1. Anthracyclines in Early Breast Cancer: Patient Perspectives and Expert Debate
Dennis J. Slamon, MD, PhD, presents his side of the debate in this program entitled, Anthracyclines in Early Breast Cancer: Patient Perspectives and Expert Debate.
In the premolecular era, breast cancer was considered to be a single disease, and the evidence clearly indicated that anthracycline-based therapy was more effective than nonanthracycline-based therapy.
This program is supported by an educational grant from 1

2. The Use of Nonanthracycline-Based Regimens in Early Breast Cancer
Dennis J. Slamon, MD, PhD
Chief
Division of Hematology/Oncology
David Geffen School of Medicine at UCLA
Los Angeles, California
In the postmolecular era, data from many clinical trials support the opposite conclusion. 2 2

3. Molecular Diversity of Human Breast Cancers
Biologic and Therapeutic Implications
HER2
BRCA1
The molecular diversity of breast cancer needs to guide the thinking regarding treatment approaches. Previously, breast cancer was treated with a “one-size-fits-all” approach, in which all patients received one of only 1 or 2 therapeutic regimens with minor changes introduced to try to adjust and improve outcomes. We have accomplished this in small but incremental numbers.

4. Breast Cancer Subtypes Are Associated With Disease Outcomex
Censored
Luminal A
Luminal B
Basal
ERBB2+
van’t Veer data set
Norway/Stanford data set 1 1 x x
0.8
0.8 x x x
0.6
0.6
Probability
Probability x
0.4
0.4 x x
0.2
0.2
These are data from some of the recent microarray analyses of gene expression profiles that demonstrate that breast cancer is not just 1 disease. All patients included had invasive breast cancer and yet their outcomes are quite different depending on the breast cancer subtype. The dark blue line indicates strongly estrogen receptor/progesterone receptor–positive tumors (ie, luminal A). The light blue line indicates changes in the hormone receptor pathway, including progesterone receptor negativity (luminal B). The red line represents the patients who have neither HER2 nor the steroid hormone receptors, also called the triple negatives. The purple line indicates those patients who have HER2-positive disease (ie, ERBB2 positive).
The graph on the left shows the natural history of human breast cancer, as these patients received no adjuvant chemotherapy. These data are from frozen samples from patients who only received surgery (some also received postoperative radiation therapy). The graph on the right represents patients who received postadjuvant therapy. The curves look remarkably similar, but there are some differences. Importantly, both graphs show that breast cancer is not a single disease.
P < .01
P < .01 24 48 72 96
120
144
168
192 24 48 72 96
Time to Distant Metastasis (Mos)
OS (Mos)
Copyright (2003) National Academy of Sciences, U.S.A.
Sørlie T, et al. PNAS. 2003;100:

5. The Meta-Analysis

6. Meta-Analysis: Relapse-Free Survival50
45.0%
CMF
Anthracycline
41.6% 40
33.9 30
31.0
Recurrence 20 10
10-yr gain: 3.4% (SE: 1.2)
Log rank 2P = .0002
CMF, cyclophosphamide, methotrexate, 5-fluorouracil; (O-E)/V, observed events minus expected events divided by the variance; SE, standard error.
Peto and colleagues conducted a meta-analysis that conclusively argued for the need to use anthracycline-based therapy. The meta-analysis showed that relapse-free survival outcomes were improved by approximately 3.5% to 4.0% with anthracycline-based therapy vs cyclophosphamide, methotrexate, 5-fluorouracil (CMF). 5 10
Yrs
Recurrence Rates (%/Yr) and Log Rank Analyses
Yrs 0-4
Yrs 5-9
Yr 10+
Anthracycline
7.80 (2136/27371)
3.41 (396/11607)
2.71 (48/1770)
CMF
8.38 (1945/23211)
3.68 (325/8822)
2.80 (25/892)
Rate Ratio, From
0.89 SE 0.03
0.89 SE 0.07
1.04 SE 0.26
(O-E)/V
-101.8/853.6
-19.0/160.2
0.7/15.4

7. Meta-Analysis: Overall Survival50 40
35.1% 30.8%
CMF
Anthracycline 30
22.5
Breast Cancer Mortality 20
19.2 10
10-yr gain: 4.3% (SE: 1.1)
Log rank 2P < .0001
CMF, cyclophosphamide, methotrexate, 5-fluorouracil; (O-E)/V, observed events minus expected events divided by the variance; SE, standard error.
Likewise, overall survival (OS) outcomes showed a 4% to 5% improvement with the use of anthracyclines. This is logical when thinking of breast cancer as a single disease, but in the postmolecular era, we know that breast cancer is not just 1 disease. 5 10
Yrs
Death Rates (%/Yr: Total Rate-Rate in Women Without Recurrence) and Log Rank Analyses
Yrs 0-4
Yrs 5-9
Yr 10+
Anthracycline
4.23 ( )
3.33 ( )
2.25 ( )
CMF
4.82 ( )
3.50 ( )
3.46 ( )
Rate Ratio, From
0.83 SE 0.04
0.89 SE 0.07
0.64 SE 0.18
(O-E)/V
-100.2/554.1
-22.6/186.8
-9.0/20.1

8. The HER2 Gene
Encodes a 185kd protein that is a member of the type I receptor tyrosine kinase family, which also contains EGFR, HER3, and HER4
Functions When Altered
Growth and proliferation increased
Differentiation decreased
Cell survival increased
Motility increased
Neoangiogenesis increased
Reduced dependency on estrogen and insensitivity to hormonal blockade
EGFR, epidermal growth factor receptor.
In 1987, research was published that showed that approximately 20% to 25% of human breast cancers have an alteration in the HER2 gene.[1] With approximately 1 million new breast cancer cases each year worldwide, this means that approximately 200,000‑250,000 of these women will have the HER2 alteration. It is an acquired, not inherited, alteration, and its cause is unknown. The functions of this gene explain why it tracks with an aggressive subtype:
It increases proliferation.
It decreases the cell’s ability to differentiate into a mature breast cell.
It increases cell motility.
It increases neoangiogenesis.
It switches the cell from its normal hormone receptor–signaling pathways to other pathways.
Reference
1. Slamon DJ, Clark GM, Wong SG, et al. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science. 1987;235:

9. How Did This All Start? Attempts to explain the differential prognosis of HER2-positive breast cancers
How did this debate start? When we originally published a paper on the HER2 alteration, it was controversial. Many people thought HER2 was associated with outcomes, but many others did not.

