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Topoisomerase-II
a Predictive Molecular Marker
in Breast Cancer:
Konecny G, Jakobson K, Pegram M,
Pauletti G, Dandekar U, Morisson J
Ramos L. He-Jing Wang, Seelig S,
and Slamon DJ
University of California, Los Angeles
Vysis Inc., Downers Grove, Illinois
Predictive Markers in Breast Cancer
Role of HER2 as a predictive marker for
response to anthracyclines is a matter
of debate
HER2 and Response to
Anthracyclines
Relative Risk of Failure for DFS
0.5
PAF vs. PF
1.0
HER2 +
HER2 -
HER2 +
AC vs. CMF HER2 -
Paik S et al.: JNCI, 1998 and 2000
1.5
p = 0.001
p = 0.74
p = 0.15
p = 0.84
HER2 and Response to
Anthracyclines
DFS Hazard Ratio
0.5
1.0
1.5
HER2 +
HER2 -
p = 0.29
p = 0.11
HER2 +
CMF vs. FEC HER2 -
p = 0.06
p = 0.61
CMF vs. EC
Di Leo et al.: Ciin Cancer Res, 2002; Pritchard K: ASCO 2002
In Vitro Sensitivity to Doxorubicin
following HER2 Transfection
100%
IC50
relative
to control
MCF-7 MDA-MB-231
BT20
MDA-MB 435
Control cells
HER2 transfected cells
Pegram M et al.: Oncogene 1998
Predictive Markers in Breast Cancer
HER2 could be a surrogate for other
markers predicting response to
anthracyclines
Comparative Genomic Hybridization
Among 38 Breast Cancer Cell lines
Forozan F. et al.: Cancer Res, 2001
Comparative Genomic Hybridization
Among 38 Breast Cancer Cell lines
Forozan F. et al.: Cancer Res, 2001
Chromosome 17q12 - q21
TRAP100
PIP5
HER2 (124kb)
ZNFN1A
GRB7
MLN51
THRA
CDC6
RARA
TOPOII
Human Genome Data Base, PubMed Nucleotides
2MB
Topoisomerase-II
• Amplifications can span large
regions of chromosome 17q
• Topo-II is close to the HER2
amplicon on chromosome 17q
• Topo-II is a key enzyme in DNA
replication and a molecular
target for Anthracyclines
Questions
• Prevalence of Topo-II gene alterations in
HER2-positive samples ?
• Correlation between the gene alteration
and protein expression ?
• Are cells with high Topo-II expression
more sensitive to anthracyclines ?
• Are cells with low Topo-II expression less
sensitive to anthracyclines ?
UCLA Study
• 300 patients with metastatic breast
cancer (MBC)
• Anthracycline based chemotherapy for
treatment of MBC
• 35% HER2 amplification (FISH)
• Clinical response data available for the
Topo-II subgroups
Topo-II Detection - FISH
• 158 kb Topo-II probe, Vysis
• SpectrumOrange labeled Topo-II probe
• SpectrumGreen chromosome 17 centromeric
alpha-satellite probe (chr.17cen)
• Topo-II /chr.17cen ratio to correct for
chromosome 17 polysomy
• Count 100 nuclei and calculate the mean ratio
of Topo-II/chr.17cen
Normal ratio < 2
Topo-II Amplification
DAPI
Cep17
Topo-II
Topo-II Deletion
DAPI
Cep17
Topo-II
Normal Topo-II Cep17 Ratio
DAPI
Cep17
Topo-II
UCLA Study
• 64/100 HER2+ patients have been evaluated
• 64 HER2+
16 (25%) Topo Ampl.
29 (45.3%) Normal
19 (29.7%) Topo Deletion
• No Topo-II alterations among HER2- patients
Topo-II Gene Alterations
in Breast Cancer
• 136 primary breast cancers
• 70/136 HER2 amplification (FISH)
• 70 HER2+
29 (41%) Topo Ampl.
11 (16%) Normal
30 (43%) Topo Deletion
• No alterations in HER2-negative samples
Jarvinen et al.: Genes, Chromosomes and Cancer, 1999
Correlation Between Topo-II Gene
Alterations and Protein Expression
Gene
amplification
+ + + - -
Protein
Gene
amplification
+ + + + + - -
Protein
Jarvinen et al.: Genes, Chromosomes and Cancer, 1999
NIH82 - EGF
NIH82 + EGF
NIH77 - EGF
NIH77 + EGF
EGFR/HER2 Stimulation Upregulates
Topo-II Protein Expression
Harris LN et al.: Clin Cancer Res.: 2001
Influence of Cell Cycle
on Topo-II Protein Expression
Topo-II expression
Topo-II expression
relative to DNA levels
Stacey DW et al.: Mol. Cell. Biol., 2000
In Vitro Experiments
• Increased Topo-II expression is
associated with drug sensitivity
• Reduced levels with drug resistance
Mechanism of Action
• Topo-II is essential for uncoiling of DNA
• Topo-II makes double-stranded breaks in DNA
• DNA molecules can pass through the break
• Topo-II religates the free ends
• Anthracyclines freeze religation
Topo-II and Response to
Anthracyclines in Breast Cancer
Clinical studies:
• Most studies are small and underpowered
• Many investigators use IHC
• Most studies do not investigate the
predictive value of Topo-II independent of
HER2 overexpression
Topo-II Gene Alterations
in Breast Cancer
• 196 patients with MBC
• Anthracycline based chemotherapy for
First-line treatment of MBC
• 32% HER2 amplification (FISH)
• 32% HER2+
34% Topo Ampl.
45% Normal
21% Topo Deletion
Isola JJ et al.: Breast Cancer Res. Treat.: 2000
Response Rates in 61 HER2+ Patients
p=0.0004
79%
80
70
60
50
35%
Normal
40
30
20
10
0
Topo-II
Deletion
17%
Topo-II
Amplified
Topo-II Gene Alterations
in Breast Cancer
• 354 primary breast cancers
• EC vs. HEC vs. CMF
• 21% HER2 amplification (FISH)
• 21% HER2+
38% Topo Ampl.
49% Normal
13% Topo Deletion
Di Leo et al.: Ciin Cancer Res, 2002;
DFS in 61 HER2+ Patients
Di Leo et al.: Ciin Cancer Res, 2002;
Summary
• 30-40% of HER2-positive patients have Topo-II
amplifications and 20-30% Topo-II deletions.
• High Topo-II levels confer anthracycline
sensitivity in vitro.
• Preliminary clinical data support this hypothesis.
• Confirmatory studies are necessary.
• At best, 10% of all breast cancer patients will
potentially benefit from using Topo-II as a
predictive marker.