1. JOURNAL CLUB
13/09/2012
DR. R. RAJKUMAR
III YR POST GRADUATE
DEPARTMENT OF MEDICAL ONCOLOGY

2. Question 1
• Important prognostic factors in breast
cancer include:
– A. Lymph node status, hormone receptor
status, and TNM stage
– B. Histologic subtype
– C. Family History
– D. Age at diagnosis

3. Question 2
• Genomic Analysis:
– A. Determines familial breast cancer risk
– B. Oncotype DX technology is useful in
ER+ and ER- breast cancers
– C. Helps determine the best adjuvant
chemotherapy regimen
– D. Has been validated in retrospective
studies

4. Question 3
HORMONAL THERAPY FOR ER+ PR –
BREAST CANCER
• 1.TAMOXIFEN
• 2.AROMATASE INHIBITORS
• 3.NOVEL TARGETED AGENTS
• 4.DONT KNOW

6. Outcomes of Adjuvant
Chemotherapy in Breast Cancer
Copyright © American Society of Clinical Oncology
Walgren et al. JCO 2005;23:7342-7349

7. Changing Portraits
Concept evolution
claudin low
Lum A Lum B Basal Her2

8. Adjuvant Systemic Therapy for
Breast Cancer: Decision Making
Prognostic Factors
– Estimate outcome independent of systemic
treatment
– Reflect tumor biology: Who should be treated?
Predictive Factors
– Reflect a relative resistance or sensitivity to
specific therapy
– What specific treatment(s) should be offered to
an individual?

9. Breast Cancer Prognostic Factors
Accepted
– TNM Stage
– Axillary Nodal Status
– Tumor Size
– Tumor Grade
– ER Content
– Oncotype DX (?)
Investigational
– Gene expression arrays
– Proteomics
– Pharmacogenetics
– Novel imaging
– Other

10. Breast Cancer Predictive Factors
Accepted
– ER status
– Grade
– HER 2 overexpression
– Oncotype DX (?)
Investigational
– Gene expression arrays
– Proteomics
– Pharmacogenetics
– Novel imaging
– Other

11. Intrinsic Breast Cancer
Subtypes described by
Perou et al.
Luminal A
Luminal B
HER2+
Basal-like
Express ↑ amounts
Express ↑ levels of EGFR,
Of luminal cyto-
c-kit, & growth factors like
Keratins & genetic
hepatocyte growth factor
Markers of luminal
and IGF
Epithelial cells of
Normal tissue
Sotiriou, C. et al. NEJM, 2009.
11

12. Figure 1a.
Sorlie T, Tibshirani R, Parker J, et al: Repeated observation of
breast tumor subtypes in independent gene expression data
sets. Proc Natl Acad Sci U S A 100:8418-23, 2003 12

13. St. Gallen 2007
Highly Endocrine Non–endocrine Incompletely
Responsive Responsive Endocrine Responsive
High ER and PgR ER and PgR Low ER and PgR
and both absent or
No HER2 PgR absent
overexpression or
and
Low Ki-67 HER2 overexpression
or
High Ki-67
ER = estrogen receptor; PgR = progesterone receptor.
Goldhirsch et al. Ann Oncol. 2007;18:1133.
13

14. St. Gallen – Endocrine Responsiveness
“Practical” Clinical Subgroups
Endocrine-
responsiveness
Absent Uncertain Sure
ER and PR absent ER and PR low/int Both receptors
and/or any of these high levels
• PgR absent No
• UPA/PAI-1 high No
• HER-2 overexpressed No
• Increased proliferation No
• High grade No
Chemo only options Chemo adds Chemo doesn’t work
to hormonal
14

15. The Level of ER Expression Is
Predictive
The higher the level of expression, the greater the benefit
from endocrine treatment
The higher the level of expression, the lesser the added
benefit of chemotherapy
15

16. Added Value of PgR Status in
Assessing Endocrine Responsiveness
Estrogens ER
Estrogen-responsive elements
Cell cycle PgR synthesis
16