10. CALGB 8541 Low c-erbB-2 Expression (< 50%) 100 100 80 80 60 60
Moderate (n = 96) 80
Moderate (n = 96) 60
High (n = 94)
High (n = 94) 60
DFS (%)
OS (%)
Low (n = 93) 40
Low (n = 93) 40 20 20
P = .46
P = .96 10 20 30 40 50 60 70 80 10 20 30 40 50 60 70 80
High c-erbB-2 Expression (≥ 50%)
100
100
High (n = 36)
High (n = 36) 80 80 60
CALGB, Cancer and Leukemia Group B; DFS, disease-free survival; OS, overall survival.
By the early 1990s, HER2-associated poor outcomes were suspected to be due to development of a resistant phenotype. As a result, Muss and colleagues conducted an analysis of the Cancer and Leukemia Group B (CALGB) 8541 study to determine, in the postmolecular era, whether HER2-altered patients have a different response to chemotherapy than HER2-normal (negative) patients. The trial was conducted at a time when anthracyclines were viewed as necessary, but the optimal dose had not yet been determined. The CALGB 8541 study was designed to evaluate 3 doses of anthracyclines—low (30 mg/m^2), moderate (40 mg/m^2), and high (60 mg/m^2). The high dose is currently the standard dose of doxorubicin.
Among the 3 doses there is no significant difference in the outcome curves for disease-free survival (DFS) or OS in HER2-normal patients (ie, those who do not have the HER2 alteration).
Regarding the approximately 20% to 25% of patients with overexpression of HER2, the surprise in the data was not that there was resistance, but that there was unique sensitivity. The higher the dose of anthracycline, the better the response was.
Several valid criticisms of the study were made: it was a retrospective analysis, there were only approximately 350 cases evaluated of the total cohort, it was a subgroup analysis, and all the patients received an anthracycline (ie, there was no nonanthracycline-containing arm).
Moderate (n = 41) 60
Moderate (n = 41)
DFS (%)
OS (%) 40
Low (n = 36) 40
Low (n = 36) 20
P < .001 20
P < .001 10 20 30 40 50 60 70 80 10 20 30 40 50 60 70 80
Mos After Enrollment
Mos After Enrollment
© Massachusetts Medical Society. All rights reserved. Muss HB, et al. N Engl J Med. 1994;330:1260.

11. CALGB (cont’d)
CALGB, Cancer and Leukemia Group B.
Dressler and colleagues addressed the question of whether the differing results were due to analysis techniques. At the time, there was a debate regarding whether immunohistochemistry (IHC) staining, fluorescence in situ hybridization (FISH), or RNA analysis was the most accurate. The investigators doubled the number of blocks in their analysis and included more than 600 blocks from the overall study cohort. All 3 techniques were used to assess the samples.
The curves on the left represent HER2-normal (negative) patients, whereas the curves on the right represent the HER2-positive patients. The data are similar. Whether FISH analysis, RNA analysis, or IHC was used, the data consistently showed that HER2-positive patients have a unique sensitivity to anthracyclines.
Although these results were thought provoking, the primary criticism was, again, that all patients received anthracyclines and there was no nonanthracycline-containing arm.
Reprinted with permission. © 2008 American Society of Clinical Oncology. All rights reserved. Dressler LG, et al. J Clin Oncol ;23:

12. Time to Treatment Failure (Yr) Time to Treatment Failure (Yr)
NSABP-B11
RFS: erbB-2 Negative Tumors
RFS: erbB-2 Positive Tumors
100 PF
98 events
100 PF
89 events
PAF
108 events 80 80
PAF
58 events
RR = .88; P = .37
Patients (%) 60 60
RR = .58; P = .002
Patients (%) 40 40 20 20 2 4 6 8 10 12 2 4 6 8 10 12
Time to Treatment Failure (Yr)
Time to Treatment Failure (Yr)
No. at Risk
PF PAF
88 107
79 92
68 84
55 56
80 83
48 58
37 48
35 46
32 43
18 34
Survival: erbB-2 Negative Tumors
Survival: erbB-2 Positive Tumors
100
100 PF
85 deaths 80 80
PAF
59 deaths
NSABP, National Surgical Adjuvant Breast and Bowel Project; PAF, L-phenylalanine, doxorubicin, 5-fluorouracil; PF, L-phenylalanine, 5-fluorouracil; RFS, relapse-free survival; RR, relative risk.
One of the anthracycline regimens had previously been tested by the National Surgical Adjuvant Breast and Bowel Project (NSABP) group. As a result, investigators decided to reanalyze the data from the NSABP B-11 study, which had compared L‑phenylalanine plus 5-fluorouracil (PF), with or without the new anthracycline, doxorubicin. In this analysis, Paik and colleagues evaluated more than 600 patient samples from the NSABP B-11 trial.
Recall that HER2-normal (negative) disease represents approximately 75% to 80% of human breast cancers. The comparison of PF to PF plus doxorubicin (PAF) showed virtually no differences in outcomes for the approximately 60% to 65% of patients with HER2-normal tumors (left graphs).
By contrast, a significant improvement in favor of the anthracycline-based therapy was seen in HER2-positive patients (right graphs). This was similar to the CALGB 8541 trial, except that the inclusion of a nonanthracycline-containing arm allowed a direct comparison. 60
RR = .66; P = .01 60
Patients (%) PF
100 deaths 40
Patients (%) 40
PAF
107 deaths 20
RR = .90; P = .47 20 2 4 6 8 10 12 2 4 6 8 10 12
Time to Death (Yr)
Time to Death (Yr)
No. at Risk
PF PAF
98 115
86 103
64 75
107 98
75 72
58 56
51 53
41 50
23 38
Reprinted with permission of Oxford University Press. Paik S, et al. J Natl Cancer Inst. 1998;90:

