1. Current concepts in Breast Cancer- Beyond TNM
Professor Ravi Kant
FRCS (England), FRCS (Ireland), FRCS (Edinburgh), FRCS(Glasgow), MS, DNB, FAMS, FACS, FICS,
Professor of Surgery

2. Applications of Genes Assay in CA Breast
To subclassify breast cancer
To estimate prognosis
To predict response to therapy

3. Applications of Genes Assay in CA Breast
To subclassify breast cancer
To estimate prognosis
To predict response to therapy

4. Gene Expression Patterns of Breast Carcinomas Distinguish Tumor Subclasses With Clinical Implications
PNAS 2001;98;

6. Molecular classification & Prognosis:
Luminal A= Best prognosis
Luminal B
Luminal C
Normal breast like
Her 2+
Basal like= Worst= Triple Negative

7. Subtype ER +, Best overall survival, Best DFS Type Importance
Luminal A
ER +, Best overall survival, Best DFS
Luminal B
ER,Her2+,Intermediate
Her 2 +ve
ER-, Intermediate
Basal like
ER-,PR-, Her2 - Worst

8. IHC profiles for the breast cancer subtypes
Basal-like: (ER−, PR−, HER2−, cytokeratin 5/6+, and/or HER1+).
HER2+/ER− subtype (HER2+, ER−, PR−).
luminal A (ER+and/or PR+, HER2−).
luminal B (ER+and/or PR+, HER2+).

9. Tumor based Gene assay Mammaprint (Amsterdam) Oncotype Dx 76 gene
Test
# of Genes
Tissue
Mammaprint
(Amsterdam) 70
Fresh
Oncotype Dx 21
Fixed
76 gene 76
Wound response
Two gene ratio 2
Intrinsic subtype

10. Tumor based Gene assay Mammaprint 70
Test
Aim
Mammaprint 70
To predict risk of distant mets in N-, To identify who will benefit from Chemo
Oncotype Dx 21
To identify N-, ER+ who will benefit by addition of Chemo to Tamoxifen
76 gene 76
To predict DFS & OS in N-, early stage
Wound response
To predict risk of mets & death
Two gene ratio 2
Intrinsic subtype
To predict clinical outcome

11. Tumor based Gene assay Oncotype Dx 21
Test
Aim
Mammaprint 70
To predict risk of distant mets in N-, To identify who will benefit from Chemo
Oncotype Dx 21
To identify N-, ER+ who will benefit by addition of Chemo to Tamoxifen
76 gene 76
To predict DFS & OS in N-, early stage
Wound response
To predict risk of mets & death
Two gene ratio 2
Intrinsic subtype
To predict clinical outcome

12. MammaPrint
• 70 gene classifier developed further by the company Agendia ( under the name MammaPrint.
• MammaPrint was approved by the FDA in February 2007 for node negative women under 61 years of age and with a tumor < 5cm.

13. Tumor based Gene assay 76 gene 76
Test
Aim
Mammaprint 70
To predict risk of distant mets in N-, To identify who will benefit from Chemo
Oncotype Dx 21
To identify N-, ER+ who will benefit by addition of Chemo to Tamoxifen
76 gene 76
To predict DFS & OS in N-, early stage
Wound response
To predict risk of mets & death
Two gene ratio 2
Intrinsic subtype
To predict clinical outcome

14. Tumor based Gene assay Wound response To predict risk of mets & death
Test
Aim
Mammaprint 70
To predict risk of distant mets in N-, To identify who will benefit from Chemo
Oncotype Dx 21
To identify N-, ER+ who will benefit by addition of Chemo to Tamoxifen
76 gene 76
To predict DFS & OS in N-, early stage
Wound response
To predict risk of mets & death
Two gene ratio 2
Intrinsic subtype
To predict clinical outcome

15. Tumor based Gene assay Two gene ratio 2 Test Aim Mammaprint 70
To predict risk of distant mets in N-, To identify who will benefit from Chemo
Oncotype Dx 21
To identify N-, ER+ who will benefit by addition of Chemo to Tamoxifen
76 gene 76
To predict DFS & OS in N-, early stage
Wound response
To predict risk of mets & death
Two gene ratio 2
Intrinsic subtype
To predict clinical outcome

16. Tumor based Gene assay Intrinsic subtype To predict clinical outcome
Test
Aim
Mammaprint 70
To predict risk of distant mets in N-, To identify who will benefit from Chemo
Oncotype Dx 21
To identify N-, ER+ who will benefit by addition of Chemo to Tamoxifen
76 gene 76
To predict DFS & OS in N-, early stage
Wound response
To predict risk of mets & death
Two gene ratio 2
Intrinsic subtype
To predict clinical outcome

17. Applications of Genes Assay in CA Breast
To subclassify breast cancer
To estimate prognosis/ prediction
To predict response to therapy

18. Conventional classification
Convential Classification assumes that women with a tumor bigger than 2 cm have a high risk to develop distant metastasis.

