Molecular Biomarkers in

breast cancer
Presenter Dr. Ankush Nayyar
 Introduction
• Breast cancer is one of the most common
type of cancer in females in India
• Breast cancers are conventionally
classified into different types by
– morphological feature,
– histological features,
– tumor grade,
– proliferation status,
– lymphovascular invasion – prognostic
variables
What is a molecular marker?

According to US National Institutes of Health’s

(NIH) Working Group and Biomarkers Consortium,
a molecular marker is a characteristic that is
objectively measured as an indicator of
pathogenic processes or a pharmacological
response to a therapeutic intervention. Although
the most of these markers are proteins, recently,
gene expression patterns and altered DNA in tumor
tissue have also taken prominence as tumor
markers
Why do we need to do molecular
testing in breast cancers ?
• Molecular testing in breast cancer is used to
– Classify tumor types,
– Recognize hereditary implications (eg,
BRCA1 mutations)
– Identify appropriate therapeutic agents (eg,
HER2+ disease or ER/PR + disease),
– Determine the prognosis of the disease by giving
the risk score,
– Identify biomarkers that can predict or monitor the
response to treatment
• The first molecular classification system uses
only hormonal receptors and HER2 which
predicts response to hormonal therapy and
anti HER2 respectively.

• As the molecular techniques have evolved

it is now possible to analyze the
expression of thousands of gene in a single
test in order to predict the outcome of
therapy
 Luminal A Luminal B Her-2/neu Basal-like
Gene Expression(LMW) Expression (LMW) High expression of High expression of
cytokeratins, and high cytokeratins, and Her-2/neu . basal epithelial
expression expression of HR’s and moderate to weak Low expression of genes, basal
pattern associated genes expression of HR’s ER and associated cytokeratins. Low
and associated genes. expression of ER and
genes. Her-2/neu
associated genes.

Clinical ~ 50% of invasive breast ~20% of invasive ~15% of invasive ~15% of invasive
cancer breast cancers breast cancers breast cancers

ER/PR status ER/PR positive ER/PR ER/PR negative Most ER/PR negative
moderate/weak
positive
Her-2/neu Her-2/neu negative Her-2/neu Her-2/neu positive Her-2/neu
status expression variable (by definition) negative(“triple
(+/-) negative”)

Biological Low proliferation High proliferation High proliferation High proliferation

features than luminal A
 Luminal A Luminal B Her-2/neu Basal-like
Grade Usually low grade Luminal B tends to be TP53 mutation TP 53 mutation
higher common common; BRCA-1
histological grade than More likely to be high dysfunction (germline
grade and sporadic)
luminal A node
positive.

Histological Tubular carcinoma IDC (NOS) High grade IDC (NOS) High grade IDC (NOS)
Cribriform carcinoma Micropapillary Metaplastic
correlation Low grade IDC (NOS) carcinoma. carcinoma Medullary
Lobular carcinoma
carcinoma

Treatment Respond to endocrine Respond to endocrine Respond to No response to

therapy therapy trastuzumab Endocrine therapy and
(tamoxifen& trastuzumab
aromatase inhibitors)

Response to Variable Variable (> in luminal Good Good

chemotherapy A) (anthracycline (platinum based
based chemotherapy
chemotherapy) )

Prognosis Good prognosis Prognosis not as good Generally poor Generally poor
as for luminal A prognosis prognosis
 Role of pathologist
• To classify the tumor on the basis of molecular
profiling
• To suggest for appropriate investigations that a
patients should undergo
• Testing for specific molecular markers
• To suggest for appropriate therapy and
management for the patients
Well-Established Prognostic and/or Therapeutic Breast Cancer Markers

1. Hormone receptors

2. Human Epidermal Growth Factor Receptor 2 (HER-2)

3. Ki 67 Antigen

4. Tumor Protein p53

5. Breast Cancer Susceptibility Genes (BRCA1 and BRCA2)

 1. Hormone Receptors (HR)

The HR best studied in breast cancer are

1. Estrogen receptor (ER) and
2. Progesterone receptors (PR).

• Expression of ER and PR have different clinical, pathological, and molecular

characteristics.
• During the 1980s, Tamoxifen became the first anti-estrogenic therapy targeted to ER
for adjuvant therapy.

• Bind to the ligand-binding domain of ER, effectively blocking the potential for
estrogen stimulation.

• Tamoxifen produced clinical remission in patients with breast cancer positive for ER,
differently from tumors with low or undetectable levels of these receptors.