13. NSABP-B11 (cont’d) DFS: 1-3 Positive Nodes erbB-2 Negative Tumors
erbB-2 Positive Tumors
100 PF
50 events
100 PF
32 events 80
PAF
68 events 80
PAF
26 events 60 60
Patients (%)
Patients (%) 40 40 20 20 2 4 6 8 10 12 2 4 6 8 10 12
Time to 1st Event (Yr)
Time to 1st Event (Yr)
No. at Risk
PF PAF
85 106
66 89
58 77
56 69
50 63
41 38
50 46
36 35
25 26
20 21
20 21
18 21
9 17
DFS: 4+ Positive Nodes
erbB-2 Negative Tumors
erbB-2 Positive Tumors
100 PF
63 events
100 PF
65 events
DFS, disease-free survival; NSABP, National Surgical Adjuvant Breast and Bowel Project; PAF, L-phenylalanine/doxorubicin/5-fluorouracil; PF, L-phenylalanine/5-fluorouracil.
At the time of the NSABP B-11 study, anthracycline-based therapies were considered necessary treatment for a patient with multiple nodes involved. Studies of anthracyclines were done in node-positive women, who at the time were considered the only patient group at high risk of recurrence. A node-negative high-risk subgroup was not identified until we started to follow patients longer and knew what the molecular markers were.
Paik and colleagues addressed the question of node positivity and found that HER2-normal (negative) patients (left graphs) experienced no difference in outcomes with PAF vs PF regardless of the number of positive nodes. However, in HER2-positive patients (right graphs), once again, a difference was seen in favor of the anthracycline in both patients with 1-3 positive nodes and those with more than 4 positive nodes. HER2-positive disease is associated with more nodal positivity. 80
PAF
67 events 80
PAF
42 events 60 60
Patients (%)
Patients (%) 40 40 20 20 2 4 6 8 10 12 2 4 6 8 10 12
Time to 1st Event (Yr)
Time to 1st Event (Yr)
No. at Risk
PF PAF
83 86
49 60
34 40
30 30
23 23
18 21
14 18
78 65
44 48
23 32
17 27
15 25
14 22
9 17
Reprinted with permission of Oxford University Press. Paik S, et al. J Natl Cancer Inst. 1998;90:

14. NSABP-B11 (cont’d) Outcome erbB-2 Status RR: PAF vs PF 0.05 1.00 1.50
P Value PAF vs PF
P Value Interaction
DFS
Negative
Positive
.74
.001
.02 OS
.47
.01
.15
RFS
.37
.002
.06
DDFS
.84
.003 +
DDFS, distant disease-free survival; DFS, disease-free survival; NSABP, National Surgical Adjuvant Breast and Bowel Project; PAF, L‑phenylalanine/doxorubicin/5-fluorouracil; PF, L‑phenylalanine/5-fluorouracil; RFS, relapse-free survival; RR, relative risk.
Taken together, the results in every category—DFS, OS, relapse-free survival, and distant DFS—showed that only the HER2-positive patients benefited from the addition of the anthracycline. Therefore, doxorubicin did not improve outcomes when added to PF, a regimen that is considered old, in the HER2-normal (negative) population. A primary criticism of this study was that it used an old regimen that is no longer in use.
Reprinted with permission of Oxford University Press. Paik S, et al. J Natl Cancer Inst. 1998;90:

15. NSABP-B15 DFS, HER2 Negative DFS, HER2 Positive
100
100 80 80 60 60
% Disease Free
% Disease Free 40 40
AC N = 487, 281 events, RR = 1.02, P =. 84
AC N = 202, 128 events, RR = 0.84, P = .15 20 20
CMF N = 464, 267 events
CMF N = 202, 134 events 2 4 6 8 10 2 4 6 8 10
Time to 1st Event (Yrs)
Time to 1st Event (Yrs)
No. at Risk
AC CMF
No. at Risk
AC CMF
110 95
94 81
86 76
71 64
Survival, HER2 Negative
Survival, HER2 Positive
100
100 80 80
AC, doxorubicin/cyclophosphamide; CMF, cyclophosphamide/methotrexate/5-fluorouracil; DFS, disease-free survival; NSABP, National Surgical Adjuvant Breast and Bowel Project; RR, relative risk.
Paik and colleagues reanalyzed the NSABP B-15 trial data, which compared doxorubicin/cyclophosphamide (AC) vs CMF vs AC followed by CMF. Data from NSABP B-15 showed that in the HER2-normal (negative) population (left graphs) CMF produced a nonsignificant improvement in outcomes vs the anthracycline-containing AC regimen. By contrast, in the HER2-positive population (right graphs), the curve reverses to show that the anthracycline-based therapy was more effective than CMF. This study provided further confirmation that the benefit of anthracyclines is restricted to the HER2-positive population. 60 60
% Alive
% Alive 40 40
AC N = 487, 210 events, RR = 1.07, P = .51
AC N = 202, 105 events, RR = 0.82, P = .14 20 20
CMF N = 464, 196 events
CMF N = 202, 118 events 2 4 6 8 10 2 4 6 8 10
Time to Death (Yrs)
Time to Death (Yrs)
No. at Risk
AC CMF
No. at Risk
AC CMF
100 97
87 79
Reprinted with permission of Oxford University Press. Paik S, et al. J Natl Cancer Inst. 2000;92:

