Hormone therapy for metastatic breast cancer in 2016 Angelo Di Leo “Sandro Pitigliani” Medical Oncology Department Hospital of Prato Istituto Toscano Tumori, Prato, Italy

Conflict of Interest Disclosure Applicability Company (1) Advisory role Yes AstraZeneca, Bayer, Lilly, Novartis, Pfizer, Roche 　 (2) Stock ownership/profit　 None (3) Patent royalties/licensing fees　 (4) Lecture fees AstraZeneca, Eisai, Genomic Health, Novartis, Pfizer, Roche (5) Manuscript fees (6) Scholarship fund (7) Other remuneration

Recommendations from the American Society of Clinical Oncology “ Endocrine therapy, rather than chemotherapy, should be offered as the standard first-line treatment for patients with HR+ advanced breast cancer, except for immediately life threatening disease or if there is concern regarding endocrine resistance ” Partridge AH et al, J Clin Oncol 32:3307 – 29, 2014

Recommendations from the ESO-ESMO ABC-2 panel “Endocrine therapy is the preferred option for hormone receptor positive disease, even in the presence of visceral disease, unless there is concern or proof of endocrine resistance, or there is disease needing a fast response” Level of evidence IA Votes: 100% yes, 29 voters (of 43 members) Cardoso F et al, The Breast 23:489 – 502, 2014, and Ann Oncol 2014

Current criteria used to support 1° line treatment choices in ER+/HER-2 negative advanced breast cancer In favour of chemotherapy Uncertain In favour of endocrine therapy Disease-free interval < 1 yr. 1-2 yrs. > 2 yrs. Visceral mets. Massive burden (visceral crisis) Moderate burden Minimal burden or absence Symptoms Heavy Moderate Minimal or absence

Evidence supporting the use of Fulvestrant at full doses

CONFIRM: a phase III trial comparing Fulvestrant 250 mg vs 500 mg in post-menopausal women with ER + advanced disease

Primary endpoint: progression-free survival 1.0 Proportion of patients progression-free Fulvestrant 500 mg Fulvestrant 250 mg 0.8 HR = 0.80; 95% CI: 0.68, 0.94; p=0.006 0.6 Median PFS (months) Fulvestrant 500 mg 6.5 Fulvestrant 250 mg 5.5 0.4 0.2 0.0 4 8 12 16 20 24 28 32 36 40 44 48 Time (months) Patients at risk: 500 mg 250 mg 362 374 216 199 163 144 113 85 90 60 54 35 37 25 19 12 12 4 7 3 3 1 2 1 0 0 CI, confidence interval; HR, hazard ratio; PFS, progression-free survival Di Leo A et al. J Clin Oncol 2010; 28: 4594-4600 8

Secondary endpoint: overall survival (first analysis at 50% maturity – full analysis set) 1.0 Proportion of patients alive Fulvestrant 500 mg Fulvestrant 250 mg 0.8 HR = 0.84; 95% CI: 0.69, 1.03; p=0.091 0.6 0.4 Median time to death (months) Fulvestrant 500 mg 25.1 Fulvestrant 250 mg 22.8 0.2 0.0 4 8 12 16 20 24 28 32 36 40 44 48 Time (months) Patients at risk: 500 mg 250 mg 362 374 330 338 285 299 251 260 223 222 165 157 116 107 74 61 46 34 29 18 16 10 6 2 0 0 Di Leo A et al. J Clin Oncol 2010; 28: 4594-4600 9

Overall survival (final analysis at 75% maturity – full analysis set) Proportion of patients alive 1.0 Fulvestrant 500 mg 0.9 Fulvestrant 250 mg 0.8 HR (95% CI) 0.81 (0.69, 0.96) p-value 0.016a 0.7 0.6 0.5 aNominal value, cannot be claimed as statistically significant 0.4 0.3 Median time to death (months) Fulvestrant 500 mg 26.4 Fulvestrant 250 mg 22.3 0.2 0.1 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80 Time (months) Patients at risk: 362 333 288 254 227 202 178 163 141 123 114 98 81 64 47 30 26 15 8 1 500 mg 374 338 299 261 223 191 164 137 112 96 87 74 64 48 37 22 14 8 3 2 250 mg Di Leo A et al, J Natl Cancer Inst, 2014; 106 (1): djt337