17. Added Value of PgR Status Assessment
Quality control of ER status assessment
– ER-/PgR+ tumors do not exist (almost!)
– ER 10%/PgR 90% is very unusual (and likely related to poor
sensitivity of ER staining)
Prognosis (among ER+ tumors)
Effectiveness of endocrine therapies (and chemotherapy
in premenopausal patients)
Response to AI?
AI = aromatase inhibitor.
Viale et al. J Clin Oncol. 2007;25:3846.
17

18. STEPP for Central PgR
(ER-Expressing) in the BIG 1-98 Trial
100
80
4-y DFS (%)
Tamoxifen
60 Letrozole
40
20
0
0 10 30 40 50 60 75 90 99
Subpopulation by PgR%
STEPP = subpopulation treatment effect pattern plot.
19

19. Is ER/PgR Status Assayed Well in
Clinical Practice?
Inconsistent allocation to “ER/PgR-negative”
– No immunoreactive cells?
– Less than 10% immunoreactive cells?
– Less than 20% immunoreactive cells?
– A different threshold for different clinical questions/settings?
Conflicting results
– >15% disagreement between different laboratories (false negative)
20

20. False-Positive Assays?
Local Central
N % N %
ER+/PgR+ 3124 71.0 3330 75.7
ER+/PgR- 965 21.9 832 18.9
ER+/PgR? 220 5.0 48 1.1
ER-/PgR+ 80 1.8 13 0.3
ER-/PgR- 5 0.1 103 2.3
ER-/PgR? 0 0 1 <0.1
ER?/PgR+ 2 <0.1 23 0.5
ER?/PgR- 0 0 7 0.2
ER?/PgR? 3 <0.1 42 1.0
21

21. Oncotype DX 21-Gene
Recurrence Score (RS) Assay
16 Cancer and 5 Reference Genes From 3 Studies
PROLIFERATION ESTROGEN RS = + 0.47 x HER2 Group Score
Ki-67 ER - 0.34 x ER Group Score
STK15 PR + 1.04 x Proliferation Group Scor
Survivin Bcl2 + 0.10 x Invasion Group Score
Cyclin B1 SCUBE2 + 0.05 x CD68
MYBL2 - 0.08 x GSTM1
GSTM1 BAG1 - 0.07 x BAG1
INVASION
Stromelysin 3 CD68 Category RS (0-100)
Cathepsin L2 REFERENCE
Low risk RS <18
Beta-actin Int risk RS ≥18 and <31
HER2 GAPDH
GRB7 RPLPO High risk RS ≥31
HER2 GUS
TFRC
Paik et al. N Engl J Med. 2004;351:2817- 22

22. Standardized Quantitative
Oncotype DX Assay
Recurrence Score in N-, ER+ patients
40%
Intermediate
Low Risk Group High Risk Group
Risk Group
35%
Distant Recurrence at 10 Years
30%
25%
20%
15%
10%
5%
0%
0 5 10 15 20 25 30 35 40 45 50
Recurrence Score
Lower RS’s Higher RS’s
•Lower likelihood of recurrence •Greater likelihood of recurrence
•Greater magnitude of TAM benefit •Lower magnitude of TAM benefit
•Minimal, if any, chemotherapy benefit •Clear chemotherapy benefit
1) Paik et al NEJM 2004, 2) Habel et al Breast Cancer Research 2006 23
3) Paik et al JCO 2006, 4) Gianni et al JCO 2005 23

23. Oncotype DX Extensively Studied:
Study Experience in >3700 Patients
Study Type No. Pts
Providence Exploratory 136
Rush* Exploratory 78
NSABP B-20 Exploratory 233
NSABP B-14* Prospective 668
MD Anderson* Prospective 149
Kaiser Permanente* Prospective Case-Control 790 Cases/Controls
NSABP B-14 Prospective Placebo vs Tam 645
Milan* Exploratory 89
NSABP B-20* Prospective Tam vs Tam+Chemo 651
ECOG 2197* Exploratory and Prospective 776
SWOG 8814 Prospective Tam vs Tam+Chemo 367
*Published studies24

24. Schema: TAILORx
Node-Neg, ER-Pos Breast Cancer
Register
Specimen
Oncotype DX Assay
banking
RS 11-25
RS <10 Randomize RS >25
Hormone Hormone Rx Chemotherapy
Therapy vs +
Registry Chemotherapy Hormone Rx
+ Hormone Rx
Primary study group
25

