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Evaluation of HR[ER/PgR] status
and correlation with Ki67
expression in BC
Gayatri Gogoi, MD
Assistant Professor
Deptt of Pathology,
Assam Medical College, India
Email id [email protected]
Co-Authors



M Borgohain, MD
Professor of Pathology
Manash P Baruah, DM ,
Consultant Endocrinologist
S A Fazal, MS
Associate Professor of Surgery
Background
 Breast cancer[BC] is a leading cause of death
in women. Hormonal Receptor status is the
most important prognostic and predictive
marker for BC.
 It has been used in management of breast
cancer since 1970s as an indicator of
endocrine responsiveness and prognostic
factor for recurrences
Management depends
• For comprehensive management testing of
BC panel is a must, namely Estrogen Receptor,
Progesterone Receptor, Her2/neu
ER-positive (ER+) tumors typically respond to
hormone therapy,
 while HER2+ tumors respond to anti-HER2
therapy, and
 no targeted therapy is currently available for
widespread use for ER−/HER− tumors
Molecular Classification
 Breast cancers are classified into 5 molecular
subtypes
 [1] Luminal A , is defined by as only Estrogen
receptor[ER] with or without Progesterone
Receptor[PR] expression
 [2] Luminal B, is defined by ER with or
without PR as well as Her2/neu expresssion
Molecular classification
3. Normal breast like
4. Her2neu enriched
5.Basal Like
including Triple negative
BC[TNBC]
ER,PR,Her2 ,all 3 markers negative is TNBC.
 BC expressing basal markers CK5/6 are called
basal like
Hormonal receptors are ER & PR
Luminal BC means arising from luminal cells
which are positive for cytokeratin 8 & 18.
It can be either Luminal A or B
Markers of proliferation, and specifically Ki-67labelling index, is also considered important
for the determination of prognosis .
This was the back ground knowledge I had at
beginning of this study in 2009-10
Aim and Objective
To evaluate the hormonal[ER/PR] receptor
status in invasive BC
and
study the
proliferative behavior of both Luminal A and B
by Ki67 expression.
Correlate the findings with prognostic
parameters
Setting and Design
 A prospective analysis of 112 BC cases, was
undertaken to study the histomorphological
features followed by immunohistochemical
study in Department of Pathology .
 The mastectomy specimens were received
from Department of Surgery and private
hospitals
of
surrounding
area.
Material and Methods:
The present study was approved by the
institutional ethical committee for Human
Research .
•
 It was carried out in the Department of
Pathology,
in
Histopathology
and
Immunohistochemistry
Sections,
Assam
Medical College & Hospital, Assam, from July
2011 to June 2012.
Material and Methods:
 All specimens were fixed in 10% buffered formalin(
PH7.2-7.4) in an adequate volume of 10 fold
immediately after operation and allowed to stay
between 12—24 hours (ASCO guidelines 6—72 hours)
 Specimens were grossed on the same day mostly or
next day, , with special emphasis to the site, size of the
tumour, margins and lymph node sampling of the
lesion and sections from representative area.
 The sections were processed under standardized
conditions for paraffin embedding.
Material and Methods:
 Histopathologic features were determined
examination.
prior to IHC
 Histopathologic types were ascertained
 Histological grade was assessed using Bloom and
Richardson's method, modified by Eltson and Ellis. by
evaluation of tubule formation, nuclear pleomorphism,
mitotic count.
 Involvement of lymph nodes and numbers.
 Comment on surgical margins,
presence of DCIS ,
LymphoVascular invasion or perineural involvement etc
-------------------------------------------------------------------------------------------------------------•
Bloom HJG, Richardson WW. Histologic grading and prognosis in breast cancer: a study of 1409 cases of which 359 have been followed for 15 years. Br J Cancer. 1957;11:359– 377
Interpretation of IHC Results:
 Interpretation of IHC results were done according
to ASCO and CAP Guideline Recommendations
for IHC Testing of ER and PR in Breast Cancer.