Overall survival: first (50% events) and final (75% events) analyses

Summary of patients who had at least one SAE during the whole treatment period (main trial plus follow-up phase) Number (%) of patients Fulvestrant 500 mg N=361 Fulvestrant 250 mg N=374 Any SAE 35 (9.7) 27 (7.2) Any causally related SAE 8 (2.2) 4 (1.1) All events occurring after first dose are summarized Patients with multiple events in the same category were counted only once in that category Patients with events in more than one category were counted once in each of those categories 12

SAEs with outcome of death during the whole treatment period Preferred term Number (%) of patients Fulvestrant 500 mg N=361 Fulvestrant 250 mg N=374 Acute myocardial infarction 2 (0.5) Acute renal failure 1 (0.3) Aspiration Cardiopulmonary failure Completed suicide Death (death cause unknown) Dyspnea 2 (0.6) Hypertension Intestinal adenocarcinoma Meningitis All events occurring after first dose are summarized Patient numbers are not mutually exclusive 13

Primary end-point = clinical benefit (CB) rate Previous data from phase II randomised studies testing Fulvestrant 500 mg: the FIRST trial anastrozole 1 mg/daily (N=103) post-menopausal ER+ advanced, 1st line fulvestrant 500 mg days 0, 14, 28, and then q 28 days (N=102) Primary end-point = clinical benefit (CB) rate F A No. CR (%) - 1 (1) No. PR (%) 32 (31) No. NC ≥ 24 wks (%) 42 (41) 36 (35) No. CB (%) 74 (72) 69 (67) Robertson JFR et al, J Clin Oncol 2009

Updated TTP data from phase II randomized studies testing Fulvestrant 500 mg: the FIRST trial Robertson JFR et al, Breast Cancer Res Treat 2012

Robertson JF et al, J Clin Oncol, 33(32):3781-7, 2015

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

Considerations on CDK4/6 inhibitor activity PD 0332991 has shown activity preferentially on ER+, luminal breast cancer cell lines with or without HER2 amplification. IC50 nM Subtype Luminal Non - luminal/post EMT HER2 Amplified luminal Immortalized 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 19

PALOMA-3: Study design N=521c Palbociclib (125 mg QD; 3 wks on/1 wk off) + Fulvestrantd (500 mg IM q4w) HR+ HER2– ABC Pre-/peri-a or postmenopausalb Progressed on prior endocrine therapy: On or within 12 mo of completion of adjuvant treatment On or within 1 mo of treatment for ABC ≤1 prior chemotherapy regimen for advanced cancer n=347 2:1 randomisation N=521c Stratification: Placebo (3 wks on/1 wk off) + Fulvestrantd (500 mg IM q4w) Visceral metastases Sensitivity to prior hormonal therapy Pre-/peri- vs. postmenopausal ABC3 Dr Crown n=174 aAll received goserelin. bMust have progressed on prior endocrine therapy (pre-/perimenopausal) or aromatase inhibitor therapy (postmenopausal). cPatients randomised. dAdministered on Days 1 and 15 of Cycle 1, then every 28 d. Randomised Phase III double-blind trial at 144 centres in 17 countries (NCT01942135) Turner NC, et al. N Engl J Med 2015;373:209–19; Turner NC, et al. ASCO 2015 (Abstract LBA502); 27

PALOMA-3: Updated investigator-assessed PFS (ITT) 100 PAL + FUL (N=347) PBO + FUL (N=174) Median PFS, months (95% CI) 9.5 (9.2, 11.0) 4.6 (3.5, 5.6) Hazard ratio (95% CI) 0.461 (0.360, 0.591) 1-sided p-value <0.000001 80 60 PFS (%) Palbociclib + fulvestrant Placebo + fulvestrant 40 20 Figure 2a 2 4 6 8 10 12 14 Time (months) Number of patients at risk PAL+FUL 347 281 247 202 91 32 7 1 PBO+FUL 174 112 83 59 22 13 2 Turner NC, et al. N Engl J Med 2015;373:209–19; Turner NC, et al. ASCO 2015 (Abstract LBA502); 28

