1. 26-30 September 2014, Madrid, Spain esmo.org Trabectedin in patients with BRCA mutated and BRCAness phenotype advanced ovarian cancer (AOC): phase II prospective MITO-15 study Lorusso D 1, Ferrandina G 2, Pignata S 3, Sorio R 4, Pietragalla A 2, Mosconi A 5, Pisano C 3, Mangili G 6, Masini C 7, Artioli G 8, Narducci F 9, Di Napoli M 3, Rigamonti C 10, Raspagliesi F 1, Scambia G 2. 1 Fondazione IRCCS National Cancer Institute Milan, 2 Catholic University of Rome, 3 National Cancer Institte Naples, 4 CRO Aviano, 5 University of Perugia, 6 San Raffaele Hospital Milan, 7 University of Modena and Reggio Emilia, 8 Mirano Hospital, 9 Santissima Trinita’ Sora hospital. 10 Pharmamar Italy.

2. 26-30 September 2014, Madrid, Spain esmo.org BACKGROUND In vitro and clinical studies have reported that mutations in BRCA repair system with functional NER machinery induce greater sensitivity to Trabectedin. We hypotize that Trabectedin will be an effective tretament in patients with BRCAness phenotype, and its activity further enhanced in those carring BRCA 1/2 mutations. Efficacy could also be affected by the presence of different NER related germ line polymorphisms.

3. 26-30 September 2014, Madrid, Spain esmo.org Aim and study design prospective phase II study (EudraCT n: 2011-001298-17) evaluate activity of trabectedin in patients with mutated BRCA and BRCAness phenotype advanced ovarian cancer (AOC) Translational sub-study: Assess germline mutations of BRCA 1 and 2 Evaluate activity of trabectedin according to BRCA and single nucleotide polymorphisms (SNPs) in DNA repair genes (XPD, XPG and ERCC1) mutations

4. 26-30 September 2014, Madrid, Spain esmo.org Patients and methods Patients AOC patients BRCA mutation or BRCAness phenotype At least 2 previous response to platinum Trabectidin 1.3 mg/m2 (3 hour iv infusion) Every 3 weeks until progression Platinum resistant (PR) Platinum sensitive (PS) Stratification Treatment

5. 26-30 September 2014, Madrid, Spain esmo.org Results 100 enrolled pts 88 evaluable pts 42 PS 46 PR 6 months follow-up NORR (%) CR (%) PR (%) SD (%) DCR (ORR + SD) (%) PD (%) PFS (median, months) OS (median, months) PR (46)15 (32.6) 0 12 (26.1) 27 (58.7) 19 (41.3) 2.79.7 PS (42)21 (50)4 (9.5)17 (40.5) 10 (23.8) 31 (73.8) 11 (26.2) 6**18.5*** All (88)36 (41)4 (9.5)32 (36.4) 22 (25)58* (66) 30 (34%) 5.214.7 ORR, overall response rate; CR, complete response; PR, partial response; DCR, disease control rate; PD, progressive disease; PFS, progression-free survival; OS, overall survivial.. * DCR vs PD:p=0.088; **PFS PRvsPS p=0.012; ***OS PRvsPS p=0.041

6. 26-30 September 2014, Madrid, Spain esmo.org Distribution of BRCA mutations in the study population BRCA mutation data available on 66 patients 43 wild type 12 BRCA 1 mut 11 BRCA 2 mut 34.8% BRCA statusPR (43)PS (42)P value WT26170.60 Mutated1211

7. 26-30 September 2014, Madrid, Spain esmo.org Response to trabectedin according to BRCA mutational status BRCA 1- 2 NORR CR+PR (%) DCR (ORR+SD) PDPFS (Months) 3- year OS (%) WT4317 (39.5) 27 (62.8)*14 (32.5)4.566.7** MUT2312 (52.2) 17 (73.9)5 (21.7)4.583.0 *P value DCR: 0.51; **P value OS: 0.041

8. 26-30 September 2014, Madrid, Spain esmo.org 5 SNPs involved in NER machinery ( ERCC1 C118T, ERCC1 C8092, XPG ASP1104HIS, XPD ASP312ASN, XPD LYS751GLN) were evaluated in 75 patients. Response to trabecteding according to mutations of single nucleotide polymorphisms (SNPs) in DNA repair genes (XPD, XPG and ERCC1) SNPsCR+PR+ SD (%)PD (%)P value ERCC1 C118T WT (26) MUT (49) 16 (61.5) 33 (67.3) 8 (30.7) 14 (28.5) 0.8 ERCC1 C8092 WT (37) MUT (38) 23 (61.3) 26 (69.3) 12 (32.4) 10 (26.3) 0.6 XPG ASP1104HIS WT (47) MUT (28) 30 (63.8) 19 (67.8) 15 (32) 7 (25) 0.6 XPD ASP312ASN WT (26) MUT (49) 15 (57.6) 34 (69.4) 9 (34.6) 13 (26.5) 0.4 XPD LYS751GLN WT (29) MUT (46) 16 (55) 33 (72) 11 (38) 11 (24) 0.2