25. 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
Van ’t Veer, L. Nature, 2002.
26

26. EORTC-BIG MINDACT TRIAL DESIGN
6,000 Node negative women
Evaluate Clinical-Pathological risk and 70-gene signature risk
N=3300 (55%) N=2100 (35%) N=600 (10%)
Clinical-pathological Discordant cases Clinical-pathological
and 70-gene both Clin-Path LOW Clin-Path HIGH and 70-gene both
HIGH risk 70-gene HIGH 70-gene LOW LOW risk
Randomize
Use Clin-Path risk to Use 70-gene risk to decide
decide Chemo or not Chemo or not
Adjuvant
Adjuvant Endocrine
Chemotherapy
therapy only
(+ endocrine Tx if ER+)
The goal of this trial is to show that MammaPrint can
spare 20-30% of patients from adjuvant chemo
Dr Martine Piccart-Gephart JBI, Brussels 27

27. 70-gene 21-gene 2-Gene Intrinsic
Signature Signature Ratio Subtypes
Analysis Supervised Supervised Supervised Unsupervised
Approach
Formalin-Fixed, Formalin-Fixed, Formalin-Fixed,
Tissue Type Fresh or Frozen Parafin- Parafin- Parafin-
embedded embedded embedded
Technique DNA microarrays Q-RT-PCR Q-RT-PCR Q-RT-PCR
Prognostic Untreated pts Untreated & TAM-treated, TAM-treated
age<60, T1-2, LN- TAM-treated ER+/LN-
ER+/LN- untreated
Predictive Benefit to TAM Response to TAM
NO +/- CMF/MF NO
Validation Retrospective Retrospective Retrospective Retrospective
Prospective
Trials MINDACT TAILORX NONE NONE
28

28. Steroid Hormone Receptor Signaling
Reading:
Handbook of cell signaling, Ed RA Bradshaw and EA Dennis, 2003.
Chapter 275
Cheskis, BJ, 2004. Regulation of cell signaling cascades by steroid
hormones
Steroid hormones are produced by endocrine glands
Essential regulators of: reproduction, secondary sex
characteristics
Development, differentiation
Glucose metabolism
Response to stress and salt balance

29. Nuclear Receptor Superfamily
 large family of structurally related ligand-inducible
transcription factors, including:
 steroid receptors (SRs),
 thyroid/retinoids receptors (TR, RARs and RXRs),
vitamin D receptors (VDR),
 estrogen receptors (ERa and ERb),
 and orphan receptors for which no ligand has
been yet identified.
 While having in common a modular structure, they
are activated by distinct lipophilic small molecules
such as glucocorticoids, progesterone, estrogens,
retinoids, and fatty acid derivatives

31. Estrogen Receptors
 ER-
 Uterus, testis, pituitary, ovary, epididymis, and adrenal
gland.
 ER- (Kuiper et al. 1996)
 brain, kidney, prostrate, ovary, lung, bladder, intestine,
and epididymis.
 88% identity with rat ER-b;
47% identity with human ER-a
 Both ERs are localized to membrane, cytosol, and
nucleus.
 ER and differ in C-terminal ligand binding domains and
N-terminal transactivation domains. Highest homology in
DNA binding domain.
 Estrogen-related orphan receptors (ERR)

32. Estrogen Receptors
http://www.bio.cmu.edu/Courses/BiochemMols/ER/#ERchime

33. Estrogen Receptor

35. ER effects on different cell types

36. Steroid receptor coactivators and
ER-dependent gene transcription
Histone
P/CAF Acetylase
CBP Activity
SRC
Family AIB1
Transcription
Estradiol-bound ER

37. Mode of Action of Estradiol
Estradiol AF2 Coactivator
FULLY
E + ER E E ACTIVATED
E RNA
E ERE TRANSCRIPTION
Receptor Nuclear POLII
Coactivator
(tumor cell
AF1dimerization localization of
AF1 division)
fully active ER
AF1 + to ERE AF1 and AF2
AF2 recruit
ACTIVE coactivators
ted from: Wakeling AE. Endocr-Relat Cancer 2000; 7: 17–28.