19. Size is an insufficient indiactor of metastasis risk
Molecular studies shows that size alone is not an indicator of high or low metastasis risk.
Small and large tumors can be either low or high risk as determined by Molecular studies
Molecular
classification

20. Molecular studies provides additional data to assess the risk of distant metastasis
classification

21. MammaPrint Discovers 34% Low Risk Patients in Adjuvant! High-Risk Group
87%
MammaPrint Low Risk
MammaPrint High Risk
N = 209
n = 137
66%
Looking at the 209 patients characterized as High Risk by Adjuvant On-line, MammaPrint identifies 72 patients, or 34% which would be re-characterized as Low Risk
n = 72
34%
Patients re-characterized as Low-Risk
MammaPrint: 34%
Buyse et al., Journal of the National Cancer Institute. 2006;98(17):

22. Gene Assay prediction > Adjuvant Online
Buyse M. Validation and clinical utility of 70 gene prognostic signatures for women with node negative breast cancer.
J Natl Cancer Inst 2006;98:

23. Different proportion of low and high risk patients
MammaPrint profiles accurately 40% as low risk compared to only 15% with St. Gallen criteria.

24. MammaPrint identifies correctly

25. MammaPrint identifies correctly
40% of patients with low risk in comparison to the 15% that are identified with conventional methods, thus preventing many unnecessary chemotherapies.
More precise in predicting the outcome of disease than St. Gallen when comparing survival rates.

26. Mets free / Survival

27. LN + or -

29. Gene Expression vs. Clinical

30. St Gallen vs NIH

33. TRANSBIG Validation: 302 Patients, Node-Neg, T1/2, Age < 61
Amsterdam
Gene expression profiling
Agilent platform
70-gene prognostic custom designed chip
RNA
Target n=400
Achieved
n=307
High or Low Gene
Signature Risk
Tissue Samples
UK (Guy’s, Oxford) 1984 – 1996
France (IGR, CRH) 1978 – 1998
Sweden (Karolinska) 1980 – 1990
Node negative, untreated
<60 years
> 5 years follow-up
T1, T2
Tumor cell % > 50%
Brussels
Comparison of clinical v gene signature assessment of prognostic risk
Endpoints
Time to Distant Metastasis
Overall Survival
Distant Metastasis-Free Survival, Disease-Free Survival
This is an overview of Agendia’s TRANSBIG multi-center validation study which is the second study that demonstrated the clinical utility of using MammaPrint to identify patients at risk of distant recurrence and which formed the basis for the FDA approval language (97% overall survival Low Risk vs. 65% High Risk, and 90% mets free Low Risk vs. 71% High Risk at 10 years). Hazard Ratios of 2.79 for overall survival, and 2.32 for time to distant mets.
Audited clinical data
Centrally reviewed path data (Milan)
<<local>> pathological data
Clinical Data
Buyse et al., Journal of the National Cancer Institute. 2006;98(17): 33

34. Three step validation strategy
Independent
validation study on archival material
N300
Agendia
70-gene
prognostic signature
N=78
N=151
Prospective clinical trial specifically addressing the gene signature’s utility
N6000
Level 1
The signature is
robust
The technology is
reproducible
Level 5 and 4
Level 2-3
Levels of evidence for biomarkers studies

35. MammaPrint® identifies early metastases risk with highest accuracy
29%
39%
50%
62%
75%
83%
96%
100%
4.52
7.54
4.68
3.24
3.5
9.14
2.13
2.33 2 3 4 5 7 10 15
none
Censoring time (in years)
0.1 1
Adjusted hazard ratio for gene signature
Cumulative proportion of events
Time to distant metastasis
HR: all endpoints strongest in first 5 years after diagnosis
MammaPrint’s ability to assign risk of distant recurrence in the first 5 years following treatment is shown here with the hazard ratios being extremely high during this initial period, and thus extremely accurate at predicting an event. As most recurrences develop in the first 5 years post treatment, a profile with the highest hazard ratios in that period attests to its power as a prognostic tool.
Buyse et al., Validation and clinical utility of a 70-gene prognostic signature for women with node-negative breast cancer, Journal of the National Cancer Institute, Vol 98, No. 17, 2006