PALOMA-3: PFS by menopausal status Pre-/perimenopausal women Postmenopausal women 100 100 HR 0.435 P=0.01 HR 0.409 P=0.000001 80 80 Palbociclib + fulvestrant n=72 Palbociclib + fulvestrant n=275 60 60 PFS PROBABILITY (%) PFS PROBABILITY (%) 40 40 20 20 Placebo + fulvestrant n=36 Placebo + fulvestrant n=138 5.6 months 9.5 months 3.7 months 9.2 months ABC3 Dr Crown 2 4 6 8 10 12 2 4 6 8 10 12 TIME (MONTHS) TIME (MONTHS) Menopausal status interaction test P=0.94 All pre-/peri-menopausal patients also received goserelin. Winer EP. ASCO 2015. ‘Highlights of the Day: Breast Cancer 29 29

PALOMA-3 final analysis: Treatment effect by PIK3CA status PIK3CA mutations were detected in 33% of patients who provided baseline plasma samples for cell-free DNA analysis (129/395) PIK3CA status did not influence the magnitude of PFS benefit from palbociclib (HR=0·45 for PIK3CA wild-type, HR=0·48 for PIK3CA mutation positive, Pinteraction = 0·83) PIK3CA-wild-type patients (n=266) Patients with PIK3CA mutations (n=129) Time (months) PFS (%) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 20 40 60 80 100 180 173 146 141 129 112 110 56 53 17 86 55 52 39 38 31 30 15 Palbociclib + fulvestrant Placebo + fulvestrant Number at risk Placebo + fulvestrant (n=86) Palbociclib + fulvestrant (n=180) HR (95% CI) 0.45 (0.31–0.64) P value <0.0001 1 2 3 4 5 6 7 8 9 10 11 12 13 14 20 40 60 80 100 HR (95% CI) 0.48 (0.30–0.78) P value 0.002 Time (months) PFS (%) Placebo + fulvestrant (n=44) Palbociclib + fulvestrant (n=85) 85 82 67 64 56 55 44 23 12 10 2 28 27 20 4 1 Palbociclib + fulvestrant Placebo + fulvestrant Number at risk PFS, progression-free survival; HR, hazard ratio; CI, confidence interval Cristofanilli M, et al. Lancet Oncol. 2016 30

An attempt to move recent trial results into clinical practice 1st line, ER+ HER-2 neg advanced disease, post-menopausal pts. prior adjuvant HT Options Naive Fulvestrant Fulvestrant + Palbociclib TAM - AI - AI + Palbociclib - Fulvestrant + Palbociclib (if relapse while on TAM) AI - Fulvestrant - Fulvestrant + Palbociclib

A functional Rb signature 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) Migliaccio I et al, Ann Oncol, 2015, 26 (suppl 3): iii15

Functional RBsig discriminates sensitive vs resistant BC cell lines AUC = 0.93 Migliaccio I et al, Ann Oncol, 2015, 26 (suppl 3): iii15

RBsig is prognostic in patients with ER+ tumors Untreated ER+ Luminal A Luminal B Endocrine treated only Migliaccio I et al, Ann Oncol, 2015, 26 (suppl 3): iii15

ESR1 mutations and response to Palbociclib in the PALOMA 3 trial ESR1 mutated ESR1 wild type Fribbens C et al, J Clin Oncol 2016 (June 6, 2016. Ahead of print)

ESR1 mutations and response to different endocrine therapy agents ESR1 mutations and response to different endocrine therapy agents. The SOFEA trial results ESR1 mutated ESR1 wild type Fribbens C et al, J Clin Oncol 2016 (June 6, 2016. Ahead of print)

Clinical trials comparing HT vs Clinical trials comparing HT vs. HT + biological agent in ER+ advanced disease. Focus on: - The BOLERO-2 trial