• Tumor cells expressing hormone receptors presented a better response to hormone

therapy and patients demonstrated higher survival, both disease free as overall
2. Human Epidermal Growth Factor Receptor 2 (HER-2)

• HER-2 is a transmembrane tyrosine kinase receptor belonging to a family of epidermal

growth factor receptors structurally related to epidermal growth factor receptor (EGFR)

• Encoded by ERBB2/HER2 oncogene located on chromosome 17q21 .

• Is amplified in 20 to 30% of breast cancers and is considered a marker of poor

prognosis.

• Its overexpression is associated with an aggressive phenotype of tumor cells, resistance

to anti-hormonal, cytotoxic therapies, and low overall survival.

• Currently, the humanized monoclonal antibody Trastuzumab, directed against the

extracellular domains of HER-2, is indicated for the treatment of HER-2 positive breast
cancer cases.

• other therapeutic strategies have been developed to target HER-2 protein, such as
tyrosine kinase inhibitor Lapatinib, which showed improved efficacy after failure of
Trastuzumab therapy
 3. Ki 67 Antigen

• The Ki-67 antigen is a labile, nonhistone nuclear protein that is tightly linked to the
cell cycle and is expressed in mid-G1, S, G2, and M phases of proliferating cells but
not in quiescent or resting cells of the G0 and early G1 phases.

• Ki-67 score is the most often measured on histological sections by IHC methodology
and is defined as the percentage of stained invasive carcinoma cells.

• Ki-67 score is the most often measured on histological sections by IHC methodology
and is defined as the percentage of stained invasive carcinoma cells
 4. Tumor Protein p53

• p53 is involved in several critical pathways including cell cycle arrest, apoptosis,
DNA repair, and cellular senescence, which are essential for normal cellular
homeostasis and genome integrity maintenance.

• Alteration of TP53 gene alter its response to cellular stress.

• In breast cancer, approximately 30% of patients display TP53 gene mutation

• Expression of mutant p53 protein is associated with high tumor proliferation rate,
early disease recurrence, and early death in node-negative breast cancer.

• IHC is used to detects indirect indicative of mutation in TP53 gene.

• P53 mutation is an indicator of a poor clinical outcome for breast cancer patients.

• Despite its prognostic value, there is still no proper treatment that takes into
account the status of this marker.
5. Breast Cancer Susceptibility Genes (BRCA1 and BRCA2)

• Approximately 80% of the familial breast cancer are associated with one gene of
hereditary susceptibility for breast and ovarian cancer, BRCA1 and BRCA2.

• The BRCA genes have been classified as tumour-suppressor genes.

• BRCA proteins play important roles in different cellular processes, including

activation and transcriptional regulation, repair of DNA damage, beyond the control
of cell cycle, cellular proliferation, and differentiation.

• somatic disease-causing mutations in BRCA1 or BRCA2 are extremely rare in sporadic

breast cancer.

• If a high-risk status of these women had been recognized, they have had the
opportunity to choose genetic counseling, testing, more effective cancer surveillance,
and potentially preventive options, such as prophylactic surgery and/or
chemoprevention
Future Candidate Markers for Prognosis and Therapeutic Responses in Breast Cancer
Evolution

1. Proliferating Cell Nuclear Antigen (PCNA)

2. Caveolin

3. Receptor C-X-C Chemokine Receptor Type 4 (CXCR4)

4. Chemokine (C-C Motif) Ligands 2 and 5 (CCL2 and CCL5)

5. Growth Factors: EGF, HGF, IGF, VEGF, and TGF-β

 Gene expression profiling(GEP)
• It is the determination of the pattern of genes
expressed, at the level of transcription, under
specific circumstances or in a specific cell to give
a global picture of cellular function.
• Done by
– Immunohistochemistry (IHC)
– Fluorescent in situ hybridization (FISH)
– Reverse transcription Polymerase chain reaction
(RT-PCR)
– Gene Microarray
– Next generation sequencing (NGS)
 IHC
• Description - Use of antibodies to
detect levels of a specific protein
• Detection - Protein expression levels
• Sample requirement - Tissue sections -
– Formalin fixed paraffin embedded (FFPE)
samples
 IHC
Advantages
• Simple, inexpensive procedure
• Processed slides can be stored for years and reassessed
• Cell morphology can be viewed
Disadvantages
• Semiquantitative, subjective score
• Fixation time can affect results
• Results dependent on quality of antibody used to
detect the protein
• Usually only 1-2 proteins can be analyzed per section
 Immunohistochemistry