16. NSABP-B15 (cont’d) DFS, HER2 Negative DFS, HER2 Positive 100 100 80 8060 60
% Disease Free
% Disease Free 40 40
AC N = 487, 281 events
AC N = 202, 128 events 20
AC → CMF N = 484, 279 events 20
AC → CMF N = 195, 117 events
CMF N = 464, 267 events
CMF N = 202, 134 events
AC, doxorubicin/cyclophosphamide; CMF, cyclophosphamide/methotrexate/5-fluorouracil; DFS, disease-free survival; NSABP, National Surgical Adjuvant Breast and Bowel Project.
The investigators also evaluated the third arm (AC followed by CMF), which represented patients who received any anthracycline during adjuvant therapy. Results confirmed that patients without the HER2 alteration received no benefit from adding an anthracycline to CMF. 2 4 6 8 10 2 4 6 8 10
Time to 1st Event (Yrs)
Time to 1st Event (Yrs) AC
AC → CMF CMF
No. at Risk AC
AC → CMF CMF
No. at Risk
464
356
288
249
222
176
202
151
148
129 95
94 89 81
86 80 76
71 71 64
Reprinted with permission of Oxford University Press. Paik S, et al. J Natl Cancer Inst. 2000;92:

17. MA.5 Trial HER2 Negative HER2 Positive
100
100 80
CEF 80
CEF 60 60
RFS (%)
CMF
RFS (%) 40 40 20 20
CMF
P = .49
P = .003 5 10 5 10
Yrs
Yrs
No. at Risk CEF CMF
No. at Risk CEF CMF
59 60
75 88 42 35
19 12
100
100
CEF 80 80
CMF
CEF 60 60
OS (%)
CEF, cyclophosphamide/epirubicin/5-fluorouracil; CMF, cyclophosphamide/methotrexate/5-fluororuacil; OS, overall survival; RFS, relapse-free survival.
The MA.5 trial was designed to evaluate the benefit of epirubicin in breast cancer by comparing CMF with cyclophosphamide/epirubicin/5-fluorouracil (CEF). The “industrial dose” of epirubicin used in this study was 120 mg/m^2, which is the highest appropriate dose.
Pritchard and colleagues showed that in the HER2-normal population CEF did not provide benefit over CMF. However, in the HER2-positive population, there was a large benefit in favor of anthracycline-based therapy.
OS (%) 40 40
CMF 20 20
P = .68
P = .06 5 10 5 10
No. at Risk
CEF CMF
Yrs
No. at Risk
CEF CMF
Yrs
237
228
71 78 75 88 49 47
20 18
© 2006 Massachusetts Medical Society. All rights reserved. Pritchard KI, et al. N Engl J Med. 2006;354:

18. Meta-Analysis: DFS Study HR 95% CI
Anthracycline Better
Nonanthracycline Better
0.90
NSABP-B11
NSABP-B15
Brussels
Milan
DBCCG-89-D
NCIC MA-5
CI, confidence interval; DBCCG, Danish Breast Cancer Cooperative Group; DFS, disease-free survival; HR, hazard ratio; NCIC, National Cancer Institute of Canada; NSABP, National Surgical Adjuvant Breast and Bowel Project.
Gennari and colleagues published a meta-analysis of the effect of HER2 on outcomes with anthracycline vs nonanthracycline-containing therapies. Red represents HER2-positive patients, and blue represents HER2-normal patients. Each study in this meta-analysis showed that HER2-positive patients do better than HER2-negative patients on anthracycline-based therapy. This meta-analysis shows a dramatic effect in HER2-positive patients and virtually no effect in the HER2-normal women. The Chi-squared test and the test for interaction were highly significant, indicating that this is not likely to be a specious observation.
Interestingly, if both populations are combined, there is still approximately a 4% to 5% improvement in benefit in favor of the anthracycline-containing regimens. Approximately 5000 patients were analyzed in this study.
Total
P = .01
P < .0001
Overall
P = 1.0
Heterogeneity c25 = 5.3; P = .38 Heterogeneity c25 = 7.6; P = .18
0.4
0.6
0.9
1.0
2.0
5.0
Test for interaction chi2 = 13.7; P < .001
HER2 positive HER2 negative
Reprinted by permission of Oxford University Press. Gennari A, et al. J Natl Cancer Inst. 2008;100:14-20.

19. Meta-Analysis: OS Study HR 95% CI
Anthracycline Better
Nonanthracycline Better
0.91
NSABP-B11
NSABP-B15
GUN3
Milan
DBCCG-89-D
NCIC MA-5
CI, confidence interval; DBCCG, Danish Breast Cancer Cooperative Group; HR, hazard ratio; NCIC, National Cancer Institute of Canada; NSABP, National Surgical Adjuvant Breast and Bowel Project; OS, overall survival.
The OS data also show that, even when the HER2-positive and HER2-negative populations are combined (ie, when breast cancer is regarded as 1 disease), there is an improvement with the use of anthracyclines. This slide shows the difference between HER2-normal (negative) and HER2-positive patients. The survival benefit accrues to the HER2-positive population.
Total
P = .056
P < .0001
Overall
P = .86
Heterogeneity c25 = 5.2; P = .39 Heterogeneity c25 = 5.5; P = .36
0.4
0.6
0.9 1 2 5
Test for interaction chi2 = 12.0; P < .001
HER2 positive HER2 negative
Reprinted by permission of Oxford University Press. Gennari A, et al. J Natl Cancer Inst. 2008;100:14-20.

20. Is this due to a unique and/or inherent sensitivity to anthracycline caused by HER2 overexpression?
The question then becomes is the benefit from anthracyclines really due to HER2 overexpression?

21. Human Breast Cancer Cells
Transfect
MCF-7
MCF-7*
HER2/neu
Single copy Low expressor
Multiple copy High expressor
Additional Human Breast Cancer Cells
MDA-435
MDA-231
BT-20
MDA-435
MDA-231
BT-20
In the process of attempting to prove that HER2 causes a more aggressive disease, we had previously engineered human breast cancer cells to overexpress HER2. When the question arose of whether HER2 was truly the root of the increased benefit from anthracyclines, we retrieved our frozen cells. These engineered cells represented a perfect tool to answer this question, as they were genetically identical to each another (ie, from the same patient) except that one line overexpressed HER2 and one did not.
Transfect
HER2/neu
Single copy Low expressor
Multiple copy High expressor
*Consistent results in 9 additional breast cancer cell lines.

