This document discusses gene expression profiling in breast cancer and its use in classifying tumor subtypes. It describes how gene expression profiling analyzes thousands of genes simultaneously to more accurately classify tumors. Breast cancer is classified into clinical subtypes based on receptor expression, including luminal, HER2-enriched, and basal subtypes. Gene signatures can provide prognostic information to help guide treatment decisions for early-stage breast cancer patients. Tests like Oncotype DX and Mammaprint analyze gene expression from tumor samples to predict the risk of recurrence.

1. GENE EXPRESSION
PROFILING IN BREAST
CARCINOMA
Dr Partha Ghosh
2nd year PGT, General Surgery
N.R.S Medical college

2. What is gene expression
profiling?
Cancer is a disease characterized by
uncontrolled cell growth and proliferation. For
cancer to develop, genes regulating cell
growth and differentiation must be altered;
these mutations are then maintained through
subsequent cell divisions and are thus
present in all cancerous cells. Gene
expression profiling is a technique used
in molecular biology to query the expression
of thousands of genes simultaneously. In the
context of cancer, gene expression profiling
has been used to more accurately classify
tumours. The information derived from gene
expression profiling often has an impact on
predicting the patient’s clinical outcome.

3. Tumor biology
IHC mRNA profiling Histology TNM Staging

4. Breast Cancer Subtypes
Breast cancer is classified into clinical
subtypes based upon receptor expression
These subtypes dictate possible therapeutic
options and vary in their prognosis
◦ Luminal: derived from the luminal cells
TYPE A:ER+, PR+,HER-2-
TYPE B:ER+,PR+,HER-2+
Can use hormonal therapy
Less aggressive
◦ Basal: derived from myoepithelial cells
ER-, PR-,HER-2-,ck 5/6+ or HER -1+
No specific target for therapies
More aggressive
◦ HER2-enriched
More aggressive
Luminal A
Luminal B
Claudin-Low
HER2-enriched
Basal

5. Luminal and Basal
Characteristics
Basal
Low ER
Low HER2
High CK5/6
c-KIT higher
High EGFR
High p53
mutation
High p53 protein
High cyclin E
Very high
vimentin
Luminal
High ER
Higher HER2
Low CK5/6
Low c-KIT
Low EGFR
Low p53
mutation
Low p53 protein
Low cyclin E
Low vimentin
Basement
membrane
Myoepithelial Cells 
Basal
Luminal Cells 
Luminal

6. What is HER 2/neu
Other names
◦ Receptor tyrosine-protein kinase erbB-
2
◦ CD340
◦ proto-oncogene Neu
◦ ERBB2 (human)
Located in chromosome 17 (17q12)
Rodent glioblastoma cell line a neural
tumour

7. Cont….
Plasma membrane bound receptor
tyrosine kinase
Positive in 15-30% cases of breast
carcinoma
Associated with increase disease
recurrence and poor prognosis
Tested by-
◦ Immunohistochemistry
◦ FISH
◦ Serum HER2 by ELISA- Trastuzumab
response

8. Cont…
Drugs targeting HER2
Trastuzumab- p27 halts cell
proliferaton
Pertuzumab- prevent dimerisation of
HER2 and HER3
NeuVax- Direct killer T cells to destroy
HER2 +ve cancer cells

9. Tamoxifen resistant breast ca
The expression of HER2 is regulated
by signaling through estrogen
receptors.
Normally tamoxifen down regulate
HER2
Ratio of coactivator AIB-3 exceed that
of corepressor PAX2 HER2
expression in presence of tamoxifen

10. Algorithm for breast cancer
subtypes
All cases
ER - PR -
ER+ or PR +
HER2 - HER2 + HER2 +
HER2 -
EGFR - CK5/6 – EGFR + or CK5/6 +
Unclassified Basal Like Luminal B Luminal AHER2+/ER-

11. Luminal A
Luminal B
HER2+
Basal-like
Intrinsic Breast Cancer
Subtypes described by
Perou et al.
Express ↑ amounts
Of luminal cyto-
Keratins & genetic
Markers of luminal
Epithelial cells of
Normal tissue
Express ↑ levels of EGFR,
c-kit, & growth factors like
hepatocyte growth factor
and IGF

