IHC evaluation of breast
cancer and recent advances:
diagnostic and prognostic
aspect
Professor Dr. Ferdousy Begum
MBBS, MD (Pathology)
Bangabandhu Sheikh Mujib Medical University
Introduction
• Breast cancer is a heterogenous disorder caused by interaction of
genetic and environmental risk factors
• Progressive accumulation of genetic and epigenetic changes results in
breast cancer
• Tumors with similar clinico-pathologic presentations may have
different behaviours
• Thus, better understanding of molecular classification leads to newer
biological insights and better therapies directed toward particular
molecular subsets
Importance of pathological
subtypes
IBC, like many other cancers, is categorized by clinical stage and
pathological subtype.
• This categorisation:
-Correlates with survival data.
-Predicts natural history of disease & treatment response
-Fundamental to therapeutic decision-making in oncology.
Histological classification of breast
cancer
Breast Carcinoma
Invasive/ Infiltrative carcinoma of no
In situ carcinoma special type
Microinvasive carcinoma
Invasive lobular carcinoma
DCIS LCIS
Tubular carcinoma
Comedo
Cribriform carcinoma
Cribriform
Mucinous carcinoma
Micropapillary
Mucinous cystadenocarcinoma
Papillary
Invasive micropapillary carcinoma
Solid
Carcinoma with apocrine
differentiation
Metaplastic carcinoma
A. Invasive lobular carcinoma
B. Tubular carcinoma
C. Mucinous carcinoma
D. Mucinous carcinoma
E. Neuroendocrine carcinoma
F. IDC with osteoclastic giant cells
G. Micropapillary carcinoma
H. Apocrine carcinoma
I. Metaplastic carcinoma
J. Medullary like carcinoma
K. Adenoid cystic carcinoma
Is histological
classification enough in
this era of personalized
medicine?
WHY?
Some cases need further work up to confirm diagnosis.
• Clinical staging and routine pathology are principle indices -for adjuvant
chemotherapy.
• However, this results in ‘over-treatment’ of many patients.
• Group of Long-term survivors in the untreated arms.
• Some patients in spite of adjuvant cytotoxic treatment develops
metastatic disease.
• 'Clinicians lack a marker that predicts who will benefit from
chemotherapy'
Uses of IHC as diagnostic and prognostic
markers
• Myoepithelial markers
• Pancytokeratin
• D2 40
• E-cadherin
• Gross cystic fluid protein 15( GCDFP-15)
• GATA 3
• Mammaglobin
• CD34
• Ki-67
Uses of IHC as diagnostic and prognostic markers
Immunohistochemical markers used for to differentiate
between benign and malignant primary carcinoma
Antibody Localization Myoepithelial Myofibroblast Carcinoma
cells
S100 Cytoplasm Weak Variable Variable
SMA Cytoplasm Strong Moderate Rare
Calponin Cytoplasm Strong Weak-mod Rare
p63 Nucleus Strong Negative Rare-nuclei
Examples of use of IHC markers in
benign breast lesion
H&E Ki-67
Examples of use of IHC markers in
benign breast lesion
P 63 SMA
Standardization for Hormone
Receptor testing
• Prompt fixation of breast tissue: 6 to 72 hours of 10% neutral buffered formalin
fixation
• Processing by conventional (not microwave enhanced) tissue processors.
• Formalin newly replenished in the processor
• Processor fluids should not exceed 37°C.
• In vitro diagnostic kits should be used that utilize one of these ER clones: 6F11,
1D5 or SP1.
• Positive and negative controls, internal and external, should be used on each
run.
• A positive cut-off of 1% - nuclear expression, and results should be semi-
quantitated with the percentage of cells staining and their intensity.
Scoring system for ER/PR expression
in breast cancer
Predictive Markers
Core biopsy of breast (H&E, 100x) ER, 100x
PR,100x HER 2 neu, 100x
Predictive Markers
Core biopsy of breast (H&E, 400x) ER,100x
PR,100x HER 2 neu,100x
H&E stain
HER2/neu (IHC: Score 1+)
HER2/neu (IHC: Score 2+)/ Equivocal
HER2/neu (IHC: Score 3+)
Molecular profiling
• Molecular profiling allows tumours to be “defined by the expression
pattern or genomic alteration of thousands of genes simultaneously”
• Techniques comes the prospect of defining individual genes or
combinations of genes whose expression level(s) can discriminate
efficiently between clinical subtypes.
• Gene expression microarrays have been used extensively to study
breast cancer.
1941 2000
Foote and Perouet al 2010
Stewart - Molecular Gene
histologic 1970 subtypes expression
subtypes-Lobular 1990
Wellings and Breast cancer- based
vs Ductal, In situ ER IHC
Jensen -all cancer Luminal A/B signatures–
vs invasive, developed
arise from TDLU Basal like Oncotype Dx,
Mucinous, Her 2 Enriched Mammaprint,
Tubular and Normal Breast PAM 50
Secretory like
Newer subtypes-
Claudin low
Molecular apocrine
Characterized variation in gene
expression patterns in a set of 65
specimens of human breast tumours
from 42 different individuals, using
complementary DNA microarrays
representing 8,102 human genes.
1.The first use of GEP to stratify breast cancer into distinct molecular classes
was introduced by Perou and colleagues in 2000.
2.The tumours show great variation in their patterns of gene expression.
3.This variation is multidimensional; i.e., different sets of genes show
independent pattern variation.
