1. Epidemiology

5. How do we translate complex molecular interactions into therapeutic benefit?

6. “Omics” Wederson Marcos Claudino et al., JCO 2007, mod.
Targetomics

7. From omics to knowmics

8. All patients with same diagnosis
Courtesy H. McLeod

9. All patients with same diagnosis
Courtesy H. McLeod

10. (CPT-11/Cetuximab Chemotherapy Combination)
Clinical Observation
(CPT-11/Cetuximab Chemotherapy Combination)
Patient Patient 2
Mucositis ++
+++ Diarrhea +
++ BM-Toxicity -
- Skin-Toxicity +++
+ Tumor Response ++++
Inter-individual Differences in
Response to Treatment and Side Effects

11. Alternate therapy Standard therapy
All patients with same diagnosis
Alternate therapy
non-responders
and toxic responders
Standard therapy
Responders and Patients
Not Predisposed to Toxicity
Courtesy H. McLeod

12. One Size Doesn't Fit All!

13. Histologically they look like, but…
Pt 39 yrs, pre-menopausal
T 2.2 cm, N (-), G1
ER (±)/PR(+), HER-2 (-)
09/2000: QUART
FEC100 x LHRH-Tam
Pt 47 yrs, pre-menopausal
T 2.4 cm, N (-), G1
ER (±)/PR(+), HER-2 (-)
12/2003: QUART
FEC100 x LHRH-Tam
10/2004: Metastatic disease
09/2005: NED
Topoisomerase II
By courtesy of S. Iacobelli, 2006

14. Overview
• A disease defined by negatives is not one disease!
• No single HER2 equivalent has been found
• A number of promising targets / driving pathways are emerging

15. Definition Biology

16. The “Triple Negative” Breast Cancer
Estrogen Receptor (ER) negative
Progesterone receptor (PR) negative
Her2neu (HER2) negative
ER/PR/HER2 -
“Basal-Like”

17. What is a triple-negative breast cancer?
Triple-negative cancers account for 10–17% of all breast carcinomas, depending on the thresholds used to define ER and PR positivity and the methods for HER2 assessment.
The main characteristics of triple negative cancers that have emerged from the literature illustrate the similarities between basal-like and triple-negative tumours, including the fact that
they more frequently affect younger patients (<50 years),
are more prevalent in African-American women,
often present as interval cancers and
are significantly more aggressive than tumours pertaining to other molecular subgroups

18. Triple-negative and basal-like breast cancers: synonyms?
Despite the great interest in basal-like cancers, there is still no internationally accepted definition of these tumours. From a scientific perspective, microarray based expression profiling analysis remains the ‘gold standard’ for the identification of basal-like breast cancers.
However, for the foreseeable future, this technology is unlikely to be introduced in the diagnostic armamentarium for breast cancer patients, and results of microarray-based expression profiling usingRNA extracted from formalin-fixed archival samples are suboptimal.
Therefore, several attempts to define an immunohistochemical surrogate for basal-like cancers have been described. Although many ‘immunohistochemical signatures’ have been described, little information about their specificity and sensitivity for the identification of basal-like cancers as defined by microarray analysis has been provided.
The best example to date is the panel proposed by Nielsen et al., where basal-like cancers are defined as those lacking both ER and HER2 expression and expressing CK5 ⁄ 6 and EGFR.

20. Gene Expression Patterns of Breast Carcinomas Predict Survival
O.S. A B C D E
Basal-like
Subgroup
= E
HER2
Subgroup
= D
Normal
breast
like
Luminal
Subtype C
Luminal
Subtype B
Luminal
Subtype A
months
Adapted from Sorlie et al. PNAS, 2001

21. Will the same type of treatment fit all types of tumor ?
Gene Expression\
ICH
Luminal-like A
ER+ and/or PgR+ HER2-
Luminal-like B
ER+ and/or PgR+ HER2+
Basal-like
ER- PgR- HER2- CK5/6+ and/or HER1+
HER2-like
ER- PgR- HER2+
Will the same type of treatment fit all types of tumor ? 21

22. Typical Histological Features of “Triple Negative” Breast CA
Grade 3, IDC, with central fibrosis
Positive Staining for EGFR
Positive Staining for CK 5/6
Turner et al. 2006

23. Triple-Negative vs Basal-Like
Conforti et al. SABCS 2007 (N=823)
Triple-Negative prevalence = 18% (150/823)
Triple-Negative Basal-Like = 66% (95/150)
Triple-negative non-Basal-Like = Luminal B

24. Triple-Negative vs Basal-Like
Lacks of ER, PgR and HER2
Prevalence = 10-15%
Mostly High-Grade
Basal-Like
No standard Definition
Lacks of ER, PgR and HER2
Stains for CK5/6/14, EGFR, p53, c-kit
Prevalence = 10-15%
Mostly High-Grade