22. Sensitivity of Human Breast Cells to Chemo Agents in Vitro
IC50 for Doxorubicin, µM
MCF7/NEO
0.39 ± 0.03
MCF7/HER2
0.34 ± 0.07
MDA-MB-435/NEO
0.60 ± 0.09
MDA-MB-435/HER2
0.60 ± 0.07
MDA-MB-231/NEO
0.30 ± 0.03
MDA-MB-231/HER2
0.20 ± 0.05
BT-20/NEO
0.17 ± 0.03
BT-20/HER-2
0.15 ± 0.02
[Peak plasma] achievable in humans
5.6
IC50, 50% inhibitory concentration; NEO, HER2 normal.
Our group looked at the sensitivity of these cells to doxorubicin by determining the IC50, which is the dose of doxorubicin that will kill 50% of the cells. Either with or without HER2, the IC50 values were essentially identical, which was puzzling. Our basic science laboratory experiment showed that HER2 overexpression did not convey increased sensitivity, yet the clinical data clearly show that anthracycline sensitivity is associated with HER2 overexpression.
It was determined that the HER2 alteration was due to an amplification event. In other words, the difference was not due to a single gene; instead, a whole region of the chromosome that contains HER2 is responsible for the alteration.

23. The Topo IIα Gene Functions Resolves topological problems in DNA
Is critical in RNA transcription from DNA
Makes transient protein-bridged DNA breaks on 1 or both DNA strands during replication
Plays critical roles in segregation, condensation, and superhelicity
Topo IIα, topoisomerase II alpha.
We studied the genes on this amplicon, which included a gene encoding topoisomerase II alpha (Topo IIα), a downstream close neighbor to HER2. Topo IIα is involved in DNA replication, RNA transcription, and other critical cell functions.

24. The Topo IIα Protein Is a Major Target of the Anthracyclines
Topo IIα, topoisomerase II alpha.
The Topo IIα protein is one of the major targets of anthracycline-based therapy.

25. Mapping the HER2 Amplicon
HER2 Core Region
TOPO II Region
Example 1
Example 2
Example 3
Example 4
Example 5
Example 6
Example 7
Topo IIα, topoisomerase II alpha.
Poletti and colleagues evaluated 7 breast cancer cases that were HER2 amplified. The red bars represent the HER2 amplicon, which varies in size. Silver on either side indicates that the chromosome is completely normal. Yellow on either side means that the chromosomal area is deleted. These differences are not unique to HER2; all amplification events do this and vary in size.
The bottom part of the slide shows all the genes in this region. The smallest HER2 amplicon contains only 5 or 6 genes (including HER2). HER2 drives the amplicon, but in some cases, the amplicon is large enough that it picks up another gene, Topo IIα, the target of anthracycline-based therapy.
TOP2A
HER2
Normal
Amplified
Deletion

26. DFS by Treatment 100 Overexpression of Topo protein P = .01 80
Probability
(%)
CEF 60 40
CMF 5 10
100
Normal expression of Topo protein
P = .6
Probability
(%)
CEF, cyclophosphamide/epirubicin/fluorouracil; CMF, cyclophosphamide/methotrexate/5-fluorouracil; DFS, disease-free survival.
Pritchard and colleagues conducted the MA.5 study, which looked at Topo IIα expression instead of HER2. The investigators found no difference between CEF and CMF in those cases that had normal expression of Topo IIα. However, in those women who had high levels of Topo IIα that resulted from amplification events, there was a dramatic difference in favor of the anthracycline for relapse-free survival and OS.
The results indicated that among the patients with tumors expressing normal levels of Topo IIα, there was no difference in DFS between the patients who received CEF and those who received classic CMF. However, among the patients with tumors that overexpressed Topo IIα, there was a statistically significant improvement in DFS in favor of the anthracycline-based therapy (P = .01). 80
CEF 60
CMF 40 5 10
Yrs
Levine MN, et al. J Clin Oncol. 2005;23:

27. OS by Treatment 100 Overexpression of Topo protein P = .02 80
Probability
(%)
CEF 60
CMF 40 5 10
100
Normal expression of Topo protein
P = .8 80
Probability
(%)
CEF, cyclophosphamide/epirubicin/fluorouracil; CMF, cyclophosphamide/methotrexate/5-fluorouracil; OS, overall survival.
A similar trend was demonstrated for OS.
CEF
CMF 60 40 5 10
Time (Yrs)
Levine MN, et al. J Clin Oncol. 2005;23:

28. These preclinical (in vitro and in vivo) as well as clinical data indicate that the increased anthracycline sensitivity is NOT due to HER2 overexpression
Instead, the current data indicate that it is the Topo IIα gene amplification and not HER2 that is responsible for improved anthracycline sensitivity
To summarize, the preclinical in vitro and in vivo data, as well as most of the recent clinical data, indicate that it is not HER2 overexpression that is driving the sensitivity to anthracyclines, but instead it is Topo IIα amplification.

