacting with VEGFR-1, seems to have an important
role in the maintenance of existing vessels, but this protein is not
well studied.
•VEGF A and B, their receptors VEGFR-1 and 2 are expressed in a
variety of normal cells, and overexpression has been described in
malignant tumors.
•The prognostic importance of VEGF in invasive breast cancer is
associated with tumour stage and ER status, and inversely
correlated with tumour grade and measurement of tumour VEGF, as
an indicator of angiogenesis, which is more reliable prognostically
than measurement of microvessel density or serum VEGF

DIAGNOSTIC MARKERS: Myoepithelial markers:
(SMA,P63,CALPONIN,SMMHC)
•Myoepithelial markers are useful in helping to
distinguish invasive carcinoma from benign proliferations
with a similar morphological appearance, benign
proliferative lesions and most preinvasive lesions with an
intact myoepithelium.
•Invasive carcinomas lack the myoepithelial cell
layer that normally surrounds benign breast glands.
There is an exception, microglandular adenosis, a benign
proliferative lesion which lacks the myoepithelial cell
layer.
•Using IHC, positive myoepithelial staining is seen in the
benign area with attenuated or absent staining in areas
of atypia or in situ carcinoma.

p63
The nuclear protein p63 - homologue of p53
that is expressed in the basal epithelia of
multiple organs.
In the breast, p63 is positive in nearly 100% of
normal MECs and those associated with benign
proliferations
Advantages
(1)Nuclear staining pattern, which removes the
interpretation difficulties that may be associated
with the cross-reactivity for myofibroblasts seen
with many of the other markers
(2) High sensitivity.

Discontinuous
staining pattern(Focal
gaps) - morphologic
impression that MECs
are absent

SMA:
•Smooth muscle actin (SMA) has long been used as a myoepithelial
marker in breast pathology diagnosis as a sensitive marker of
myoepithelial differentiation, even if it is not specific, because any
cell with substantial expression of actin is positive for SMA
(myofibroblasts and blood vessels are positive for SMA).
•This becomes problematic in lesions where there are either
myofibroblasts or blood vessels in close proximity to the epithelial
lesion.
•One pitfall is the presence myofibroblasts within desmoplastic
stroma adjacent to nests/ glands of invasive carcinoma being
misinterpreted as myoepithelial cells, resulting in a false-negative
diagnosis.
•This is why the use of a panel of markers (p63, calponin, smooth
muscle myosin, CD10, S100) or a more specific marker such as p63
are recommended.

Smooth Muscle Myosin Heavy Chain
 SMMHC is a structural component of myosin - specific
for smooth muscle cells,terminal smooth muscle
differentiation.
•SMMHC is associated with contractile elements and is
present in all cells with such properties.
•It is expressed primarily in myoepithelial cells, but is also
expressed in blood vessels.
•An advantage of SMMHC is that it demonstrates less
crossreactivity in myofibroblasts than calponin and SMA.
•Overall, the studies so far suggest that among smooth
muscle markers, SMMHC provides the best results, in terms
of both sensitivity and specificity.

Ideal panel Combination of
SMMHC
and p63

Detecting Absence
Detecting the absence – more problematic than
detecting its presence.
When IHC fail to reveal myoepithelial cells around
tumor- the diagnosis of stromal invasion is
supported.
Truly absent or whether they are merely markedly
attenuated and out of the plane of section.
Reassuring features - Medium to large tumor nests
without detectable myoepithelial cells, multiple
tumor nests without detectable myoepithelial
cells, and lack of reactivity with two different
myoepithelial markers.

Avoidance of Pitfalls
p63 and SMM-HC complement each other well.
P63 - sensitive and specific nuclear myoepithelial
marker but staining can be discontinuous
Cytoplasmic myoepithelial marker, such as
SMMHC or calponin, as well will aid
interpretation.
If these two stains yield unclear results, the
slightly more sensitive but less specific markers
calponin and SMA can be used..
Combination of the sensitive marker smooth
muscle actin, and two more specific markers
such as smooth muscle myosin heavy chain
and p63.