25. How do we identify “Basal-Like” Breast Cancers in the clinical setting?

26. Definition of “Basal-Like” Breast Cancers
Multiple immunohistochemical markers have been used to identify Basal-Like differentiation
No universally agreed upon criteria or precise set of basal markers
Nielsen et al.  ER/PR/HER2 negativity in addition to either EGFR + and/or CK5/6 +
Smith et al.  high molecular weight cytokeratins CK5, 14 and 17
Carey et al.  ER/PR/HER2 negativity

27. 100 Patients with “Triple Negative” Breast Cancer
ER/PR/HER2 -
80 to 90% of these will be
“Basal-Like” Breast Cancers
20-10% Adenoid cystic
and Medullary Typical

28. “Triple Negative” Breast Cancers
Comprise approximately 15% of all invasive cancers
More common in:
Younger patients
African Americans
BRCA1 mutation carriers
Unique Morphologic Attributes
Pushing border
high grade
central scarring/acellular zone
Stromal/peritumoral lymphocytic infiltrate

29. BRCA1 and BLC phenotype
BRCA1 is a tumor suppressor gene that functions in DNA repair
These tumors are often ER-, HER2- (up to 80% of BRCA1 associated tumors have been shown to be “basal-like”)
Though…most “basal-like” tumors have normal BRCA1
Hereditary BRCA1 breast tumors and “basal-like” sporadic tumors have a similar phenotype and gene expression signature
Suggesting involvement of BRCA1 in the pathogenesis of sporadic “basal-like” cancer

30. BRCA1/BLC
BRCA1 mutations are rarely found in sporadic breast cancer, but BRCA1 expression is often reduced, especially in “basal-like” breast cancers
Why?
BRCA1 promotor methylation has been demonstrated in 7 to 31% of sporadic cancers (Catteau et al)
LOH of the BRCA1 locus which occurs in 15 to 45% of sporadic breast cancers (Rio et al)
However, studies looking at the relationship between BRCA1 methylation, BRCA1 LOH and clinical features of breast tumors have been inconsistent
ID4, a negative regulator of BRCA1, was found to be expressed at a 9-10 times higher in BLC (Turner et al)

31. Shared Features with BRCA-1 Cancers
BRCA1 associated breast cancer
“Basal-Like” Breast Cancer
Estrogen receptor Negative 
HER2 negative
Express Cytokeratin 5/6
EGFR overexpression and mutation
High grade
p53 mutation
Cytogenic abnormalities

32. Immunohistochemical Features:
Other Breast CA
n (%)
“Basal-like”
p value
ER expression
517/665 (78%)
3/21 (14%)
HER2
23/87 (26%)
0/21 (0)
Cytokeratin 5/6
105/761 (14%)
13/21 (62%)
EGFR
14/521 (8%)
12/21(57%)
< 0.001
C-KIT
67/605 (11%)
6/21(30%)
P53 mutation
13%
9/11 (82%)
P53 protein
27/124 (22%)
32/63 (51%)
< 0.006
Vimentin
2/28 (7%)
17/18 (94%)
0.0001
Livasy et al, Sorlie et al, Foulkes et al, Nielsen et al

33. Phenotype changes during breast development
Laakso, Clin Canc Res, 2006

39. Clinical features

40. “Triple-Negative” Breast Cancer: Clinical Features and Patterns of Recurrence
HBBC database ( )
1601 (80%) of patients had details on hormone receptors/HER2 and were eligible for the study
180 (12%) of the 1601 patients were defined as “triple negative” breast cancers
Mean follow up was 8.1 years
Dent, R. et al. Clin Cancer Res 2007

41. Characteristics of “Triple Negative” vs. Other Breast Cancers
number (percent)
“Triple Negative”
(N=180)
Significance
p value *
Mean Age at Diagnosis (yrs)
57.7 53
p <
Mean Tumor Size
2.1 cm
3.0 cm
Tumor Size
T1 (≤ 2 cm)
880
(62.7) 65
(36.5)
T2 (>2cm to ≤ 5cm)
461
(32.8) 99
(55.6)
T3 (>5cm) 64
(4.6) 14
(7.9)
Missing 16 2
Lymph Node Status
Positive
510
(45.6) 87
(54.4)
p = 0.02
Negative
609 70
(44.6)
Missing or Not Tested
302 23
Tumor Grade I
237
(19.9) 15
(9.8) II
616
(51.8) 37
(24.2)
III
336
(28.3)
101
(66.0)
* p values were calculated with the use of the chi-square test
Dent, R. et al. Clin Cancer Res 2007