12. Mammaprint: Development of the 70-
Gene Signature
 DNA microarray analysis of 78 breast primary tumors
(untreated)
 Pts were <55 years of age with T1-2/N0 disease
 Pts selected based on outcome: Distant metastases within 5
years
 Statistical analysis, “supervised classification,” identified 231
genes correlated with disease outcome  Top 70 genes
selected
 Genes that regulate cell cycle, invasion, metastasis, &
angiogenesis
 Patients categorized as “good prognosis” or “poor prognosis.”
 Found to be a better predictor of distant metastases within 5
years than all clinical variables in this study
 Odds ratio (distant metastases): poor to good prognosis

13. Does 70-gene Signature have
Independent Prognostic Value?
 Gene signature adds independent prognostic information to that provided by various risk
classifications
 The signature remained a statistically significant prognostic factor for time to distant
metastases & OS even after adjustment for various risk classifications (HR 2.15 & 2.15,
respectively)

14. The 21-gene Recurrence
Score: Oncotype DX

15. Oncotype Dx: The 21-Gene
Assay
Designed to quantify the risk of distant recurrence in
patients with LN(-), ER(+) tumors receiving tamoxifen
RT-PCR was used to quantify gene expression from
fixed, parafin-embedded tumor tissue
250 candidate genes selected based on published
literature, genomic databases, & experiments based on
DNA arrays on fresh-frozen tissue
Analyzed data from 3 independent studies (447
patients) including tamoxifen-only arm of NSABP trial B-
20 to test relation b/w 250 genes and recurrence of
breast cancer
From these studies, 16 genes (+5 reference genes)
were selected that correlated with proliferation and
endocrine response

16. Levels of Gene Expression
Determine Recurrence Score
21-gene assay = 16 outcome-related genes + 5 reference genes
Higher expression levels of
“favorable” genes = ↓ RS
Higher expression levels of
“unfavorable” genes = ↑ RS
A risk score is calculated from 0 -100
Cutoff points chosen based on
Results of NSABP trial B-20

17. Prospective Validation of
Oncotype DX:TAILORX Trial
Low RS:
Hormonal
Therapy
High RS:
Chemo +
Hormonal
Therapy
Hormonal Therapy Chemo + Hormonal
11,248 ER+/LN- patients

18. TN VS BASAL Subtypes
Triple negative
◦ ER,PR,HER2
receptor negative
Basal Subtypes
◦ ER,PR,HER2
receptor negative
◦ Over expression of
breast basal cell
epithelium gene
◦ EGFR+,CK5/6+

19. Basal like subtypes of breast
cancer
No proven therapeutic target
ER,PR negative-cannot use tamoxifen
or anti estrogen
HER2 negative-cannot use Herceptin

20. Targeted therapy
ER + ER-
Hormonal Herceptin EGFR BRCA 1 C-
kit
Therapy Geftinib, erlotinib, DNA damage
Imatinib
lapatinib PARP inh
Luminal
A
Luminal
B HER 2 +
Basal Like

21. Potential treatment options in
TNBC

22. Taxane and Anthracycline Based
Therapy
Typical regimens:
AC-T: doxorubicin plus cyclophosphamide
every 2 weeks for four cycles followed by
docetaxel every 2 weeks for 4 cycles
TAC: docetaxel, doxorubicin, and
cyclophosphamide every 3 weeks for 6
cycles
 Dense dosing is better

23. Platinum Agents
Platinum agents can bind to DNA and
cause cross-linking to occur  cell
death
Cisplatin, carboplatin and oxaplatin
are approved for some types of
cancers and are being studied as
treatments for TNBC

24. PARP Inhibitors
PARP: poly ADP ribose polymerase
◦ Involved in DNA repair by detecting
single-strand breaks
◦ Can be activated in cells with damaged
DNA
Several types of cancer are more
dependent on PARP, so it can be a
good therapeutic target
PARP inhibitors prevent breaks from
being repaired, causing cell death

25. Anti-EGFR
EGFR is overexpressed in 45-70% of
TNBC
Cetuximab is an anti-EGFR antibody
used to treat metastatic cancer
◦ Breast cancer patients with metastatic
disease respond twice as well when
Cetuximab is added
Other treatments include tyrosine kinase
inhibitors (erlotinib, gefitinib)
◦ Gefitinib is the only one currently approved
for breast cancer, but the others are in clinical
trials
Inhibits an important signaling pathway

26. Angiogenesis in Cancer
Angiogenesis: formation of new blood
vessels.
◦ Tumors need blood vessels to grow and
spread.
Angiogenesis inhibitors prevent the
formation of new blood vessels, thereby
stopping or slowing the growth or spread
of tumors.