 According to the guidelines it is recommended
that ER & PR assays be considered positive if at
least 1% positive tumour nuclei in the sample on
testing in presence of expected reactivity of
internal control (normal epithelial element) and
external control.
IHC scoring for ER,PgR
Her2+
• More than 30% of cells showing strong
complete membrane + in invasive BC
component to be interpreted as over
expressed
Ki 67 or Mib 1 Antibody :
 Only showing distinct nuclear staining by Ki67 of
invasive carcinoma component were selected for
counting.
 Though various authors used different criteria for
counting in various malignancies but purpose of breast,
it was counted like mitosis counting formula of BRG, so
that it is easily comparable to mitotic count and can be
analyzed effectively (Trihia H et al, 2003).
 ------------------------------------------------------------------------ Trihia H, Murray S, Price K, et al: Ki-67 expression in breast carcinoma. Cancer
97:1321-1331,2003.
Results & observations
• Table 1.1: Age
AGE DISTRIBUTION
33.04
PERCENTAGE (%)
35
27.68
30
25
20.54
20
15
9.82
10
5.36
3.57
5
0
< 29
30—39
40—49
50—59
AGE GROUP (IN YEARS)
60—69
> 70
• Table–1.1 shows more than 66.08% of the
invasive breast carcinoma cases belonged to
younger than 50 years of age group out of
which 33.04% from 40—49 years age range
and 26% from less than 39 years age range.
The youngest case was 24 years and the oldest
one was 75 years with a mean age of
presentation is 44.53  11.24.
Histology Grade I [BRG]
IDC with apocrine change
BRG II
IDC –BRG III
Table 1.2 Bloom Richardson Grade
BLOOM RICHARDSON GRADE
44.64
45
40
32.12
PERCENTAGE (%)
35
30
23.21
25
20
15
10
5
0
I
II
BRG GRADING
III
Table 1.2 Bloom Richardson Grade
The histological grading showed that 44.64%
of invasive breast carcinoma cases belonged
to grade II category.
 Next 32.12% cases of breast carcinoma
having poorly differentiated features belonged
to grade III. Last 23.21% cases show well
differentiated feature with histologic grade I.
 So it is seen that best prognostic category
have least numbers of cases in the study.
Table 1.3 ER/PgR profile
54.47
56
52.68
PERCENTAGE (%)
54
52
50
47.32
46.56
48
46
44
42
ER
PgR
MARKERS
POSITIVE CASES
NEGATIVE
ER/PgR profile
• This
study
of
112
invasive
BC
immunohistochemical evaluation showed
47.32% are ER positive and 46.58% are PgR
positive.
• There are ER+ cases where histologic grades
are high, Her2 overexpressed, Ki67 high.
• We tagged them as Luminal B
HR positive : Luminal A
Micropapillary type
Her2 +
ER+, Allred score 8/8
PgR+ but low 2-5% cells
Ki 67 high grade- Luminal B
Luminal B example
 Micropapillary case belonged to Luminal B .
ER + strong 100%
PR + low 2-5%,
overexpression of her2/neu
High expression of Ki67
Table 1.4 Luminal A and Luminal B
35
31.25
30
PERCENTAGE (%)
25
16.04
20
15
10
5
0
ER and or PgR expression
ER and or PgR with Her2neu over
expression
Total HR expression (47.29%)
Table 1.4 Luminal A and Luminal B
• Total Hormonal Receptor positive in 47.29% of
cases, but pure ER and or PR positive cases are
only 31.25% which is grouped as Luminal A
category .
• The subgroup with Her2neu over expression
is 16% which is categorized as luminal B
according to available literature
Table 1.5 ER+ groups correlation to
Ki67 expression
HR GROUPS CORRELATION TO Ki67 EXPRESSION
41.06
45
35.71
40
PERCENTAGE (%)
35
23.21
30
25
20
15
10
5
0
Low
Moderate
Ki67 EXPRESSION
High
Table :1.7 Luminal A , B with Ki67
38
40
35
30
26
NUMBER (n)
24
25
20
15
12
10
10
2
5
0
Luminal A
Luminal B
SUB GROUPS
Low Ki67
Moderate Ki67
High Ki67
Correlation in subgroups significant
ER+ expression profiles were correlated , no
significant correlation found.
