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.