36. FDA Clearance of MammaPrint®
Study
Purpose
Details
Comments 1
Nature Development 70-gene profile
patients, LN0, <55yrs 6.4% adjuvant treatment
Within 5 year metastasis risk by profile multivariate OR 18 2
NEJM Validation 70-gene profile
patients 5.2% adjuvant treatment
Metastasis-free at 10 yrs: low risk 87%,high risk: 44% 5 yrs: low risk 93%,high risk 56% 3
MammaPrint Development MammaPrint
2006 reproducibility of (1) and (2) on MammaPrint
Highly reproducible MammaPrint as a diagnostic tool 4
TRANSBIG Independent European validation
patients no adjuvant treatment
Metastasis-free at 10 yrs: low risk 88%,high risk: 71% 5 yrs: low risk 96%,high risk 83%
MammaPrint was developed and validated in several different studies that have been published in the following journals. The MammaPrint result generates a result that is either Low Risk or High Risk of distant recurrence at 10 years. The FDA cleared patient population is Stage I or II, ER+ or ER-, node-, and less than 61 years old. Agendia has submitted clearance to add patients 61 years and older. None of the patients in the studies had been treated with any form of adjuvant chemotherapy, giving a true natural biology of the patient’s tumor aggressiveness and your ability to use any form of hormonal or chemo therapy available. 36

37. TRANSBIG, the Translational Research Network of the Breast International Group (BIG), conducted an
independent validation study of both the Amsterdam and Rotterdam gene signatures in a series of 302 patients
Although there was only a 3-gene overlap between the two signatures, both were validated on the same patient cohort

38. So time to learn basics again

40. MammaPrint interrogates critical genomic pathways
MammaPrint interrogates all of the critical genomic pathways associated with tumor progression and the metastatic cascade
Catabolism and tumor hypoxia related metabolism
Cell cycle and cytoskeleton related biogenesis
Extracellular matrix adhesion and remodeling
General signal transduction and intracellular transport
Growth factor
Immune response
Cellular mobility or actin filament related
We are all aware of Weingberg’s Criteria for Malignancy or Metastasis which is shown here. As you will see in the next slide, it is important to capture as many of these pathways as possible in the analysis of the tumor to provide the most accurate assessment of metastasis potential.

41. Applications of Genes Assay in CA Breast
To subclassify breast cancer
To estimate prognosis/ prediction
To predict response to therapy 41

43. 71 Gene assay predictive value
Van de Vijver MJ,He YD, van’t Veer IJ, et al. A gene expression signature as predictor of survival in breast cancer. Amsterdam
N Engl J Med 2002; 347:

44. Oncotype Dx-21 gene predictive value
Palk S, Tang G, Shak S et al. Gene expression and benefit of chemotherapy in women with node negative, ER + breast cancer. J Clin Oncol 2006;24:
Can be done on fixed tissue.

45. 21 gene analysis-Oncotype Dx
Independent of
Tumor size
Age
Park S. NEMJ 2004;351:
> Accurate than Adjuvant Online
Goldstein RP. Abstract #63. San Antonio 2007

46. 21 gene analysis can predict
breast cancer related mortality
Habel LA. Breast Cancer Res 2006
NSABP- B14 trial

47. 21 gene Predictive value

48. Newer prognostic Indicators
Wound response gene– risk of mets and death
2 gene recurrence score –adding chemotherapy to tamoxifen in ER+ ve, N- ve
Chang HY . Gene signature of fibroblast serum predicts cancer progression:similAarities between tumors and wound.
PloS Biol 2004; 2:

49. Wound response gene expression profile
Activation of fibroblasts
Active wound healing predicts ▲ risk of
metastases
Death
( in Breast, Lung & Gastric cancer)
Cong HY. PLoS Biol 2004;2:206-14

50. 2 gene expression profile
60, ER+, Early stage (MaXJ.Cancer Cell 2004)
Expression of
Homeobox 13
IL-17B
▲ ratio= poor outcome
≡Tamoxifen alone will not do in such patients

51. Six models All are different
Great concordance in five out of six gene expression profile models
21 gene (Oncotype Dx) and 70 gene (Mammaprint) are popular
81% concordance between 21 & 70 gene.
Perou, Fan C. NEMJ 2006

52. Breast Cancer: The Treatment Dilemma
Choices of 40 experts world-wide
61 y-old, fit,
postmenopausal
Node negative
pT = 0.9 cm
ductal cancer
ER and PR negative
HER2 negative
Grade 2
Courtesy: Martine Piccart

53. Of 100 women with breast cancer53

54. Only 25% will develop distant metastases54

55. But we treat over 75% of all patients
with chemotherapy 55

56. Which means that 50% of all breast cancer patients get a toxic chemotherapy that they did not need!56

57. Applications of Genes Assay in CA Breast
To subclassify breast cancer
To estimate prognosis
To predict response to therapy

