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Topoisomerase-II  a Predictive Molecular Marker in Breast Cancer:

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.

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Topoisomerase-II  a Predictive Molecular Marker in Breast Cancer:

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  1. 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

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

  3. HER2 and Response to Anthracyclines Relative Risk of Failure for DFS 0.5 1.0 1.5 p = 0.001 p = 0.74 HER2 + HER2 - PAF vs. PF AC vs. CMF p = 0.15 p = 0.84 HER2 + HER2 - Paik S et al.: JNCI, 1998 and 2000

  4. HER2 and Response to Anthracyclines DFS Hazard Ratio 0.5 1.0 1.5 p = 0.29 p = 0.11 HER2 + HER2 - CMF vs. EC CMF vs. FEC p = 0.06 p = 0.61 HER2 + HER2 - Di Leo et al.: Ciin Cancer Res, 2002; Pritchard K: ASCO 2002

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

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

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

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

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

  10. Topoisomerase-II • Amplifications can span large regions of chromosome 17q • Topo-II is close to the HER2 amplicon on chromosome 17q • Topo-II is akey enzyme in DNA replication and a molecular target for Anthracyclines

  11. 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 ?

  12. 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

  13. 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

  14. Normal ratio < 2

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

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

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

  18. 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

  19. 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%) Normal 30 (43%) Topo Deletion Jarvinen et al.: Genes, Chromosomes and Cancer, 1999

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

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

  22. 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

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

  24. 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

  25. 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

  26. 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% Normal 21% Topo Deletion Isola JJ et al.: Breast Cancer Res. Treat.: 2000

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

  28. 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% Normal 13% Topo Deletion Di Leo et al.: Ciin Cancer Res, 2002;

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

  30. 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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