1. Jo Anne Zujewski, MD Cancer Therapy Evaluation Program Division of Cancer Diagnosis and Treatment National Cancer Institute May, 2011 Targeted Therapy for Breast Cancer

2. No surgery mastectomy chemoTx + antiER chemoTx + antiER + targeted Incremental Benefit

3. No surgery mastectomy chemoTx + antiER chemoTx + antiER + targeted Incremental benefit Each incremental step assumed that no pt is cured with the previous step Significant overtreatment Necessity to conduct large trials to demonstrate small benefit Significant overtreatment Necessity to conduct large trials to demonstrate small benefit

4. PNAS 2005 Molecular profiling

5. Subtypes and Prognosis Sorlie T et al, PNAS 2001

6. Oncotype DX 21 Gene Recurrence Score (RS) Assay PROLIFERATION Ki-67 STK15 Survivin Cyclin B1 MYBL2 ESTROGEN ER PR Bcl2 SCUBE2 INVASION Stromolysin 3 Cathepsin L2 HER2 GRB7 HER2 BAG1GSTM1 REFERENCE Beta-actin GAPDH RPLPO GUS TFRC CD68 16 Cancer and 5 Reference Genes From 3 Studies CategoryRS (0 – 100) Low riskRS < 18 Int riskRS ≥ 18 and < 31 High riskRS ≥ 31

7. B-20 Summary Patients with tumors that have high Recurrence Scores have a large absolute benefit of chemotherapy (similar results with CMF and MF) Patients with tumors that have low Recurrence Scores derive minimal, if any, benefit from chemotherapy RS < 18 RS 18-30 RS ≥ 31

8. Recurrence Score as a Continuous Predictor My RS is 30, What is the chance of recurrence within 10 yrs? 95% CI

9. Schema: TAILORx 17 Node Neg, ER (+), Breast Cancer RS < 10 Hormone Therapy Registry N=1625 RS < 10 Hormone Therapy Registry N=1625 RS 11 – 25 Randomize Hormone Rx vs. Chemotherapy + Hormone Rx N=6908 RS 11 – 25 Randomize Hormone Rx vs. Chemotherapy + Hormone Rx N=6908 RS > 25 Chemotherapy + Hormone Rx N=1731 RS > 25 Chemotherapy + Hormone Rx N=1731 21-gene RS n=11,233 21-gene RS n=11,233 Register Specimen banking Accrual complete as of 10/06/2010

10. Breast Cancer: Stable from Preneoplasia to Metastasis 245 DCIS in population-based study: Livasy, Human Pathol 2007 SubtypeN (%) Basal-like19 (8%) Luminal A149 (61%) Luminal B23 (9%) HER2+/ER- 38 (16%) Unclass.16 (6%) Molecular subtype persists before and after therapy and in metastases: * * * Weigelt et al., Cancer Res, 2005 4 studies find basal-like present but uncommon in DCIS (5-10%)

11. HER2 cluster Basal gene cluster Luminal (hormone receptor-related) cluster Proliferation cluster Basal-like Breast Cancer Comprise 15-20% of tumors Low ER (and related genes) expression Low HER2 cluster expression  usually “triple negative” High basal cluster –basal cytokeratins –EGFR –c-kit –others… Very proliferative Often p53 mutant Evidence of genomic instability

12. Surrogates: Clinical Phenotypes versus Molecular Subtypes Triple negative and Basal-like Basal but not triple negative 15-40% are ER+, PR+, or HER2+ Triple negative but not basal 10-30% Can also include “claudin-low”, a subtype notable for high expression of stem cell markers “Triple negative” (ER negative, PR negative, and HER-2 negative) breast cancer is mostly the basal-like subtype

13. Defining the biology of ER - breast cancer Aberrant expression of transcription factors and growth factor receptors has been correlated with basal-like (triple negative) subtype of breast cancer Mechanisms of ER loss can vary: – ER promoter methylation in ER - breast cancer (25%) – Src activated ER degradation ER - tumors share similarities with BRCA-1 associated breast cancer – Clinical & pathological features – Gene profiling data

14. Hereditary Basal-likeSporadic Basal-like BRCA1 Xiso XIST Loss Triple Neg Cancer Genomic Instability ? ? BRCA1 Xiso XIST Loss Triple Neg Cancer Genomic Instability Courtesy J. Garber BRCA1-Associated and Sporadic Basal-like Breast Cancer

15. CharacteristicsHereditary BRCA1Triple Negative/Basal-Like 1,2,3 ER/PR/HER2 statusNegative TP53 statusMutant BRCA1 statusMutational inactivation*Diminished expression* Gene-expression patternBasal-like Tumor histology Poorly differentiated (high grade) Poorly differentiated (high grade) Chemosensitivity to DNA- damaging agents Highly sensitive TNBC Shares Clinical and Pathologic Features with BRCA-1-Related Breast Cancers 3 Sorlie et al. Proc Natl Acad Sci U S A 2001;98:10869-74 4 Miyoshi et al. Int J Clin Oncol 2008;13:395-400 *BRCA1 dysfunction due to germline mutations, promoter methylation, or overexpression of HMG or ID4 4 1 Perou et al. Nature. 2000; 406:747-752 2 Cleator et al.Lancet Oncol 2007;8:235-44 15

16. PARP Inhibitor Treatment Strategies 1.Sensitization to DNA damaging therapy 2.‘ Chemical synthetic lethality ’ in genetically susceptible tumors BRCA1, BRCA2, others?