9. 26-30 September 2014, Madrid, Spain esmo.org CONCLUSIONS  This study shows an increased percentage of BRCA 1/2 mutations in the BRCAness phenotype compared to published data in AOC patients. BRCAness profile and, in particular, a history of higher number of previous responses to platinum, is associated with increased responses to trabectedin  Clinical response was higher although non statistically significant in patients carrying germline BRCA 1/2 mutations. The fact that BRCA driven HR is not the only repair mechanism available in tumor cells, can explain the limited sensitivity of germline BRCA mutations as predictive factors in this study.  In this clinical study NER polymorphisms did not correlate with responses to trabectedin.

10. 26-30 September 2014, Madrid, Spain esmo.org Next step Paper finalized Possible journals: JCO, Lancet Oncology, Annals of Oncology Clinical substudies: Emesis (Dr Di Napoli), Analisys of subsequent treatments (Dr Salutari) Translational substudy: evaluation of immoreactive signature in MITO 15 specimens (Dr Gourley)

11. MITO 15 Emesi AGGIORNAMENTO Marilena Di Napoli

12. Progetto di valutazione del PFS2 dopo Yondelis nei pazienti arruolati in MITO15 CENTRIAGGIORNAMENTI Centro 001-RomaSi Centro 002-MilanoSi Centro 011-PerugiaSi Centro 014-MilanoSi Centro 021-MiranoSi Centro 043-CatanzaroSi Centro 004-RomaNo Centro 009-FerraraNo Centro 013-ModenaNo Centro 022- Rionero in VultureNo Centro 030-NapoliNo Centro 039-AvianoNo Centro 047-SoraNo

13. Patients (N°)59 N° (%) Next therapy43 (73) Not other chemotherapy13 (22) Screening failure1 (2) Not evaluable2 (3) CentroN° pz arruolateN° pz sottoposte ad ulteriore CT N° (%) Centro 001-Roma2116 (76) Centro 002-Milano1813 (72) Centro 011-Perugia74 (57) Centro 014-Milano75 (71) Centro 021-Mirano44 (100) Centro 043-Catanzaro21 (50)

14. Regime di chemioterapiaN° (%) Paclitaxel settimanale9 (20) Cisplatino d1q216 (14) Carboplatino d1q215 (12) Doxorubicina Liposomiale Peghilata d1q28 4 (9) Ciclofosfamide p.o.3 (7) Gemcitabina2 (5) Carboplatino-Paclitaxel d1q212 (5) Cisplatino intraperitoneale1 (2) Adriamicina1 (2) Cisplatino-Paclitaxel d1q211 (2) Carboplatino settimanale1 (2) Carboplatino-Gemcitabina1 (2) Vinorelbina1 (2) Tamoxifene1 (2) Carboplatino-PLD d1q281 (2) NV3 (7)

15. Next chemotherapy N% No. total patients43100 No. of evaluable patients266 Median PFS (month) Range Progression event 4 (1-14) 25

16. Randomized phase III trial on Trabectedin (ET 743) vs clinician’s choice chemotherapy in recurrent ovarian, primary peritoneal or fallopian tube cancers of BRCA mutated or BRCAness phenotype patients MITO – 23

17. MITO - 23 MITO-23: TRANSLATIONAL STUDIES DNA sequencing in order to evaluate mutation/genetic aberration profile in selected panels of genes associated to tumor sensitivity to trabectedin. Evaluating the impact of altered gene and microRNA (miRNA) expression on trabectedin efficacy with the aim of identify which miRNAs/genes are involved in the so called BRCAness phenotype; Analysis of cellular infiltrate present on tumor specimens of patients treated with trabectedin;

18. MITO - 23 TRANSLATIONAL STUDIES: Specimens Tumor histological blocks (FFPE material): samples will be collected at primary surgery and/or surgery nearest to trabectedin treatment (or before trabectedin treatment by dedicated biopsies). Storage and analysis will be centralized at Fondazione Istituto Nazionale dei Tumori. Characterization of tumor infiltrate by IHC (2 slides per marker & 1 HE slide & 3 backup slides); Extraction of RNA from tissue sections for miRNA/gene expression; Extraction of DNA from tissue sections for targeted sequencing of “Panel cancer related genes”. Blood samples: Blood samples will be collected at baseline (registration), at third cycle of treatment and at progression. Storage and analyses will be centralized at Istituto Mario Negri, Milan. 10 ml blood sample will be taken at each time point, centrifuged in EDTA, processed to obtain 5 ml plasma collected and stored at -20°C or at -80°C when possible.