Topoisomerase-II a Predictive Molecular Marker
in Breast Cancer:
Konecny G, Jakobson K, Pegram M, Pauletti G, Dandekar U, Morisson JRamos L. He-Jing Wang, Seelig S,
and Slamon DJ
University of California, Los AngelesVysis Inc., Downers Grove, Illinois
Role of HER2 as a predictive marker for response to anthracyclines is a matter of
debate
Predictive Markers in Breast Cancer
PAF vs. PF
AC vs. CMF
HER2 +HER2 -
HER2 +HER2 -
0.5 1.0 1.5
p = 0.001p = 0.74
HER2 and Response to Anthracyclines
p = 0.15p = 0.84
Relative Risk of Failure for DFS
Paik S et al.: JNCI, 1998 and 2000
CMF vs. EC
CMF vs. FEC
HER2 +HER2 -
HER2 +HER2 -
0.5 1.0 1.5
p = 0.29p = 0.11
p = 0.06p = 0.61
DFS Hazard Ratio
Di Leo et al.: Ciin Cancer Res, 2002; Pritchard K: ASCO 2002
HER2 and Response to Anthracyclines
In Vitro Sensitivity to Doxorubicin following HER2 Transfection
Pegram M et al.: Oncogene 1998
MCF-7 MDA-MB-231 BT20 MDA-MB 435
100%
IC50relative to control
HER2 transfected cells
Control cells
HER2 could be a surrogate for other markers predicting response to anthracyclines
Predictive Markers in Breast Cancer
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
TRAP100PIP5HER2 (124kb)ZNFN1AGRB7MLN51THRACDC6RARATOPOII
2MB
Human Genome Data Base, PubMed Nucleotides
• 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
Topoisomerase-II
• 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 ?
Questions
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
Cep17DAPI Topo-II
Topo-II Deletion
Cep17DAPI Topo-II
Normal Topo-II Cep17 Ratio
Cep17DAPI Topo-II
UCLA Study
• 64/100 HER2+ patients have been evaluated
• 64 HER2+
• No Topo-II alterations among HER2- patients
16 (25%) Topo Ampl.29 (45.3%) Normal19 (29.7%) Topo Deletion
Topo-II Gene Alterationsin Breast Cancer
• 136 primary breast cancers
• 70/136 HER2 amplification (FISH)
• 70 HER2+
• No alterations in HER2-negative samples
29 (41%) Topo Ampl.11 (16%) Normal30 (43%) Topo Deletion
Jarvinen et al.: Genes, Chromosomes and Cancer, 1999
Correlation Between Topo-II Gene Alterations and Protein Expression
Protein
Gene amplification + + + - -
Jarvinen et al.: Genes, Chromosomes and Cancer, 1999
Protein
Geneamplification + + + - -+ +
EGFR/HER2 Stimulation Upregulates Topo-II Protein Expression
Harris LN et al.: Clin Cancer Res.: 2001
NIH
77
- E
GF
N
IH77
+
EG
F
NIH
82
- E
GF
N
IH82
+
EG
F
Influence of Cell Cycle on Topo-II Protein Expression
Stacey DW et al.: Mol. Cell. Biol., 2000
Topo-II expression
Topo-II expression relative to DNA levels
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 Alterationsin 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% Normal21% Topo Deletion
Isola JJ et al.: Breast Cancer Res. Treat.: 2000
Response Rates in 61 HER2+ Patients
0
10
20
30
40
50
60
70
80
Topo-IIDeletion
Topo-IIAmplified
17%
35%
79%
p=0.0004
Normal
Topo-II Gene Alterationsin Breast Cancer
• 354 primary breast cancers
• EC vs. HEC vs. CMF
• 21% HER2 amplification (FISH)
• 21% HER2+38% Topo Ampl.49% Normal13% 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.
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