58. To predict response to therapy
Selection of the therapy based on attributes of the
Tumor
Host

59. 21 gene analysis can predict
Responsiveness to Chemo/ Hormone
Gianni L. J Clin Oncol.2005
Palk S. J Clin Oncol 2006;24:
NSABP- B14 trial

60. TAILORx = Trial Assigning Individualized Options for Treatment

61. TAILORx trial
LN-, HER-, ER+
HRx
HRx+ / - Chemo
Chemo ►HRx
Oncotype Dx

62. MINDACT study design 55% 10% 35% BOTH HIGH RISK DISCORDANT RISK
6000 patients, <70 YRS, 1-3 POS NODES
ASSESS clinical RISK AND MammaPrint RISK
(adjuvant!online & MammaPrint)
55%
10%
35%
BOTH HIGH RISK
DISCORDANT
RISK
BOTH LOW RISK
RANDOMIZE
decision-making
Use clinical risk
Use MammaPrint
high
low
high
low
Chemotherapy
No chemotherapy

63. MINDACT Microarray in Node Negative Disease may avoid Chemo
LN-
70 gene assay & adjuvant online
Low risk
HRx if Er+
Discordant
Chemo or HRx
High risk
Chemo + HRx

64. Her2 positive MammaPrint low risk patients have an excellent survival
Knauer SABCC 2008

65. Benefit of adjuvant chemotherapy in MammaPrint high risk patients
88% Endocrine & Chemo (n= 265)
benefit
69% Endocrine (n=184)
HR 0.28 ( ; p=0<0.001)
MammaPrint low risk (n=126)
HR 1.99 ( ; p=non significant)
Distant metastasis-free survival (years)
Median Follow-up 5.2 years
Knauer et al, 2008 unpublished

66. To predict response to therapy
Selection of the therapy based on attributes of the
Tumor
Host

67. To predict response to therapy based on attributes of the host
Drugs metabolized by
CYP450 encoded enzymes
CYP 2
CYP 3
CYP2D6
CYP2C19

68. To predict response to therapy based on attributes of the host
Drugs metabolized
AmpliChip CYP450 by Roche

69. Prognostic Signature and Clinical Benefit
-the chemotherapy choice-
MammaPrint prognosis signature
Assigns patients to risk categories with high
specificity and sensitivity
(low risk vs high risk for recurrence)
Low risk sufficiently low to forego chemotherapy
High risk identifies patients with early relapse
and shows chemo benefit (predictiveness) 69 69

70. Clinical applications of microarrays
Who to treat:
Prognosis profiles as diagnostic tool
-> improved selection for adjuvant therapy
How to treat:
Predictive profiles for drug response
-> selection of patients who will benefit most
WHO
NEEDS
THERAPY?
WHICH
THERAPY WILL WORK BEST?
Prognostic factors
Predictive factors

71. What does “low risk” mean?
MammaPrint® “Low Risk”: 90% metastasis-free without any adjuvant treatment over the following 10 years (NEJM 2002/JNCI 2006)
Most of the “Low Risk” patients are ER+
With ER+ patients receiving hormonal therapy, a further 50% risk reduction can be achieved in the “Low Risk” group, thus MammaPrint “Low Risk” means >95% 10 year metastasis-free survival
The cut-off point chosen in the development study was based on having only 10% of the patients having a recurrence in the Low Risk group (3 out of 34). Again this is with no hormonal therapy, so with the addition of hormonal therapy, and an assumed benefit of approximately 50%, the low-risk patients are more in the ~5% risk of distant recurrence at 10 years and you should feel confident this is an extremely Low Risk patient. MammaPrint does not have an intermediate group with as patients in the studies that were classified as High Risk, had a risk of distant recurrence of 30%-50% (~20 patients out of 40 had a recurrence in the development study).

72. Summary : Poor risk Basal like Luminal B HER2+/ER-
Poor 70 gene profile
High 21 gene recurrence score
Activated wound response

73. Summary : New Decision aid
21 gene (Oncotype Dx) & 70 gene (Mammaprint) is better than Adjuvant Online
Personalised treatment
Less toxicity, less cost

74. Contrast of Appearance vs. Expression Phenotyping
Microscope
Low Grade
High Grade
Treatment Advice
The Microscope is used to review an H&E for grading purposes however studies have shown significant discordance especially in the stage 2 grade. Today’s latest technologies for staging using molecular diagnostic tools include DNA Microarrays and RT-PCR.
Microarray
Low Risk
High Risk

75. Triple Negative ER-, PR-, and HER2 – Basal like on gene profiling
Adverse prognosis
→ new Rx

76. Thanks