27. Anti-Angiogenesis
Bevacizumab (Avastin)
◦ Monoclonal antibody to VEGF
◦ Improves survival in breast cancer patients with
combined with Taxol
◦ Approved for metastatic breast cancer but benefit
isn’t subtype specific – this has since been
revoked because it slowed progression but didn’t
extend length or quality of life and had many
adverse effects
Metronomic chemotherapy: repeated, low,
less than toxic doses can destroy endothelial
cells and prevent angiogenesis, slowing
tumor growth – works in clinical trials

28. Androgen Receptor
Nuclear receptor activated by binding
testosterone or dihydrotestosterone
◦ Closely related to PR
Expressed in 75% of breast cancer and 10-20%
of TNBC
◦ TNBC that express AR are molecularly similar to
prostate cancer and could potentially be treated
similarly.
Bicalutamine: anti-androgen used to treat
prostate cancer
17-DMAG: semi-synthetic antibiotic derivative,
has shown promise in clinical trials
Enzalutamide: androgen agonist used to treat
prostate cancer; is in Phase II for TNBC

29. RTK Inhibitors
Suninitib (Sutent)
◦ Multiple-target RTK inhibitor
All PDGFRs and VEGFRs
KIT (CD17) which drives the majority of all GI
stromal tumors & several others
Imatinib (Gleevec)
◦ Prevents phosphorolation of BCR-Abl,
inhibiting signaling pathways necessary for
cancer cell growth
BCR-Abl: Exists only in cancer cells!
Worked in vitro; no effect on metastatic breast
cancer patients in Phase II

30. Src Tyrosine Kinase
Src is overexpressed in breast cancer
Dasatinib: multiple tyrosine kinase
inhibitor approved for CML
◦ Possible efficacy in breast cancer - small
effect seen in Phase II
◦ In vitro: basal breast cancer cells were
more sensitive!
Several others in trials also seem to
have promising preclinical activity

31. mTOR
Cell cycle regulator and a downstream
effector in the PI3K/PTEN/AKT pathway
PTEN is often mutated in TNBC, leading
to increased AKT and mTOR activation
Everolimus and temsirolimus block
mTOR function and inhibit proliferation
◦ Everolimus is approved for some types of
cancers - currently in clinical trials for TNBC
in combination with chemotherapy
◦ Temsirolimus is approved for renal cell
carcinoma and completed a Phase II trial with
promising results

32. Conclusions
Gene signatures augment current
clinicopathological variables in assessing
risk of recurrence
Gene expression profiles may be both
prognostic and predictive for patients with
early breast cancer
NCCN guidelines suggest that Oncotype
DX is an option for risk evaluation in 0.6-1
cm tumors with unfavorable characteristics
or in >1 cm LN-, ER+/HER2 negative
tumors

33. NCCN guidelines include Oncotype DX® testing in
the treatment-decision pathway for node-negative
and micrometastatic disease
33
• Tumor 0.6-1.0 cm,
moderately or
poorly
differentiated,
intermediate or
high grade, or
vascular invasion
• Tumor > 1 cm with
favorable or
unfavorable
pathologic
features
Consider
Oncotyp
e DX
Hormone receptor-positive, HER2-negative disease
pT1, pT2, or pT3 and pN1mi
No test
RS <
18
RS 18-
30
RS ≥
31
Adjuvant endocrine
therapy
± adjuvant chemotherapy
Adjuvant endocrine
therapy
endocrine therapy
± adjuvant chemotherapy
Adjuvant endocrine
therapy
+ adjuvant chemotherapy

34. THANK YOU