 But when correlated in subgroup as luminal A
and B seperately, it shows a statistically
significant relationship with P value<.005, high
expression in Luminal B type
Luminal A group show mostly low and
moderate Ki67 expression
Table 1.6 :Lymph node in ER + groups
LYMPH NODE IN HR POSITIVE GROUPS
44
45
40
NUMBER (n)
35
30
23
25
20
20
15
15
10
5
5
5
0
0
1—3
4 or more
LYMPH NODES
Luminal A
Luminal B
Lymph node status in ER + groups
Luminal A have maximum node negative
breast cancer , which is statistically
significantWith P value less than .005
Luminal B have maximum number node
positive cases of 1-3 node followed by more
than 4 nodes.
Discussion & review literature
 we have found in Luminal B cases are generally
 High histologic grades ,
 High expression of ki67 ,
 PR score is low or absent
 more likelihood of axillary node positive
More commonly seen in less than 50 years woman.
Besides Her2 overexpression which is criteria to
include that category.
PR role in clinics
 PR another molecular marker that may be used in the clinic,
as loss of PR in ER+ tumors is thought to be predictive for lack
of response to hormone therapy.
 In our study while ER+ subgrouping were done with help of
protein expression taking the criteria of ER & Her2 +, or ER,
PR&her2+
 In that situation, PR downregulation was not taken into
consideration for favour of Luminal B as most literature or
consensus for clinical setting doesnot talk much on it.
 ---------------------------------------------------------------------------------------- -Cui X, Schiff R, Arpino G, Osborne CK, Lee AV. Biology of progesterone
receptor loss in breast cancer and its implications for endocrine therapy. J
Clin Oncol. 2005;23(30):7721–7735. [PubMed]
Predictive value of PR
 The predictive value of PR has long been attributed to
the dependence of PR expression on ER activity, with
the absence of PR reflecting a nonfunctional ER and
resistance to hormonal therapy.
 However, recent clinical and laboratory evidence
suggests that ER-positive/PR-negative breast cancers
may be specifically resistant to selective ER
modulators,
 whereas they may be less resistant to estrogen
withdrawal therapy with aromatase inhibitors, which is
a result inconsistent with the nonfunctional ER theory
Pathophysiology of Luminal B
The designation of luminal B first came about with
the early gene expression profiling studies of breast
cancer near the start of the millennium, it has long
been understood that there exists a more aggressive
form of ER+ breast cancer.
 Earlier studies had noted that ∼30% of ER+ invasive
breast cancers showed no benefit from hormone
therapy, suggesting that these cancers either
augment or entirely bypass the classical estrogenstimulated mitogenic pathway

---------------------------------------------------------------------------------------------------------------------------------------------------------------------

. Allred DC, Brown P, Medina D. The origins of estrogen receptor alpha-positive and estrogen receptor
alpha-negative human breast cancer. Breast Cancer Res. 2004;6(6):240–245. [PMC free article] [PubMed
ER modulator resistance
 Novel alternative molecular mechanisms
potentially explaining SERM resistance in ERpositive/PR-negative
tumors
have
been
suggested by recent experimental indications that
growth factors may downregulate PR levels.
 Thus, the absence of PR may not simply indicate
a lack of ER activity, but rather may reflect
hyperactive cross talk between ER and growth
factor signaling pathways that downregulate PR
even as they activate other ER functions
Alternate pathway of ER function
• Loss of PR expression is thought to represent a
surrogate for a more aggressive disease
phenotype that is less dependent upon estrogen
signaling; in correlative studies
• PR loss has been associated with lower ER levels,
more positive nodes, aneuploidy, larger tumor
size, higher proliferation, and expression of
growth factor receptors (GFRs) including EGFR
and HER2
Diagnosis of Luminal B
 Assays for diagnosing luminal B subset of ER+ in the
clinic are currently available, though it will likely take
some time for these assays to become routine as new
clinical practices are often gradually adopted. EgOncotype Dx, Mammaprint etc. Cost?