DR. NOOPUR S. PATIL
IMMUNOHISTOCHEMISTRY IN
BREAST PATHOLOGY-II

MEP MARKER LOCATION MEP
CELLS
MYOFIBROBLAST VESSELS
P63 Nuclear +++ _ _
CALPONIN Cytoplasmic +++ + ++
SMMHC Cytoplasmic +++ + ++
SMA Cytoplasmic +++ +++ +++
BASAL TYPE CK Cytoplasmic
+Membranous
+++ _ _

Recommendations for a
Diagnostic Panel
The optimal antibody depends upon the type of
lesion being evaluated.
Reactive stroma - p63 is an excellent choice
because it does not stain myofibroblasts or
blood vessels.
p63 is less adroit at highlighting architecture in
small glandular proliferations such as sclerosing
adenosis, and in these cases a cytoplasmic
marker such as SMA may be easier to interpret.

MYOEPITHELIAL CELL MARKERS IN THE
EVALUATION OF BENIGN SCLEROSING LESIONS:
To assess intraductal proliferative lesions, high-molecular-weight
cytokeratins (cytokeratin 14 and cytokeratin 5/6) can be helpful.
Radial scar and complex sclerosing lesion -
Proliferation of benign glands and tubules within a
fibrous/fibroelastotic stroma.
MECs associated with sclerosing lesions may
have different immunophenotypic characteristics
from the MEC layer.
Reduced expression of CK5/6 in 32% of cases,
SMMHC in 20%, CD10 in 15%, p63 in 10%, and
calponin in 6%, compared to that of normal
MECs

Central sclerosis with fibroelastotic stroma and entrapped glands radiating outward.

Benign lesions without MEP cells include microglandular adenosis, which is an infiltrative,
benign breast lesion consisting of small, round, open glands formed by a single layer of
flat to cuboidal epithelial cells with fibrous or fatty stroma. Microglandular adenosis,
which is surrounded by an often-thickened basement membrane, lacks MEP cells and
contains periodic acid–Schiff diastase–positive secretions. Microglandular adenosis is a
mimic of tubular carcinoma. Microglandular adenosis is positive for collagen IV; negative
for ER, PR, and p63; and often strongly positive for S100, whereas tubular carcinoma is
ER+/PR+ and p63-ve and S100-ve.

•MEP markers serve as important adjuncts for distinguishing between radial scars,
invasive ductal carcinoma, and DCIS involving sclerosing adenosis.
•Basal-type cytokeratins, smooth muscle myosin heavy chain, smooth muscle actin,
calponin, and p63 that are the frequently used MEP markers with varying
specificities and sensitivities that are used to make the distinction.
•Clinical practices often use more than one MEP marker.
•Collagen IV and laminin have been used for distinguishing invasive carcinoma from
in situ carcinoma.
•Benign or in situ lesions should demonstrate positive staining for MEP markers,
whereas invasive carcinomas should show a loss of MEP staining at the periphery of
each glandular structure .
• EXCEPTION:
•There are invasive carcinomas that express MEP markers; this group of cancers
consist of tumor cells that stain positive for MEP markers.
• They include Adenoid cystic carcinoma and Metaplastic carcinoma, In such cases
location of MEP marker( Non peripheral and liner) must be taken into consideration
Invasive Carcinoma Versus In Situ Carcinoma

Detection of microinvasion
Stromal invasion occurs when malignant epithelial
cells extend beyond the myoepithelial cell layer
and
Earlier investigators- antibodies to basement
membrane components - collagen IV and laminin
to differentiate between in situ and invasive
carcinomas.
•Cytokeratin markers may be helpful in highlighting the presence of
malignant cells of a microinvasive carcinoma (1 mm) arising in a
background of DCIS.