29. Update of the DBCG Trial 89D: CMF vs CEF
CEF, cyclophosphamide/epirubicin/fluorouracil; CMF, cyclophosphamide/methotrexate/5-fluorouracil; DBCG, Danish Breast Cancer Cooperative Group.
Neilsen and colleagues from the Danish Breast Cancer Cooperative Group (DBGG) also conducted a study whose results supported those of the previously discussed studies: Patients with Topo IIα amplification (ie, HER2 positive) had a dramatic difference in response to anthracycline-containing therapy.
Reprinted by permission of Taylor & Francis, Ltd. Neilsen K, et al. Acta Oncol. 2008;47:

30. 6 x Docetaxel and Carboplatin
BCIRG 006
4 x AC 60/600 mg/m2
4 x Docetaxel
100 mg/m2
AC → T
HER2+
(central FISH)
N+ or
high-risk N-
(N = 3222)
4 x AC 60/600 mg/m2
4 x Docetaxel
100 mg/m2
AC → TH
1 yr of Trastuzumab
AC → T, doxorubicin/cyclophosphamide followed by docetaxel; AC → TH, doxorubicin/cyclophosphamide followed by docetaxel and trastuzumab; BCIRG, Breast Cancer International Research Group; FISH, fluorescent in situ hybridization; HER2+, HER2 positive; N+, node positive; N-, node negative; TCH, docetaxel/carboplatin/trastuzumab.
The Breast Cancer International Research Group (BCIRG) 006 trial evaluated the use of 3 regimens in more than 3000 HER2-positive patients: AC followed by docetaxel (AC → T) vs AC followed by docetaxel and trastuzumab (AC → TH) vs docetaxel/carboplatin/trastuzumab (TCH).
For more information, go online to:
6 x Docetaxel and Carboplatin
75 mg/m AUC 6
TCH
Stratified by nodes and hormonal receptor status
1 yr of Trastuzumab
Slamon D, et al. SABCS Abstract 52.

31. HER2 and Topo IIα in BCIRG 006
2990 of 3222 patients analyzed
17 q 12
17 q 21.1
17 q 21.2
HER2
Core Region
Topo IIα Region
N = 2990
Topo IIα non- coamplified
1788 pts (60%)
145 pts (5%)
BCIRG, Breast Cancer International Research Group; Topo IIα, topoisomerase II alpha.
Results showed that in approximately 35% of the HER2-positive breast cancers the amplicon is large enough to pick up the Topo IIα gene. In 65% of cases, HER2 is amplified but Topo IIα is not. The question is whether this makes a difference in outcome.
For more information, go online to:
Coamplified
1057 pts (35%)
Most recent analysis
Normal
Amplified
Deletion
Permission granted by author to print. Slamon D, et al. SABCS Abstract 52.

32. DFS: BCIRG 006 2nd Interim Analysis
1.0
93%
0.9
87%
92%
83%
87%
86%
0.8
82%
81%
Disease Free (%)
77%
0.7
AC → T, doxorubicin/cyclophosphamide followed by docetaxel; AC → TH, doxorubicin/cyclophosphamide followed by docetaxel and trastuzumab; BCIRG, Breast Cancer International Research Group; DFS, disease-free survival; TCH, docetaxel/carboplatin/trastuzumab.
This slide shows the DFS results of BCIRG 006, which included more than 1000 HER2-positive patients in each arm. Overall, both experimental arms did much better than the control arm, with the AC → TH arm doing slightly better than the TCH arm.
For more information, go online to:
Patients
Events
0.6
1073
192
AC → T
1074
128
AC → TH
HR (AC → TH vs AC → T): 0.61 ( ; P < .0001)
1075
142
TCH
HR (TCH vs AC → T): 0.67 ( ; P = .0003)
0.5 1 2 3 4 5
Yr From Randomization
Permission granted by author to print. Slamon D, et al. SABCS Abstract 52.

33. DFS Non-coamplified Topo IIα by Arm: BCIRG 006 2nd Interim Analysis
1.0
91%
0.9
90%
85%
83%
0.8
84%
83%
81%
Disease Free (%)
78%
0.7
71%
AC → T, doxorubicin/cyclophosphamide followed by docetaxel; AC → TH, doxorubicin/cyclophosphamide followed by docetaxel and trastuzumab; BCIRG, Breast Cancer International Research Group; DFS, disease-free survival; TCH, docetaxel/carboplatin/trastuzumab; Topo IIα, topoisomerase II alpha.
After looking only at HER2-positive patients whose amplicon was not large enough to pick up the Topo IIα gene, the differences in DFS rates were shown to be even greater.
For more information, go online to:
Patients
Events
0.6
643
146
AC → T
642 87
AC → TH P < .001
618 92
TCH P < .001
0.5 1 2 3 4 5
Yr From Randomization
Permission granted by author to print. Slamon D, et al. SABCS Abstract 52.

34. DFS Coamplified Topo IIα by Arm: BCIRG 006 2nd Interim Analysis
1.0
95%
89%
0.9
94%
92%
87%
83%
0.8
87%
83%
Disease Free (%)
85%
0.7
AC → T, doxorubicin/cyclophosphamide followed by docetaxel; AC → TH, doxorubicin/cyclophosphamide followed by docetaxel and trastuzumab; BCIRG, Breast Cancer International Research Group; DFS, disease-free survival; TCH, docetaxel/carboplatin/trastuzumab; Topo IIα, topoisomerase II alpha.
Considering only the cases with coamplification of HER2 and Topo IIα, patients who received only the anthracycline did approximately as well as those who received trastuzumab.
For more information, go online to:
Patients
Events
0.6
328 42
AC → T
357 35
AC → TH P < .336
359 42
TCH P < .648
0.5 1 2 3 4 5
Yr From Randomization
Permission granted by author to print. Slamon D, et al. SABCS Abstract 52.

35. Patients From the Registrational Trial for Trastuzumab in HER2+ First-Line Metastatic Disease
The registration trial for trastuzumab in patients with HER2-positive, first-line metastatic disease confirmed the benefit of trastuzumab in this population.

