“Sandro Pitigliani” Medical Oncology Department Hospital of Prato

1. Nuovi pathways nuovi farmaci: quali prospettive? “Sandro Pitigliani” Medical Oncology Department Hospital of Prato Istituto Toscano Tumori, Prato, Italy Angelo Di Leo

2. Conflict of Interest Disclosure

3. Focus on ER+ / HER-2 negative breast cancer • CDK 4-6 inhibitors • PI3K inhibitors

4. Cooperation between ER and Cyclic D1 pathways enhances proliferation in luminal breast cancer Cyclin D1 ER Inhibitory control Inhibitory control p Rb CDK4-6 Cell Cycle: G1 S • Inhibitors: • Palbociclib • Abemaciclib • Ribociclib proliferation

5. Considerations on CDK4/6 inhibitor activity Subtype Subtype Luminal Luminal Non Non - - luminal/post EMT luminal/post EMT HER2 Amplified HER2 Amplified Non Non - - luminal luminal Immortalized Immortalized IC50 nM PD 0332991 has shown activity preferentially on ER+, luminal breast cancer cell lines with or without HER2 amplification. • RB1, cyclin D1, and CDKN2A (p16) were differentially expressed - with higher levels of RB1 and cyclin D1, and lower levels of p16, in the sensitive group. • Resistance to PD in many of the nonluminal breast cancer cell lines may be explained by the absence of pRb. Recent publications highlighted the lack of pRb in basal-like breast cancer tissue and observed that pRb depletion can result in the characteristic epithelial-to-mesenchymal transition changes • The lack of activity of a CDK4/6 inhibitor in cell lines and tumors that lack pRb can be explained by the fact that cyclin D1 does not offer G1 control in the absence of pRb. Finn et al, BCR 2011

6. We have results from two Phase III trials testing CDK 4-6 inhibitors in the first-line treatment of HR+/HER-2 advanced breast cancer patients: - PALOMA 2 - MONA LEESA 2

11. MONA LEESA 2: Study design N= 668 2 1 Hortobagyi GN et al, New Engl J Med, 2016 (published online on October 8)

12. PFS (investigator-assessed) – ITT population Hortobagyi GN et al, New Engl J Med, 2016 (published online on October 8)

13. MONA LEESA 2: Adverse events Hortobagyi GN et al, New Engl J Med, 2016 (published online on October 8)

14. Do we have strong evidence that the combination CDK 4-6 inh. + endocrine therapy should be the upfront option??

15. (In my opinion) not yet … • Cross-over would have been important to address this question CDK 4-6 inh + endocrine therapy endocrine therapy CDK 4-6 inh + endocrine therapy • In addition, the clinical activity of endocrine therapy after PD to CDK 4-6 inhibitors is not clear

16. First-line therapy for HR+/HER-2 negative advanced breast cancer: Back-home message (my personal view) • Life-threatening disease: chemotherapy • Visceral involvement and/or symptomatic disease: CDK 4-6 inhibitor + endocrine therapy (HT) • Non visceral involvement and non symptomatic disease (previously treated or untreated with HT): Fulvestrant CDK 4-6 inhibitor + aromatase inhibitor

17. Perspectives: Identifying patients with primary resistance to CDK 4-6 inhibitors. The Rb Sig E2F1 and E2F2 high vs low breast cancers in the TCGA Expression data Differential gene expression analysis Functional RBsig • Correlation with palbociclib activity (in-vitro) • Prognostic value (in-silico analysis) Malorni L et al, Oncotarget, 13:68012-22, 2016

18. Functional RBsig discriminates sensitive vs resistant BC cell lines AUC = 0.93 Malorni L et al, Oncotarget, 13:68012-22, 2016

19. Luminal A Luminal B ER+ ER+ Luminal A Luminal B RBsig is prognostic in patients with ER+ tumors Untreated Endocrine treated only Malorni L et al, Oncotarget, 13:68012-22, 2016

20. New agents currently tested in Phase III trials

22. PI3K Pathway Inhibitors in Clinical Development in Breast Cancer ClinicalTrials.gov. From: www.clinicaltrials.gov. Accessed August 2013.

23. Baselga J et al, proc. SABCS, 2015

24. Baselga J et al, proc. SABCS, 2015

25. Baselga J et al, proc. SABCS, 2015

26. IGFR1 PI3K Phospho AKT IRS1 Phospho Short loop Long loop mTORC2 mTORC1 S6 inhibition inhibition Pre-clinical rationale for a PI3K inhibitor after progression to mTORC1 inhibitors • mTORC1 inhibition elicits AKT phosphorylation (feedback activation) • PI3K inhibitors abrogate or attenuate AKT phosphorylation elicited by mTORC1 inhibition • Any role for PI3K inhibitors after progression to Everolimus? Sun SY et al, Cancer Res 2005; O’Reilly KE et al, Cancer Res 2006; Breuleux M et al, Mol Canc Ther 2009; O’Brien NA et al, Clin Cancer Res 2014; Wander SA et al, J Clin Invest 2011; Mayer IA et al, Annu Rev Med 2016

27. BELLE-3 trial design and Endpoints Post menopausal women with HR+, HER2-, locally advanced or metastatic breast cancer, who received Everolimus + AIs as last line of therapy, N= 432 • Primary Endpoint • PFS in the full population • Key secondary Endpoint • OS in the full population • Other Secondary Endpoints • PFS by PIK3CA status based on ctDNA • OS by PIK3CA status based on ctDNA • ORR and CBR in the full population and by PIK3CA status based on ctDNA • Safety, Pharmacokinetics, Quality of Life Randomization (2:1) Stratification by visceral disease status Buparlisib (100 mg/day) + Fulvestrant (500 mg) n=289 Placebo + fulvestrant (500 mg) n=143 90% power to detect a 33% reduction in the risk of progression (α one-sided= 0.025) (313 events required)

28. Primary tumor vs. Circulating Tumor Cells (CTC) vs. circulating tumor DNA (ct DNA) Analysis of single CTCs, ct DNA and primary tumor tissue from pt 11, pt 12, and pt 18 Pt18 Pt11 ct DNA ct DNA Primary Primary Pt12 ct DNA Primary De Luca F et al, Oncotarget, 2016; 7: 26107-119

29. Acknowledgments