Irregularly shaped nests of neoplastic cells in a sclerotic
background
SMMHC highlights the myoepithelial cells surrounding
the DCIS within the complex sclerosing lesion. Small foci
of invasive ductal carcinoma - apparent in the
immunostained section

•A papillary lesion of the breast is characterized by an epithelial
proliferation arising within the ductal/lobular system, supported by
fibrovascular cores, with or without an intervening MEP cell layer.
•Papillary lesions consist of a range of benign, atypical, in situ, and
even invasive lesions.
•Papillary lesions are among the most challenging in diagnostic
breast pathology, and IHC analysis is often used in making the
diagnosis.
•The most commonly used markers are CK5 (or CK5/6) for the
presence or absence of UDH and MEP cells, and p63 for the
presence or absence of MEP cells

p63 - highest sensitivity and lowest cross
reactivity, and the nuclear staining is easy to
interpret.
CK5/6 appears to have a better sensitivity and
specificity than other markers.
Neuroendocrine markers are useful in
differentiating solid papillary carcinoma (spindle
cell type, neuroendocrine type) from papilloma
with extensive florid epithelial hyperplasia.

•Intraductal Papilloma.—Intraductal papilloma (IP) is a benign
papillary lesion characterized by fibrovascular cores lined by MEP
and epithelial layers.
•Immunohistochemistry analysis for MEP markers highlights the
presence of MEP cells along the fibrovascular cores and at the
periphery of the lesions.
•Intraductal papilloma with atypia is characterized by the presence
of a population of monotonous cells with cytologic and architectural
features of LG-DCIS.
•The MEP cells are typically scant or absent in atypical areas.
•Lack of CK5 or ADH-5 (the breast marker cocktail-CK8/18,
CK5/14,P63) staining and uniform ER expression are helpful clues
for the diagnosis of IP with ADH/LG-DCIS.
•An IP with atypia can be classified as IP with ADH or IP with LG-
DCIS, depending on the size of the atypical proliferation.
•If the size of the atypical area within an IP is smaller than 3 mm,
the lesion is classified as IP with ADH; if the atypical area is 3 mm or
larger, it is called IP with LG-DCIS.

Papilloma
with ADH
vs
Papilloma
with DCIS
Both show
absent MEC
markers
within
Both show
presentMEC
around the ducts
Positive 34βE12,
CK5/6is within
proliferative
component is
indicative of ADH

•Intraductal Papillary Carcinoma.—Intraductal papillary carcinoma lesions are
considered a de novo, in situ papillary malignant process without a recognizable
benign IP in the background.
•Intraductal papillary carcinoma consists of slender fibrovascular cores covered by
a single layer of monotonous neoplastic cells without the presence of MEP cells;
however, the MEP cells are retained at the periphery of the lesions but, often, in a
more-attenuated form .
•The neoplastic cells are usually low to intermediate nuclear grade and are often
strongly positive for ER, PR, and luminal cytokeratins.
•Solid Papillary Carcinoma.—Solid papillary carcinoma (SPC) is a distinctive variant
of papillary carcinoma with a solid growth pattern and inconspicuous fibrovascular
cores.
•It typically has a single, large, expansile mass or multiple solid, closely opposed
nodules, and it may show spindle cell morphology and/or mucin production.
•The neoplastic cells are strongly positive for ER and PR,few neuroendocrine
markers and are negative for CK5, CK5/6, and MEP markers.
•Solid papillary carcinoma should be differentiated from UDH, which is positive for
CK5 and CK5/6.
•Regardless of the presence or absence of peripheral MEP cells, SPC is staged as an
in situ tumor (Tis) if it has a smooth, nodular tumor border.
•A lesion is considered invasive SPC only with the presence of a geographic
‘‘jigsaw’’ pattern with ragged, irregular borders in the absence of MEP cells.