The same group:
ER-positive luminal group -separated to at least
2 subgroups: luminal A and luminal B.
Diagnostic modalities for
molecular classification of
breast cancer
• IHC (Protein)
• Fluorescent in situ hybridization (FISH)
• Gene expression (m RNA ):
• Comparative genomic hybridization
• Gene expression microarray
• qRT-PCR
• Next generation sequencing (NGS) (Somatic alterations)
Molecular subtyping of breast cancer using IHC surrogates
Prognostic multigene
signatures:(Microarray & RT-PCR)
• 21 gene signature (Oncotype Dx)
• 70 gene signature (Mamma Print) 1st generation
• 76 gene signature (Rotter dam)
• 50 genes: Risk of Recurrence (ROR) (PAM-50) 2nd generation
• 12 genes (Endo-predict) & Epclin
• 97 gene: Genomic grade index (Map Quant Dx)
• 14 genes (Breast Onc Px)
• 14 gene signature (Celera Metastasis Score™)
• 7 gene assay (THEROS The Breast Cancer Index)
Prognostic multigene
signatures:(Microarray & RT-PCR)
When to ask?!:
• ER +, Her 2-, Node-ve tumours
Why to ask?!:
• To asses patients who will benfit from additional chemotherapy and who will
not.
• Risk of recurrence
What to consider?!:
• Financial constraint
Oncotype DX and Mammaprint
Oncotype DX and Mammaprint–FDA approved!!
• Oncotype DX -incorporated in NCCN Guidelines and St Gallen criteria
(management decision models)
• Recommended by ASCO and ESMO as useful diagnostic tool –with
additional information to complement pathology report.
Where do we stand??(At least in
our Setup)
• ER, PR, HER2 and Ki-67 status are major drivers for clinical decision.
• Considered as Surrogates for Intrinsic subtypes.
Experience in BSMMU
• Immunohistochemistry started in 2004 at research level.
• Commercially started from 2009 (manually)
• Automation started from 2011.
• So far 5386 cases have been reported up to December, 2022.
• FISH test has been introduced in October, 2021
Breast cancer cases ( ER, PR
and Her2) from 2009 to 2022
No. of cases
800
700 687
645
600 567 576
530
500
441
400
320
300
250
216
200 190
100 83
59
37
0
2009 2010 2011 2012 2013 2014 2015 2016 2017 2018 2019 2020 2021
No. of cases
IHC equivocal HER2/neu Cases Received in Pathology Department of BSMMU For
FISH Test (From October’2021- September’2023)
1. Age distribution (n=197)
Age groups of the patients
Cumulative
Frequency Percent Valid Percent Percent
Valid 20-40 years 43 21.8 21.8 21.8
old
41-60 years 113 57.4 57.4 79.2
old
61-80 years 37 18.8 18.8 98.0
old
> 80 years 4 2.0 2.0 100.0
Total 197 100.0 100.0
IHC equivocal HER2/neu Cases Received in Pathology Department of BSMMU For
FISH Test (From October’2021- September’2023)
2. Specimen Type (n=197)
2%
33.5%
64.5%
IHC equivocal HER2/neu Cases Received in Pathology Department of BSMMU For
FISH Test (From October’2021- September’2023)
3. Results of FISH test (n=197)
32.5%
67.5%
HER-2 FISH HER-2 CEP-17 HER2/CEP-17 ratio REFERENCE RANGE
(Red signal) (Green signal) (HER-2 gene amplification)
TOTAL SCORE (in Ratio < 2: Not amplified
20 tumour cells nuclei)
39 33 1.18 Ratio ≥ 2: amplified
Green Signal- CEP17, Red Signal-HER2
HER-2 FISH HER-2 (Red CEP-17 HER2/CEP-17 ratio REFERENCE RANGE
signal) (Green signal) (HER-2 gene amplification)
TOTAL SCORE Ratio < 2: Not amplified
(in 20 tumour cells
229 51 4.49 Ratio ≥ 2: amplified
nuclei)
Green Signal- CEP17, Red Signal-HER2
IHC equivocal HER2/neu Cases Received in Pathology Department of BSMMU For
FISH Test (From October’2021- September’2023)
4. Molecular subtypes: before and after FISH test. (n=197)
Molecular subtype Frequency percent(%)
Molecular subtype before FISH
before FISH
100
Luminal type A 20 10.2 90
80
Luminal type B 19 9.6 70
60
Luminal type ( Ki67 93 47.2
Frequency
50
unknown) 40
30
HER2 Enriched 4 2
20
Triple negative 61 31 10
0
Luminal A Luminal B Luminal ( Ki67 HER2 Enriched Triple negative
Unknown)
Total 197 100
Molecular subtype before FISH
Continued..
IHC equivocal HER2/neu Cases Received in Pathology Department of BSMMU For
FISH Test (From October’2021- September’2023)
4. Molecular subtypes: before and after FISH test. (n=197)
Molecular subtype Frequency percent(%)
Molecular subtype after FISH
after FISH
100
Luminal type A 20 10.2 90
80
Luminal type B 19 9.6 70
60
Luminal type ( Ki67 93 47.2
Frequency
50
unknown) 40
30
HER2 Enriched 26 13.2 20
10
Triple negative 39 19.8 0
Luminal A Luminal B Luminal type HER2 Enriched Triple Negative
( Ki67 Unknown)
Total 197 100 Molecular subtype after FISH
Thank
you