36. 0648 Registrational Trial: Trastuzumab in First-Line HER2+ MBC
Analysis of Survival Outcomes as Related to Topo IIa
Treatment Arm
Topo IIa Status
Survival, Mos
P Value
AC alone (n = 77) (med survival)
Topo normal (n = 50) Topo co-amplified (n = 27)
18.2
38.5
.004
AC + Tras (n = 85)
(med survival)
Topo normal (n = 54)
Topo co-amplified (n = 31)
29.3
29.4
.66
AC, doxorubicin/cyclophosphamide; Topo IIα, topoisomerase II alpha; Tras, trastuzumab.
In the registration trial that resulted in the approval of trastuzumab in metastatic breast cancer, patients were randomized to AC vs AC plus trastuzumab, or for the women who had already received an anthracycline in the adjuvant setting, paclitaxel with or without trastuzumab. Results showed a substantial survival advantage in the patients who received AC alone if they were Topo IIα coamplified (shown in green): the median survival time nearly doubled, from 18 months to 38 months. The addition of trastuzumab improved survival in both groups, even among patients without Topo IIα amplification.

37. 0648 Registrational Trial: Trastuzumab in First-Line HER MBC (cont’d)
Analysis of OS Outcomes as Related to Topo IIa
Treatment Arm
Topo IIa Status
Survival, Mos
P Value
Paclitaxel alone (n = 59) (median OS)
Topo normal (n = 43) Topo co-amplified (n = 16)
20.6
18.4
.96
AC + Tras (n = 85)
(med survival)
Topo normal (n = 33)
Topo co-amplified (n = 25)
35.7
30.7
.22
AC, doxorubicin/cyclophosphamide; Topo IIα, topoisomerase II alpha; Tras, trastuzumab.
Importantly, virtually no difference was seen whether Topo IIα was amplified or not in patients who received paclitaxel alone. This is an anthracycline-specific phenomenon; without the anthracycline, there is no benefit to other chemotherapy.

38. BCIRG 006: Cardiac Deaths and CHF as per Independent Review Panel
First Analysis/Second Analysis
AC → T
(n = 1050)
AC → TH
(n = 1068)
TCH
(n = 1056)
Cardiac-related death
0/0
Grade 3/4 cardiac left ventricular function (CHF)
3/4
17/20
4/4
P = .0015
AC → T, doxorubicin/cyclophosphamide followed by docetaxel; AC → TH, doxorubicin/cyclophosphamide followed by docetaxel and trastuzumab; BCIRG, Breast Cancer International Research Group; TCH, docetaxel/carboplatin/trastuzumab.
The incidence of congestive heart failure increases approximately 4- to 5‑fold with the use of trastuzumab, and this was seen in all the trastuzumab trials.
For more information, go online to:
Permission granted by author to print. Slamon D, et al. SABCS Abstract 52.

39. BCIRG 006: Patients With > 10% Relative LVEF Decline
First Analysis/ Second Analysis
AC → T
(n = 1012/1014)
AC → TH
(n = 1040/1042)
TCH
(n = 1029/1030)
Patients, n
91/102
180/189
82/89
Patients, %
9/10
17.3/18.0
8.0/8.6
P = .002
P < .0001
P < .0001
P < .0001
P = .5
P = .5
AC → T, doxorubicin/cyclophosphamide followed by docetaxel; AC → TH, doxorubicin/cyclophosphamide followed by docetaxel and trastuzumab; BCIRG, Breast Cancer International Research Group; LVEF, left ventricular ejection fraction; TCH, docetaxel/carboplatin/trastuzumab.
In the BCIRG 006 study, up to 18% of patients experienced subclinical loss of cardiac function, resulting in a left ventricular dysfunction rate of greater than 10%.
For more information, go online to:
Permission granted by author to print. Slamon D, et al. SABCS Abstract 52.

40. BCIRG 006: Mean LVEF—All Obs: Second Interim Analysis66
AC → T (n = 1014)
AC → TH (n = 1042) 65
TCH (n = 1030) 64 63
LVEF Points (%) 62 61
AC → T, doxorubicin/cyclophosphamide followed by docetaxel; AC → TH, doxorubicin/cyclophosphamide followed by docetaxel/trastuzumab; BCIRG, Breast Cancer International Research Group; LVEF, left ventricular ejection fraction; TCH, docetaxel/carboplatin/trastuzumab.
Importantly, the trastuzumab-associated cardiac dysfunction was not transient. The data from the BCIRG 006 trial show that the drop in left ventricular function in the AC → TH arm lasts at least 2.5 years, whereas in the nonanthracycline arm there is reasonable recovery of cardiac function.
For more information, go online to: 60 59 58
100
200
300
400
500
600
700
800
900
1000
Time Since Randomization (Days)
Permission granted by author to print. Slamon D, et al. SABCS Abstract 52.

41. Therapeutic Index: Most Recent BCIRG 006 Data
AC → TH
TCH
Breast cancer recurrence 93 98
Breast cancer deaths 44 47
Grade 3/4 CHF 20 4
Acute leukemia 4*
Total
161
149
*In both anthracycline-based arms.
AC → TH, doxorubicin/cyclophosphamide followed by docetaxel and trastuzumab; BCIRG, Breast Cancer International Research Group; CHF, cardiac heart failure; TCH, docetaxel/carboplatin/trastuzumab.
The therapeutic index from BCIRG 006 shows that the number of events—including breast cancer recurrence, breast cancer death, congestive heart failure, and acute leukemia—favors the nonanthracycline-containing regimen.
For more information, go online to:

42. Onset of Heart Failure: 66-70 Years of Age
1.0
0.8
0.6
Proportion Free of CHF
0.4
Adjuvant anthracycline
Nonanthracycline
CHF, cardiac heart failure.
Giordano and colleagues from M. D. Anderson Cancer Center in Houston, Texas, evaluated data from the Surveillance, Epidemiology and End Results (SEER) database, representing approximately 42,000 US women with breast cancer who were treated with anthracycline vs nonanthracycline regimens. The yellow line represents the incidence of congestive heart failure over time in women who received no chemotherapy. This is the natural decline in cardiac function in women with breast cancer. Women who received nonanthracycline chemotherapy are represented by the red line. The blue line represents women who received anthracycline-based chemotherapy. Long-term follow-up results show that their incidence of congestive heart failure has increased by as much as 30%.
0.2
No adjuvant chemotherapy
0.0 24 48 72 96
120
144
Mos
Permission granted by author to print. Giordano S, et al. ASCO Abstract 521.