•Encapsulated papillary carcinoma (EPC) is a variant of intraductal papillary
carcinoma and is characterized by fine fibrovascular cores covered by neoplastic
cells surrounded by a fibrous capsule.
•Encapsulated papillary carcinoma lacks MEP cells, both at the periphery of the
lesion and within the fibrovascular cores.
•Most of the EPCs are low- to intermediate-grade, usually have a favorable
prognosis, and are currently staged and managed as in situ carcinoma (pTis).
•Some authors consider EPCs to be slow-growing, invasive lesions or lesions in
transition from in situ to invasion.
•High-grade EPCs are rare and account for approximately 3% of the EPCs. A high-
grade EPC is defined by marked nuclear pleomorphism and increased mitotic
activities. It is also often negative for hormone receptor, tends to be larger, and is
more frequently associated with stromal invasion as compared with low- to
intermediate grade EPCs.
• A recent study reported that 1 of 10 patients with pure, high-grade EPC
developed recurrence and died of the disease.
•Because of its aggressive clinical behavior, those authors suggest managing high-
grade EPC in a similar fashion to invasive breast carcinoma.

Residual benign intraductal
papilloma with solid and cribriform
pattern, indicative of DCIS.
p63 – MECs are present within the
papillae of the residual papilloma
are greatly reduced in number in the
portion of the papilloma occupied by
DCIS/atypia

Intrinsic Breast Cancer Subtypes:
Breast cancers are classified as follows:
1) Luminal-like Breast Cancer Types
– LuminalA
– Luminal B
2) HER2 enriched breast cancer subtype
3) Basal-like breast cancer subtype
4) Claudin-low breast cancer subtype.

Luminal A
• Derives its name from its similarity to the expression
profile of normal luminal breast epithelium.
• Overexpression of ER-regulated genes
• Underexpression of an HER2 gene cluster
• Underexpression of proliferation-related genes.
• Sensitive to endocrine manipulation( hormonal
therapy).
• Less sensitive to cytotoxic agents in both the
neoadjuvant and metastatic settings.
• Approximately 40% of all breast cancers are classified
as luminalA.
• They have favorable prognosis

Luminal B
• Have lower expression of ER-related genes
• Variable expression of an HER2 cluster of genes,
• Relatively higher expression of proliferation-
related genes.
• They represent about 20% of breast cancers.
• They also been shown to have genomic
instability, and to harbor mutations in TP53.
• less sensitive to cytotoxic chemotherapy, sensitive
to hormonal therapy
• Associated with a relatively higher risk of relapse.

HER2 enriched breast
cancer subtype
• It is characterized by high expression of
– HER2
– Proliferation genes and
• low expression of luminal clusters.
• Constitute 20% to 30% of all breast tumors.
• Clinically, they are associated with a poorer
prognosis

Basal-like breast cancer subtype
• Constitute about 15% of invasive ductal breast
cancers.
• Its name is derived from shared gene expression
patterns with normal basal epithelial cells.
• They are considered ER/PR and HER2 negative
(“triple negative”)- TNBCs
• This subtype is also characterized by relatively
high frequency of BRCA1 mutations, increased
genomic instability, high expression of the
proliferation cluster of genes, and a high
histologic grade

Claudin-low breast cancer
subtype
• Is characterized by overexpression of genes
associated with epithelial-to-mesenchymal
(EMT) transition.
• Have no expression of luminal differentiation
markers, are HER2 and hormone-receptor-
negative by IHC
• Frequently exhibit metaplastic and medullary
differentiation, and are often part of the basal
intrinsic subgroup.

Luminal A Luminal B Her-2/neu Basal-like
Gene
expressio
n pattern
Expression(LMW)
cytokeratins, and high
expression of HR’s and
associated genes
Expression (LMW)
cytokeratins, and
moderate to weak
expression of HR’s
and associated
genes.
High expression of
Her-2/neu .
Low expression of
ER and
associated
genes.
High expression of
Basalepithelial
genes, basal
cytokeratins.
Low expression of
ERand Her-2/neu
associatedgenes.
Clinical ~50% of invasivebreast
cancer
~20% of invasive
breast cancers
~15% of invasive
breast cancers
~15% of invasive
breast cancers
ER/PR status ER/PR positive ER/PR positive ER/PRnegative Most ER/PRnegative
Her-2/neu
status
Her-2/neu negative Her-2/neu
Expression
variable (+/-)
Her-2/neu positive
(by definition)
Her-2/neu
Negative
(“triple
negative”)
Biological
features
High proliferation
than luminalA
Highproliferation Highproliferation