The BOLERO-2 Phase III trial: Everolimus in advanced disease EVE 10 mg daily + EXE 25 mg daily (n = 485) Placebo EXE 25 mg daily (n = 239) R 2:1 N = 724 Postmenopausal ER+ Unresectable locally advanced or metastatic BC Recurrence or progression after letrozole or anastrozole 84% of patients sensitive to prior endocrine therapy (i.e. DFI > 24 mos. if relapse while/after adjuvant AI or clinical benefit to the prior line of endocrine therapy for advanced disease) 84% of patients received the study drugs as ≥ 2° line of therapy for advanced disease BOLERO-2 is an acronym for Breast cancer trials of OraL EveROlimus-2. BOLERO-2 is a pivotal, international, multicenter, randomized phase III study evaluating EVE in combination with exemestane (EXE), conducted in postmenopausal women with ER+, refractory, advanced BC (with recurrence or progression after prior therapy with LET or anastrozole [ANA]). Women (N = 724) were randomized 2:1 to receive either EVE + EXE or placebo + EXE. Treatment continued until disease progression, unacceptable toxicity, or consent withdrawal. No crossover after disease progression was allowed. Randomization was stratified by documented sensitivity to prior hormonal therapy (yes vs no) and by the presence of visceral metastasis (yes vs no). Sensitivity to prior hormonal therapy was defined as either (1) documented clinical benefit (complete response, partial response, stable disease ≥ 24 weeks) from at least 1 prior hormonal therapy in the advanced setting or (2) ≥ 24 months of adjuvant hormonal therapy before recurrence. This study is ongoing and remains blinded; all patients without documented disease progression continue to be followed for progression free survival (PFS). All patients are being followed for overall survival (OS) in a blinded manner, even when they have disease progression and go off study. Baselga J, et al. N Engl J Med. 366:520-529, 2012 38

PFS (based on local assessment at 18 mos. follow-up) and OS EVE 10 mg + EXE PBO + EXE Number of patients still at risk 20 40 60 80 100 Time (week) 6 12 18 24 30 36 42 48 54 66 72 78 84 90 96 102 108 114 120 485 436 366 304 257 221 185 158 124 91 50 35 22 13 10 8 2 1 239 190 132 67 39 21 15 5 3 Censoring times EVE 10 mg + EXE (n/N = 310/485) PBO + EXE (n/N = 200/239) HR = 0.45 (95% CI: 0.38-0.54) Log-rank P value: < .0001 Kaplan-Meier medians EVE 10 mg + EXE: 7.8 months PBO + EXE: 3.2 months Probability (%) of Event OS** HR = 0.89 (95% CI, 0.73-1.10) Log-rank P = .14 Kaplan-Meier medians EVE+EXE: 30.98 months PBO+EXE: 26.55 months Censoring times 232 109 248 113 266 120 279 130 292 145 311 153 330 162 347 170 373 182 399 194 414 201 429 211 448 220 471 485 239 EVE+EXE PBO+EXE No. at risk 11 5 23 8 39 18 58 28 91 41 118 56 154 77 196 98 216 102 1 * Baselga J et al, N Engl J Med. 366: 520-29, 2012; ** Piccart M et al, Ann Oncol 25: 2357-62, 2014

Safety profile everolimus: most common adverse events % G3-G4 (%G1-G4) Placebo N = 238 Stomatitis 8 (56) 1 (11) Anemia 6 (16) 1 (4) Dyspnea 4 (18) 1 (9) Hyperglicemia 4 (13) 1 (2) Fatigue 4 (33) 1 (26) Pneumonitis 3 (12) - (-) Baselga J et al, New Engl J Med 366: 520-9, 2012. 40 40

The TAMRAD trial post-menopausal ER+/HER-2 neg No. = 111 pts. pre-treatment with AI TAM + Everolimus TAM TAM TAM + Everolimus Clinical benefit rate at six months 42% (95% CI: 29-56%) 61% (95% CI: 47-74%) Bachelot T et al, J Clin Oncol 30: 2718-24, 2012

Primary resistance Secondary resistance The TAMRAD trial: TTP results by level of response to prior aromatase inhibitor therapy Primary resistance Secondary resistance Bachelot T et al, J Clin Oncol 30: 2718-24, 2012

An attempt to move recent trial results into clinical practice Pts. progressing on endocrine therapy + CDK 4-6 inhibitors chemotherapy HT + everolimus HT + everolimus = last attempt of “endocrine therapy” before moving to chemothrapy