43. Implications for HER2-Negative and HER2-Positive Breast Cancers
The superior efficacy benefits for anthracyclines (when present) derives from their effects on Topo IIa amplification and/or overexpression
Given that, to date, Topo IIa amplification occurs ONLY in 35% of the 25% of breast cancer patients with HER2 amplification, ie, a subset of a subclass (tested in > 5500 patients for which Topo IIa was determined) . . .
What data support their preferential use in the HER2-normal breast cancer population which is ~ 75% to 80% of all breast cancers
Moreover, for HER2-positive breast cancers, we now have trastuzumab and lapatinib, which appears to replace the gained efficacy of anthracyclines in the one third of patients (which is ~ 8% of all breast cancers) with coamplification of HER2 and Topo IIa without risking their known and well-established toxicities
Is there an alternative?
Topo IIα, topoisomerase II alpha.
The implications for patients with either HER2-negative or HER2-positive disease are that the superior benefit of anthracycline, when present, derives from the alteration of the Topo IIα gene. Therefore, Topo IIα amplification appears to occur in only HER2-positive patients and in only one third of those cases, which accounts for approximately 8% of human breast cancers. For patients receiving targeted therapy, such as trastuzumab and probably lapatinib, the data shows that it more than makes up for the anthracycline benefit.

44. DFS by Treatment 1.00 0.95 0.90 TC 0.85 81% Proportion DFS 0.80 AC
0.75
75%
0.70
P = .033
HR = .74
AC, doxorubicin/cyclophosphamide; DFS, disease-free survival; HR, hazard ratio; TC, docetaxel/cyclophosphamide.
The US Oncology Group has demonstrated that TC is superior to AC in terms of DFS and OS. Although this was a small study of only 1000 patients, the differences are highly statistically significant in all subgroups, including node-positive vs node-negative disease.
0.65
0.60 12 24 36 48 60 72 84 96
Mos
Reprinted with permission. © 2008 American Society of Clinical Oncology. All rights reserved. Jones SE, et al. J Clin Oncol. 2006;24:

45. OS by Treatment 1.00 0.95 TC 0.90 87% 0.85 AC Proportion Surviving
0.80
82%
0.75
0.70
P = .032
HR = .69
AC, doxorubicin/cyclophosphamide; HR, hazard ratio; OS, overall survival; TC, docetaxel/cyclophosphamide.
This slide shows the OS curves and demonstrates that the presence of multiple involved nodes does not necessarily require treatment with an anthracycline.
0.65
0.60 12 24 36 48 60 72 84 96
Months
Reprinted with permission. © 2008 American Society of Clinical Oncology. All rights reserved. Jones SE, et al. J Clin Oncol. 2006;24:

46. TC x 4 vs AC x 4 Subgroup HR (95% CI)
All patients (TC = 506, AC = 510)
Age < 50 yrs (TC = 210, AC = 214)
Age ≥ 50 yrs (TC = 296, AC = 296)
ER-/PR- (TC = 137, AC = 157)
ER+ or PR+ (TC = 369, AC = 383)
Node neg (TC = 239, AC = 248)
Node pos (TC = 267, AC = 262)
0.67 ( )
0.64 ( )
0.73 ( )
0.64 ( )
0.71 ( )
AC, doxorubicin/cyclophosphamide; CI, confidence interval; ER, estrogen receptor; HR, hazard ratio; PR, progesterone receptor; TC, docetaxel/cyclophosphamide.
This slide shows that treatment with an anthracycline is not required for better outcomes, regardless of age, hormone receptor status, or nodal status.
0.73 ( )
0.67 ( )
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
HR Log Scale
Reprinted with permission. © 2008 American Society of Clinical Oncology. All rights reserved. Jones SE, et al. J Clin Oncol. 2006;24:

47. Do We Need Another Study?
The preceding clinical data are derived from a total of more than 10,150 patients for which HER2 status is available
All enrolled in multiple randomized anthracycline vs nonanthracycline studies
Spanning a period of time of more than 3.5 decades
Conducted by a total of 8 separate US, Canadian, and European cooperative groups
ALL SHOWING THE EXACT SAME THING!
Do we need another study? The preceding clinical data are derived from more than 10,150 women for whom HER2 status is available. All were enrolled on multiple randomized anthracycline vs nonanthracycline trials over 3 decades. The analyses are retrospective, but the HER2 gene was not identified when many of these trials were launched. Importantly, these studies were conducted by 8 US cooperative groups, and all showed exactly the same result regarding anthracyclines in the adjuvant treatment of human breast cancer.

48. Critical Question Considering
BCIRG 006 with > 3200 patients ( > 1000 patients/arm) shows that the difference in DFS efficacy events and breast cancer deaths in favor of AC→TH in HER2-positive breast cancer is statistically insignificant and is exceeded by the number of critical adverse events including grade 3/4 CHF, AC-related leukemias as well as a significant AND sustained loss of LVEF for 18% (189 patients) both of which are highly statistically significant and . . .
The recently published data from US Oncology1 showing a highly statistically significant DFS and OS superiority (HR = 0.67) of TC over AC in terms of breast cancer efficacy
What is the role of anthracyclines in the adjuvant treatment of breast cancer?
AC, doxorubicin/cyclophosphamide; BCIRG, Breast Cancer International Research Group; CHF, cardiac heart failure; DFS, disease-free survival; LVEF, left ventricular ejection fraction; OS, overall survival; TC, docetaxel/cyclophosphamide.
This does not imply that anthracyclines are not effective. It only implies that they provide no incremental benefit for any women except those who have the alteration of the Topo IIα gene, which represents approximately 8% of the human breast cancer population. Approximately 92% of women with breast cancer receive a drug that gives them no incremental benefit and confers significant toxicity.
1. Jones SE, et al. J Clin Oncol. 2006;24:

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