Luminal A Luminal B Her-2/neu Basal-like
Luminal B tends to be TP53mutati on TP53 mutation
Higher histological common common; BRCA-1
grade than luminalA More likely to behigh dysfunction (germline
grade and node sporadic)
positive.
Histological
correlation
Tubularcarcinoma
Cribriform carcinoma
Low grade IDC(NOS)
IDC(NOS)
Micropapillar
y carcinoma.
High grade IDC(NOS) High grade IDC (NOS)
Metaplasticcarcinoma
Medullary carcinoma
lobular carcinoma
Treatment Respond to endocrine
therapy
Respondto endocrine
therapy(tamoxifen &
aromataseinhibitors)
Respondto
trastuzumab
No responseto
Endocrine therapy and
trastuzumab
Response to
chemotherapy
variable variable (> in luminal
A)
Good
(anthracycline
based
chemotherapy)
Good
(platinum based
chemotherapy )
Prognosis Good prognosis Prognosis not as good
asfor luminal A
Generally poor
prognosis
Generally poor
prognosis

Prognostic and Predictive Values of Androgen Receptor in TNBCs:
•Androgen receptor (AR) expression has been shown to have prognostic
implications in breast cancers, and higher AR expression levels have been
associated with higher expression of ER or PR, lower nuclear grade, and smaller
tumors, with lower risk of recurrence and death.
•Significant differences in AR protein expression have been detected in the
various molecular subtypes of breast cancers.
•Triple-negative breast cancers, which are by nature ER-ve, characteristically have
much lower frequency of AR expression compared with ER+ breast cancers.
•The AR signaling pathway may represent a molecular driver that can be
therapeutically targeted in Ar expressing TNBCs by using an AR inhibitor as a
novel therapeutic agent(Bicalutamide)
•Therefore, knowing the AR expression status of a TNBC is necessary for selecting
patients who could benefit from antiandrogen therapy.
•Antiandrogen therapy may be of particular interest to those who do not
respond to conventional chemotherapy, or it could even be used as an additional
first line drug.

Lobular or ductal carcinoma: E-cadherin, CK8
•Determining whether an in situ lesion is lobular carcinoma or ductal carcinoma has
clinical management implications and is another situation in which IHC proves its
worth.
•Generally, ductal and lobular carcinomas, either invasive or in situ can be
distinguished in hematoxylin-eosin stained sections.
•In cases with non-specific morphologic characteristics, categorization can be
performed through IHC, and E-cadherin is currently used to differentiate between
the two.
•The majority of ductal carcinomas express cytoplasmic E-cadherin, whereas most
lobular carcinomas lack expression of E-cadherin.
•In addition, the differences in CKs expression may be used: high molecular-
weight CK (clone 34βE12) is usually expressed by lobular carcinomas, but is absent
or expressed at low levels in most cases of DCIS.
•In the same context, CK 8 is stained in ductal carcinoma cells in the peripheral
cytoplasm, while perinuclear staining is characteristic of lobular carcinoma.

E-cadherin
• Strong E-cadherin (ECAD)membranous staining has
been long used to define ductal carcinoma
• Ductal carcinomas, both in situ and invasive,
retainmembranous ECAD
• Lobular carcinomas studied at the genetic level
have oftenshown mutation that accounts for
the loss ofcohesiveness
• Thiscorrelates with either acomplete absenceof
the ECADprotein or abnormal localization (apical
or perinuclear)

p120ctn
• When ECADis absent, the cytoplasmic pool of
p120increases.
• Therefore in normal ducts and in ductal
carcinomas, p120 showsa membranous pattern
of staining
• In contrast, lobular carcinomas, with absent or
non functionalE- cadherin, show strong
cytoplasmic p120 immunoreactivity