38. Fig.15-12

39. HREs are short cis-acting sequences located within the promoters or
enhancers of target genes.
HREs: inverted repeats of AGGTCA (ER and ERRs)
inverted repeats of AGAACA (for GR, MR, PR, and AR)

45. Hall et al.
J. Biol. Chem., Vol. 276, Issue 40, 36869-36872, October 5, 2001

47. Genomic versus Non-
genomic
 changes in gene  changes in existing
expression enzyme activity and/or
protein structure
 delayed (hrs-days)
 rapid (sec-min)
 requires nuclear
 unknown cytosolic
receptor
mechanisms
 prevented by
 not affected by
transcription and
transcription and
translation inhibitors
translation inhibitors

49. Cross-talk between signal transduction
and endocrine pathways
Growth factor
Estrogen IGFR
HER2 Trastuzumab
Plasma
P P
membrane P P
P
AI P SOS
PI3-K RAS
RAF
Cell
P
survival Akt MEK
P
ER
p90RSK P
MAPK
P
Cytoplasm Cell
P P P Basal growth
P transcription
ER p160 CBP machinery
ER
Nucleus ERE ER target gene transcription
Adapted from Johnston

50. Ligand
E
ErbB ErbB
P P
P E
p85
p110 Ras ER
Akt MAPK
P E
P ER
Transcription
ER-Responsive Element

53. CHARACTERISTICS OF ER+ PR-
BREAST CANCER
• MORE AGGRESSIVE PHENOTYPE
• LARGER IN SIZE
• OLDER PATIENTS >60 YRS
• HIGHER BMI
• HIGHER S PHASE FRACTION
• GREATER GENOMIC INSTABILITY
• HIGHER LEVELS OF EGFR & HER1 HER2
• TAMOXIFEN RESISTANCE

58. ER+/PR tumors are resistant to
tamoxifen (ATAC)
From Cui et al. JCO 23:7721, 2005

59. Negative PR is a marker of high HER1/HER2
levels and tamoxifen resistance
ER+/PR+
ER+/PR+
Arpino et al. JNCI 97:1254, 2005

60. Negative PR is a marker of high HER1/HER2
levels and tamoxifen resistance
ER+/PR+ ER+/PR
ER+/PR+ ER+/PR
Arpino et al. JNCI 97:1254, 2005

63. The Molecular Portrait Hypothesis
You can recognize the
Mona Lisa by her smile
and her nose and her eyes and even her hands – if you are really good,
but not the sky or the trees

64. The Promise of Personalized
Medicine in Breast Cancer
Tamoxifen
Postmenopausal
Women with HR+ Aromatase
breast Cancer Inhibitor
Chemotherapy
Biologic agents Anth, Taxane,
Platimun
Her2, EGFR, VEGF, Parp

65. Question 1
• Important prognostic factor in breast
cancer include lymph node status,
hormone receptor status, and TNM
staging
– Histologic subtype and family history have
not been independently validated
prognostically, and age at diagnosis is
neither prognostic nor predictive
Stearns et.al., BCRT 1998; 52: 239-259
Harris, L et.al., J Clin Oncol 2007 Nov 20; 25 (83) 5287-312

66. Question 2
• Genomic analysis has been validated in
retrospective studies
– Available genomic analytic assays
(Oncotype DX, Mammaprint) do not
determine familial risk. Oncotype DX has
been validated only in ER+ breast cancers.
Neither assay determines type of adjuvant
chemotherapy.
Paik, S et.al., N Eng J Med 2004 Dec 30; 351(27): 2817-26
Paik, S et.al., J Clin Oncol 2006 Aug 10; 24(23): 3726-34
Albain, K et.al., SABCS 2007 abstr #10

67. Question 3
• HORMONAL THERAPY FOR ER+ PR –
BREAST CANCER
1. AI - 52% lower risk for recurrence
2. EGFR INHIBITORS , m TOR INHIBITORS,
PI3K INHIBITORS, IGF INHIBITORS
anastrozole plus gefinitib- 49% clinical benefit.