2. 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

3. Role of HER2 as a predictive marker for response to anthracyclines is a matter of debate Predictive Markers in Breast Cancer

4. PAF vs. PF AC vs. CMF HER2 + HER2 - HER2 + HER2 - 0.51.01.5 p = 0.001 p = 0.74 HER2 and Response to Anthracyclines p = 0.15 p = 0.84 Relative Risk of Failure for DFS Paik S et al.: JNCI, 1998 and 2000

5. CMF vs. EC CMF vs. FEC HER2 + HER2 - HER2 + HER2 - 0.51.01.5 p = 0.29 p = 0.11 p = 0.06 p = 0.61 DFS Hazard Ratio Di Leo et al.: Ciin Cancer Res, 2002; Pritchard K: ASCO 2002 HER2 and Response to Anthracyclines

6. In Vitro Sensitivity to Doxorubicin following HER2 Transfection Pegram M et al.: Oncogene 1998 MCF-7 MDA-MB-231 BT20 MDA-MB 435 100% IC50 relative to control HER2 transfected cells Control cells

7. HER2 could be a surrogate for other markers predicting response to anthracyclines Predictive Markers in Breast Cancer

8. Comparative Genomic Hybridization Among 38 Breast Cancer Cell lines Forozan F. et al.: Cancer Res, 2001

9. Comparative Genomic Hybridization Among 38 Breast Cancer Cell lines Forozan F. et al.: Cancer Res, 2001

10. Chromosome 17q12 - q21 TRAP100 PIP5 HER2 (124kb) ZNFN1A GRB7 MLN51 THRA CDC6 RARA TOPOII  2MB Human Genome Data Base, PubMed Nucleotides

11. 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 

12. 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

13. 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

14. 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

15. Normal ratio < 2

16. Topo-II  Amplification Cep17 DAPI Topo-II 

17. Topo-II  Deletion Cep17 DAPI Topo-II 

18. Normal Topo-II  Cep17 Ratio Cep17 DAPI Topo-II 

19. 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%) Normal 19 (29.7%) Topo Deletion

20. Topo-II  Gene Alterations in 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%) Normal 30 (43%) Topo Deletion Jarvinen et al.: Genes, Chromosomes and Cancer, 1999

21. Correlation Between Topo-II  Gene Alterations and Protein Expression Protein Gene amplification +++ -- Jarvinen et al.: Genes, Chromosomes and Cancer, 1999 Protein Gene amplification +++ -- ++

22. EGFR/HER2 Stimulation Upregulates Topo-II  Protein Expression Harris LN et al.: Clin Cancer Res.: 2001 NIH77 - EGF NIH77 + EGF NIH82 - EGF NIH82 + EGF

23. Influence of Cell Cycle on Topo-II  Protein Expression Stacey DW et al.: Mol. Cell. Biol., 2000 Topo-II  expression relative to DNA levels

24. In Vitro Experiments Increased Topo-II  expression is associated with drug sensitivity Reduced levels with drug resistance

25. 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

26. 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

27. 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

28. Response Rates in 61 HER2+ Patients 0 10 20 30 40 50 60 70 80 Topo-II  Deletion Topo-II  Amplified 17% 35% 79% p=0.0004 Normal

29. 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;

30. DFS in 61 HER2+ Patients Di Leo et al.: Ciin Cancer Res, 2002;

31. 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.