New agents currently tested in Phase III trials

Combined LEE011, Fulvestrant and PI3K Inhibition in ER+ Breast Cancer Triplet combination therapies of LEE011 with fulvestrant and PI3K inhibitors demonstrate potent tumor regressions in PIK3CA-wild-type and PIK3CA-mutant ER+ breast cancer models Activity of LEE011 + fulvestrant + PI3Ki in a PIK3CA-mutant ER+ BC model (MCF7) Activity of LEE011 + fulvestrant + PI3Ki in a PIK3CA-wild-type ER+ BC model (KPL1) Tumor Volume mm3 Tumor Volume mm3 Triplet combination therapies of LEE011 with fulvestrant and PI3K inhibitors demonstrate potent tumor regressions in PIK3CA wild-type and PIK3CA-mutant ER+ breast cancer models Reference 1. O’Brien NA, et al. AACR 2014;abst 4756. PI3Ki, PI3K inhibitor. 1. O’Brien NA, et al. AACR 2014; Abstract 4756 49

PI3K Pathway Inhibitors in Clinical Development in Breast Cancer Drug Source Target(s) GDC-0032 Genentech PI3Kα MLN-1117 Millenium BYL719 Novartis GS-1101 Gilead PI3Kd XL-147/SA245408 Exelixis/Sanofi Pan-PI3K BKM120 GDC-0941 PF-05212384/PKI-587 Pfizer XL-765/SAR245409 PI3K/mTOR BEZ235 GDC-0980 MLN-128/MLN0128 TORC1/2 OSI-027 OSI Pharma AZD2014 AstraZeneca AZD5363 AKT (catalytic) MK2206 Merck AKT (allosteric) GDC-0068 ClinicalTrials.gov. From: www.clinicaltrials.gov. Accessed August 2013. 50

The FERGI trial: a phase II randomised trial post-menopausal ER+/HER-2 negative No. = 168 pts. prior AIs Fulvestrant 500 mg + Pictilisib Fulvestrant 500 mg + Placebo Cross-over allowed ! 42% of pts. with PIK3CA mutation Krop I et al, Lancet Oncol, 17(6):811-21, 2016

Krop I et al, Lancet Oncol, 17(6):811-21, 2016

Potential (non-mutually exclusive) explanations for the lack of interaction between PIK3CA gene status and clinical activity of PI3K/MTOR inhibitors Pictilisib is a pan-PI3K inhibitor. Different results with an α-specific PI3K inhibitor?

Sensitivity of PIK3CA-mutant breast cancer to single-agent BYL719 in vivo In vivo, BYL719 shows statistically significant antitumor efficacy in PIK3CA-mutant breast tumor xenograft models T/C 100% 1.5% T/C 100% –62.6% Vehicle 10 ml/kg po q24h NVP-BYL719-NX-7 50 mg/kg po q24h po, per os (by mouth); q24h; once every 24 hours; SEM, standard error of mean; T/C, treatment over control. Novartis data on file. 2013.

Potential (non-mutually exclusive) explanations for the lack of interaction between PIK3CA gene status and clinical activity of PI3K/MTOR inhibitors Pictilisib is a pan-PI3K inhibitor. Different results with an α-specific PI3K inhibitor? PI3K pathway down-stream effectors might be more informative than PIK3CA gene status (Loi S et al, Proc Natl Acad Sci USA 107:10208-13, 2010)

Potential (non-mutually exclusive) explanations for the lack of interaction between PIK3CA gene status and clinical activity of PI3K/MTOR inhibitors Pictilisib is a pan-PI3K inhibitor. Different results with an α-specific PI3K inhibitor? PI3K pathway down-stream effectors might be more informative than PIK3CA gene status (Loi S et al, Proc Natl Acad Sci USA 107:10208-13, 2010) Differences in PIK3CA gene status between the primary and the matched metastatic sites

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 57

Baselga J et al, proc. SABCS, 2015

Baselga J et al, proc. SABCS, 2015

Baselga J et al, proc. SABCS, 2015

Baselga J et al, proc. SABCS, 2015

Acknowledgments