42. Tumor Size by Nodal Status according to “Basal-Like” Group
Non “Basal-like” Group
(N=1421)
“Basal-like” Group
(N=180)
Tumour Size
Lymph Node Positive
Number (percent)
<1.0 cm 38
(19.3) 5
(55.6)
1 - 2 cm
180
(39.3) 25 cm
238
(59.5) 43
(48.9)
>5.1 cm 53
(91.4) 12
(92.3)
p<0.0001
p=0.042
* p values were calculated with the use of the chi-square test
Dent, R. et al. Clin Cancer Res 2007

43. Probability of being recurrence-free
Distant Recurrence
Other (290 of 1421)
“Triple-negative” (61 of 180)
1.0
0.9
0.8
0.7
0.6
Probability of being recurrence-free
0.5
p< (log-Rank test)
0.4
0.3
0.2
0.1
0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Years after diagnosis
Dent, R. et al. Clin Cancer Res 2007;13:

44. Probability of survival
Overall Survival
Other (261 of 1420)
“Triple-negative” (62 of 180)
1.0
0.9
0.8
0.7
0.6
Probability of survival
0.5
p< (log-Rank test)
0.4
0.3
0.2
0.1
0.0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Years after diagnosis
Dent, R. et al. Clin Cancer Res 2007;13:

46. Non-random distribution
X² statistic
42.78 p 
0.0003
Subtype
Smid et al, Cancer Res, in press

47. Patterns of Metastatic Spread
More likely to spread to brain, lung and possibly liver and less likely to spread to bone and soft tissues
Tsuda et al Am J of Surgical Pathology
Rodriguez-Pinilla et al. Clinical Cancer Research 2006
Fulford et al. Breast Cancer Research and Treatment 2007
Hicks et al Am J of Surgical Pathology
More likely to present with visceral metastases versus bone metastases as first site of metastases
70% vs 37%, p < (Dent et al. SABCS 2007)

48. Median Time from Distant Relapse to Death
“Triple Negative” Breast CA
9 months
Other Breast CA
22 months
Dent R, Trudeau M, Pritchard K, Hana W, Narod S. et al. Clinical Cancer Res 2007

49. Treatment

50. Current treatment for triple-negative breast cancers
There is currently no specific systemic regimen recommended for the treatment of triple-negative breast cancers, and little data on which to base treatment selection.

51. Current treatment for triple-negative breast cancers
There are several features inherent to basal-like cancers that have consistently been shown to be associated with clinical and pathological responsiveness to neoadjuvant chemotherapy in general
ER negativity
high expression of Ki-67
In a prospective study to document the chemo sensitivity of breast-cancer subtypes, a complete pathological response rate to preoperative paclitaxel and doxo rubicin chemotherapy was seen in 45% of basal-like cancers, 45% of ERBB2-positive cancers, and only 6% of luminal cancers.

52. Current treatment for triple-negative breast cancers
The genetic instability of basal-like cancers is expected to lead to an increased potential for resistance to treatments.
The use of combination and sequential regimens seems a sensible option for basal-like cancers, although there is no evidence to support this idea.

53. Triple-negative breast cancer: therapeutic options
Because of the absence of specific treatment guidelines for this subgroup, triple-negative breast cancers are managed with standard treatment.
However, such treatment leaves them associated with a high rate of local and systemic relapse.
S Cleator, W Heller, R C Coombes, The Lancet, 2007

54. Triple-negative breast cancer: therapeutic options
S Cleator, W Heller, R C Coombes, The Lancet, 2007

56. Response of Brca1/p53 Mammary Tumors to Doxorubicin or Cisplatin/Carboplatin in vivo

57. Characteristics of retrieved studies - I
Study
Comparison
HER2 status
determined (%)
NSABP B11
Paik S et al, JNCI 1998
PF vs PAF
638/ (94%)
NSABP B15
Paik S et al, JNCI 2000
CMF vs AC
2.034/ (89%)
GUN 3
De Laurentiis M et al, ASCO 2001
CMF vs CMF/EV
123/ (56%)
Belgian
Di Leo A et al, Clin Cancer Res 2002
CMF vs HEC/EC
354/ (46%)
Milan
Moliterni A et al, J Clin Oncol 2003
CMF vs CMF→ A
506/ (92%)
Danish
Knoop AS et al, J Clin Oncol 2005
CMF vs FEC
805/ (82%)
NCIC MA5
Pritchard KI et al, NEJM 2006
CMF vs CEF
628/ (88%)
Total (determined/randomised)
5.088/ (82%)
Gennari et al, JNCI 2008

58. HER2 positive/screened
Characteristics of studies - II
Study
Method
HER2 positive/screened %
NSABP B11
IHC
239/638
37%
NSABP B15
599/2.034
29%
GUN 3
30/123
24%
Belgian
FISH
73/354
21%
Milan
95/506
19%
Danish
IHC/FISH
246/805
33%
NCIC MA5
IHC/FISH/PCR
163/628 (FISH)
26%
Total (positive/screened)
1.445/5.088
28%
Gennari et al, JNCI 2008