• Hormonal receptors (HR) and HER2 are

prognostic marker and therapeutic target
for breast cancer
• The techniques for identifying HER2 & HR
– Immunohistochemistry (IHC) and
– Fluorescence in situ hybridization (FISH) - lack of
morphologic details
– Chromogenic ISH (CISH)
 Scoring system for ER/PR
Score for propotion Score for intensity

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
Total score ranges from 0 to 8.
Tumors scoring ≤2 are regarded as ER negative and have a negligible chance of response.
IHC for ER/PR
 Her 2 Neu Scoring
Her- Staining pattern Her-2/neu protein
2/neu overexpression
0 No reactivity seen Negative
1 Weak incomplete staining in any proportion of Negative
tumour cells

2 Non uniform or weak to moderate complete Equivocal

membranous reactivity in >10% of the tumour
cells
OR
Intense complete staining of <30% of the invasive
tumour cells.

3 Uniform, intense, complete membranous Positive

reactivity in >30% of the invasive tumour cells.
 IHC HER2

HER2 immunohistochemical staining with a score of 3+

 FISH

• Description - Use of a fluorescently labeled

DNA probe to detect specific DNA
sequences in chromosome
• Detection –
– Gene number alterations,
– DNA rearrangements
• Example assays - HER2 FISH pharmDX Kit
• Sample requirement - Tissue sections
 FISH
Advantages
• High sensitivity and specificity
• The resolution is better.
• Can be applied to both dividing and non-
cells.
dividing
Disadvantages
• costly fluorescence microscope required;
• results must be captured and stored within a short
period (fluorescent signal decays within a few
weeks);
• Only 1-2 DNA regions analyzed per experiment
• There are two types of bright-field chromogenic
HER2 ISH assays:
1) single color ISH for the HER2 gene only –
– six or > HER2 positive,
– four-six HER2 signals considered equivocal and
– less than four signals considered HER2 negative
2)Dual color ISH for the HER2 gene and CEN17
(chromosome 17 centromere). - the ratio of
HER2 gene copy numbers to CEN17 copy
numbers
– negative: HER2/CEN17 ratio <1.8;
– equivocal: HER2/CEN17 ratio 1.8-2.2;
– positive: HER2/CEN17 ratio >2.2
HER2 Testing using fluorescence in-situ hybridization (FISH).
Normal HER2 gene status is observed with 1-2 copies of HER2
gene (black dots) and CEN17 (red dots) targets in each nucleus.
B) Amplified HER2 gene status is observed with multiple HER2
gene copies.
 Gene Microarray technique
• Description - Detection of specific DNA
sequences or cDNAs (for RNA analysis)
by hybridization to an array of DNA
probes
• Detection - Gene number alterations, DNA
rearrangements, gene expression levels, RNA
editing
• Sample requirement - As low as 75 ng DNA
from FFPE sample can be used
Gene Microarray technique
 Gene Microarray technique
Advantage
• Predict the disease behavior
Disadvantage
• Dependent on the sensitivity and specificity
of the probes
• Rare sequences not necessarily detected
 Next generation DNA
sequencing
• Description - Sequencing of thousands or
millions of DNA sequences in 1 reaction
• Detection –
– DNA amplification
– DNA rearrangements,
– DNA mutations
– DNA deletion
– RNA editing
• Sample required - > 100 ng DNA/RNA
from fresh frozen specimens more ideal or
FFPE
 Next generation DNA sequencing
Advantages
• Whole genome sequencing
• Targeted genome sequencing
• Facilitate the sequencing at a greater depth (at base
pair level)
• Enable the detection of rare gene sequences
• Large panels (a few hundred) of cancer-specific genes
are selectively sequenced at a time.
Disadvantage
• Costly
• complicated data analysis
• length of time (weeks) for results
• High-tech lab, not routinely done
 Next generation sequencing
• NGS-based assays that can detect gene mutations
from small amounts of DNA are also in development;
– DNA from fine needle aspirates or
– circulating tumor DNA (ctDNA) from blood samples
• Several studies have shown a high degree of
concordance between mutations in ctDNA (detected
using NGS techniques) and mutations from the primary
tumors
• NGS panels gene sets are available in companies
such as Foundation Medicine, Life Technologies, and
Illumina etc.
 Conclusion
• Molecular testing is becoming
increasingly important in the precise
classification, diagnosis and treatment of
breast cancer

• In spite of so many assays the

molecular profiling experiments still
evolving

• With the increasing use of molecular profiling

sequencing, the identification of novel
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