17. DNA Damage Repair PathwaysDouble-strand breaks (DSBs) Recombinationrepair ATMBRCA DNA-PK HR NHEJ Type of damage: Repair pathway: Repair enzymes: Bulky adducts Insertions & deletions O6- alkylguanine Nucleotide- excision repair Mismatch repair Direct reversal XP, polymerases MSH2, MLH1 AGT

18. PARP-1 Inhibition Increases DNA DS Damage PARP Inhibition of PARP-1 prevents recruitment of repair factors to repair SSB XRCC1 LigIII PNK 1 pol β Replication (S-phase) DNA DSB DNA SSB

19. Cell survival Base Excision Repair Synthetic Lethality: Selective effect of PARP-1 inhibition on cancer cells with BRCA1 or BRCA2 mutation Homologous Recombination DNA Damage PARP Inhibitor BRCA Mutation Cancer cell death

20. HER-2 as a Target for Therapy: NeoALTTO HER-2 nucleus cancer cell cell division Trastuzumab (Herceptin) Anti-HER-2 Antibody Lapatinib (Tykerb) Dual HER-1/HER-2 Tyrosine Kinase Inhibitor Growth factor receptor: Overexpressed in 20-25% of breast cancersGrowth factor receptor: Overexpressed in 20-25% of breast cancers Neo-ALTTO: pre-operative study of trastuzumab; lapatinib; or the combinationNeo-ALTTO: pre-operative study of trastuzumab; lapatinib; or the combination pCR: COMBINATION 51.3% Trastuzumab 29.5% Lapatinib 24.7%

21. Another HER-2 Targeted Therapy in Development Trastuzumab-DM1 (T-DM1) Trastuzumab Mertansine: anti-tubulin

22. The Truth About Targeted Therapy Cancers have redundant, modular pathways Answers will not come from purely clinical trials in unselected populations. Real understanding of how to target will require more tissue-based studies and global collaborations Citri and Yarden, Nat Rev Mol Cell Biol 2006 EGFR Clean preclinical model Messy clinical situation Multiple ligands Heterodimerization Mutated receptors Cleaved receptors Horizontal activation Kinase alterations Epigenetic alterations Alternate signaling pathways…. Courtesy of Lisa Carey

23. Copyright ©2008 American Association for Cancer Research Tan, S.-H. et al. Clin Cancer Res 2008;14:8027-8041 Fig. 2 Predicting response to Endocrine therapy

24. The Host: Metabolic factors Body Size in Operable Breast Cancer

25. Obesity – Breast Cancer Interaction of Estrogen and Insulin / IGF Mechanisms Adipose Tissue  estrogens  insulin  IGFBP-1  IGF-I   SHBG ER/PgR IGF-IR IR α, β (free) + ++ + + + + + +    * * Proliferation Anchorage Independent Growth Reduced Apoptosis * PI3K, ras-raf-MAP Kinase signalling pathways + inflammatory markers adipocytokines (e.g. leptin, TNF  ) ++

26. Molecular Action of Insulin Adapted from Vigneri P et al., Endocr Relat Cancer 2009 Jul 20 (epub ahead of print)

27. Potential Treatment Targets Lifestyle Weight Loss – LISA Physical Activity Diet – fat (WHI / WINS / WHEL) – calories Physiologic Mediators Insulin Glucose Adipocytokines (e.g. leptin) Cellular Mediators AMPK PI3K / AKT/ mTOR pathway ras / raf / MEK pathway Insulin / IGF receptors

28. Goodwin P J et al. J Clin Oncol 2009; 27:3271-3273 Mechanism of Metformin Action

29. Pathologic Complete Response Between Study Groups (Metformin, No Metformin, Non-Diabetic) Jiralersprong S et al. J Clin Oncol 2009; 20:3297-3302

30. NCIC CTG MA.32 Multicentre Phase III Randomized Double-Blind Placebo Controlled Trial in Early Stage Breast Cancer Metformin 850 mg po bid X 5 years () Identical Placebo One caplet po bid X 5 years RANDOMIZERANDOMIZE FUNDED BY: NCI (US), CCS, BCRF, Apotex Canada

31. Biological Information Flow Epigenetics Gene Expression Genetics MICROENVIRONMENT