59. Adjuvant Anthracyclines and HER2: Disease Free Survival
Study
HER2 status
Anthra better
Non anthra better HR
(95% CI) +
0.60
(0.44 to 0.82)
NSABP B11 -
0.96
(0.75 to 1.23) +
0.84
(0.65 to 1.08)
NSABP B15 -
1.02
(0.86 to 1.20) +
0.65
(0.34 to 1.26)
Belgian -
1.35
(0.93 to 1.97) +
0.83
(0.46 to 1.49)
Milan -
1.22
(0.91 to 1.64) +
0.75
(0.53 to 1.06)
DBCG 89D -
0.79
(0.60 to 1.05) +
0.52
(0.34 to 0.80)
NCIC MA5 -
0.91
(0.71 to 1.17)
Overall
0.90
(0.82 to 0.98) +
0.71
(0.61 to 0.83)
HER2 specific -
1.00
(0.90 to 1.11)
0.00
0.50
1.00
1.50
2.00
Test for interaction: 2 13.7, p< .001
Gennari A. et al. JNCI 2008 59

60. Adjuvant Anthracyclines and HER2 : Overall Survival
Study
HER2 status
Anthra better
Non anthra better HR
(95% CI) +
0.66
(0.42 to 1.01)
NSABP B11 -
0.90
(0.69 to 1.18) +
0.82
(0.63 to 1.06)
NSABP B15 -
1.07
(0.88 to 1.30) +
0.85
(0.27 to 2.69)
GUN -
1.64
(0.85 to 3.15) +
0.61
(0.32 to1.16)
Milan -
1.26
(0.89 to 1.79) +
0.73
(0.50 to 1.05)
DBCG 89D -
0.82
(0.59 to 1.13) +
0.65
(0.42 to 1.01)
NCIC MA5 -
1.06
(0.80 to1.40) +
0.71
(0.32 to 1.55)
GOIRC -
1.25
(0.58 to 2.67)
Overall
0.91
(0.79 to 1.04) +
0.73
(0.62 to 0.85)
HER2 specific -
1.03
(0.92 to 1.16)
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
Test for interaction: 2 12.6, p< .001
Gennari A. et al. JNCI 2008 60

61. Efficacy summary HER2 positive HER2 negative HR 1.00 (0.90-1.11)
Risk of relapse
29%
HR 0.71 ( )
Risk of death
27%
HR 0.73 ( )
Risk of relapse
anthra ≈ non anthra
HR 1.00 ( )
Risk of death
HR 1.03 ( )
p <0.001
p <0.001
Gennari et al, JNCI 2008

62. Highly hormon-sensitive
Moderately hormon-sensitive
HER-2 amplified
Triple negative

64. Pathological Complete Response to Chemotherapy Differs by Subtipes
AC → T
Carey CCR 07
T → FAC
Rouzier CCR 05
Luminal A/B
4/62 (7%)
2/30 (7%)
Normal-like NA
0/10 (0)
HER2+ and ER-
4/11 (36%)
9/20 (45%)
Triple negative
9/34 (27%)
10/22 (45%)

65. The largest date set available (1118 pts) 23% TNBC, pCR 15%
Neoadjuvant Chemotherapy in Triple Negative Patients. MD Anderson Experience
The largest date set available (1118 pts) 23% TNBC, pCR 15%
Regimens
pts
TNBC
non-TNBC
FAC/FEC/AC
308
20% 5%
TFAC/TFEC
588
28%
17%
Taxanes 58
12% 2%
Other
164
14% 7%
Total
1118
22%
11%
p 0.034
Liedtke, M. et al. J Clin Oncol; aheadof print on Febr 4, 2008

71. Neoadjuvant Chemotherapy in TNBC Survival by Pathological Response
Liedtke, M. et al. J Clin Oncol; aheadof print on Febr 4, 2008

72. Metastatic breast cancer
Ixabepilone+Capecitabine a Phase III Trial
Previous Anthra
Taxane Resistant
Ixabepilone +
Capecitabine
N = 375
N = 377
Metastatic breast cancer
N = 752
Vahdat LT et al: ASCO2007

73. Proportion Progression Free
Ixabepilone+Capecitabine a Phase III Trial
1.0
0.0
Proportion Progression Free
Months
0.6
0.8
0.4
0.2
Median
5.8 mo
4.2 mo
95 % Cl
( )
( )
HR: 0.75 ( )
p = 4 8 12 16 20 24 28 32 36
Ixabepilone
+ Capecitabine
Capecitabine
Vahdat LT et al: ASCO2007

74. Ixabepilone+Capecitabine a Phase III Trial
Rugo et al: SABCS 2007

75. Perspectives