METASTATIC
ADENOCARCINOMAS
The breast itself is an uncommon site of metastatic
disease.
Cutaneous melanoma is the most common
extramammary solid malignancy to metastasize to
the breast.
Pulmonary, ovarian, gastric, and renal carcinomas
are also common sources of metastases to the
breast, as is prostatic carcinoma in males
In 24% to 40% of cases the breast lesion is the first
presentation of an occult malignancy.
Absence of an in situ component in a tumor that
is negative for ER, PR, and HER2/neu is a ‘‘red-
flag’

Ovary- WT-1 antibody together with a negative
reaction for GCDFP-15 and/or mammaglobin,
provides strong evidence for metastases.
Negativity for pan-CK, coupled with
immunoreactivity to HMB-45 and Melan-A, is
consistent with metastatic melanoma.
Positivity for S100 protein alone is of limited value.
Metastasis from a gastric signet ring cell carcinoma vs
invasive lobular carcinoma - IHC panel of ER, PR,
and CDX-2 (caudalrelated homeobox gene 2)
antibodies is helpful.

Breast Versus Lung
Solitary lung lesions in patients with a history of
breast cancer and in the workup of metastases of
unknown primary.
GCDFP-15 and TTF-1
Breast Versus Ovary
Same patient population, particularly in those
women who harbor BRCA mutations.
GCDFP-15 and sometimes WT-1
WT-1, being expressed in 96% of ovarian serous
ca and 2% in all breast carcinoma

Breast Versus Stomach
GCDFP-15, ER, and CK20, + for GCDFP-15 is
consistent with a breast primary
An ER+ signet-ring cell carcinoma is more likely to be
of breast origin, and a CK20+ tumor is more likely to
be of gastric origin.
A CK20+/ ER+ signet ring cell carcinoma is more
likely to be of breast origin
Breast Versus Melanoma
Positive reactions for HMB-45 and MART-1, and a
negative reaction for cytokeratin
S-100 is of limited value

Sentinel lymph node Rationale:
• Historically, complete axillary lymph node dissection
wasperformed with lumpectomy or mastectomy
specimen, primarily for staging purposes, providing
information that was used to determine adjuvant
chemotherapy
• Themorbidity associated with this procedure is
substantial in terms of arm pain, limitation of arm
motion, and chronic lymphedema
• TheSLNis identified by injecting aradioisotope and
blue dye before planned surgical excision, Identified
by acombination of visualinspection for blue dye
and intraoperative scanning forradioactivity
• Therationale for this approach is that for patients who
are SLNnegative, a further morbid procedure of
axillary cleanout is unnecessary

SLN
invasion
• SLNsare histologically examined
on multiple levels and by
cytokeratin stains (AE1/AE3)onat
least two levels
• If the primary breast carcinoma is
of the ductal type, most
pathologists would agree that they
would be able toidentify micro
metastases (0.2- 2mm) on HE.
Therefore cytokeratin stains on
SLNdo not add any significant
information in a primary ductal
cancer
• However, when the primary breast
tumorshows alobular
morphology; Becauseof single-cell
infiltration, small metastases (micro
metastases) of lobular carcinoma
requireIHC

Paget’sdisease
• Thepresence of malignant epithelium
with breastcancer immunophenotype
(CK-7+)within the nipple epidermis
• Themajority (>90%)of the underlying breast
carcinomas arehigh grade ductal carcinoma in
situ with apocrine differentiation and comedo-
necrosis, they are her 2 neu positive and ERPR
negative
• Accordingly the Pagetcells are also her 2 neu
positive and ER,PRnegative
• Thedifferential for Pagetdisease includes
melanoma and squamous cell carcinoma in situ
aka(Bowen disease)

Immunohistochemistry of breast lesions(IHC)

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Immunohistochemistry of breast lesions(IHC)