 PR is one marker that is frequently measured in the
clinic along with ER, in order to further subdivide ER+
breast cancers by prognosis or anticipated therapeutic
response.
 ----------------------------------------------------------------------- . Cui X, Schiff R, Arpino G, Osborne CK, Lee AV. Biology of progesterone
receptor loss in breast cancer and its implications for endocrine therapy. J
Clin Oncol. 2005;23(30):7721–7735. [PubMed
Down PR ,up Her2, EGFR?
 the association of PR with hormone response has not
been observed everywhere, which may be an indicator
of a single biomarker having insufficient information as
compared to a biomarker panel
 Clinical variables such as grade or Ki-67 can help
distinguish the subset of ER+ breast tumors with
expected worse outcome.
Her2 over expression is well established but EGFR
expression in Luminal B require further study
– ------------------------------------------------------------------Cheang MC, Chia SK, Voduc D, et al. Ki67 index, HER2 status, and prognosis of patients with
luminal B breast cancer. J Natl Cancer Inst. 2009;101(10):736–750. [PMC free article]
[PubMed
Clinical potential and future research
At present, luminal B breast cancer is routinely
treated as an ER+, hormone-sensitive disease.
While often considered as simply a more
aggressive form of the ER+ subtype, luminal B
might be considered more in the future as a type
of breast cancer that is entirely distinct in many
ways from luminal A.
•
Prat A, Ellis MJ, Perou CM. Practical implications of gene-expressionbased assays for breast oncologists. Nat Rev Clin Oncol. 2011;9(1):48–57.
[PMC free article] [PubMed]
What best can be done in clinical
setting?
Having that background knowledge, we can
sum up
 ER+ positive BC have two different entities
 pathophysiologic, treatment, molecular
profiling point of view,
What best can be done in our clinical setting
respect to our limited facility ?
Stratification ER+ type of BC
Ki67 expression can be used document
proliferative behavior as the previous studies
applied to test aggressive behavior in long term
management of early BC.
This ki67 is also can be used to tag Luminal B in
clinical setting and managment
---------------------------------------------------------------Molecular profile of Luminal B cancer, Chad J
Creighton, Biologics 2012, 6:289-297
Ki67 in BC clinics
• Ki67 can be used in ER+ Breast Cancer sub
grouping In Clinical Setting
• Our study too find its higher expression is
directly proportional to poor prognostic
parameters .
Conclusion & Future perspective
• Ki67 expression can significantly add into
understanding of aggressive behaviour of BC
• It is a valuable tool in stratification of
ER+[Luminal tumours] into likely SERM
responsive or resistant or Luminal A or B
• It also has a definitive role in long term
management of early BC .
• Testing of Ki67 in both primary tumour and
lymph node together may help in understanding
its prognostic role.
57
•
Acknowledgement:
Assam Medical College &Hospital
Srimanta Sankardev University& Health Science
Assam, India
• . But the Beginning of the Brown Era of IHC
•
At AMCH
• So, everyone of us to give the best to the
the Dream
realize
References
• Molecular profile of Luminal B cancer, Chad J
Creighton, Biologics 2012, 6:289-297
• Cui X, Schiff R, Arpino G, Osborne CK, Lee AV. Biology of
progesterone receptor loss in breast cancer and its
implications for endocrine therapy. J Clin Oncol.
2005;23(30):7721–7735. [PubMed]
. Sørlie T, Perou CM, Tibshirani R, et al. Gene expression
patterns of breast carcinomas distinguish tumor
subclasses with clinical implications. Proc Natl Acad Sci
U S A. 2001;98(19):10869–10874. [PMC free article]
[PubMed]