1. Evaristo Maiello U.O.C. Oncologia IRCCS Casa Sollievo della Sofferenza San Giovanni Rotondo (FG) Napoli, 16 ottobre 2015

2. Molecular alterations in cancer cells offer potential for therapeutic intervention with "target-based agents" Hanahan & Weinberg Cell 2011

3. Meric-Bernstam JCO 2013 Target-based agents + predictive biomarkers: PERSONALIZED MEDICINE

4. Predictive biomarkers and personalized medicine Biomarkers that are associated with response to drugs (positive selection) – EGFR mutations in NSCLC – BRAF mutations in melanoma – ERBB2 gene amplification in breast/gastric cancer Biomarkers that are associated with resistance to drugs (negative selection) – RAS mutations and resistance to EGFR monoclonal antibosies in CRC

6. Oncogene-addicted vs NO BIOMARKERS FRANCE: driver mutations in 10,000 patients with non-squamous NSCLC Lung Cancer Mutation Consortium USA

7. Oncogene-addicted vs NO BIOMARKERS FRANCE: driver mutations in 10,000 patients with non-squamous NSCLC Lung Cancer Mutation Consortium USA

8. Kris M et al JAMA. 2014;311(19):1998-2006 Survival ComparisonsALK indicates anaplastic lymphoma kinase gene; EGFR(s), epidermal growth factor receptor gene (sensitizing); EGFR(o), epidermal growth factor receptor gene (other); KRAS, Kirsten rat sarcoma; NA, not applicable.A, Median survival (95% CI): oncogenic driver + no targeted therapy, 2.38 (1.81-2.93); oncogenic driver + targeted therapy, 3.49 (3.02-4.33); no oncogenic driver, 2.08 (1.84-2.46). B, Survival by oncogenic driver detected for patients with the 5 most frequent oncogenic drivers and targeted treatment. Median survival (95% CI): EGFR(s), 3.78 (2.77-NA); EGFR(o), 2.70 (1.42-NA); ALK, NA (2.80-NA); KRAS, 4.85 (1.30-NA); doubletons (oncogenic drivers in 2 genes), 2.69 (1.94-NA). Vertical tick marks are censoring events. Figure Legend : Using Multiplexed Assays of Oncogenic Drivers in Lung Cancers to Select Targeted Drugs It is worthwhile finding an actionable genetic alteration in Lung Cancer Schiller et al, NEJM 2002 ECOG 1594

9. Kamalakaran S. et al, 2015

13. Heinemann V et al. ASCO 2013 (Abstract No. LBA3506); Stintzing S et al. ECC 2013 (Abstract No. LBA17) KRAS wt (exon 2) population RAS wt population (KRAS and NRAS wt) ~85% of KRAS wt (exon 2) population 0.75 1.0 0.50 0.25 0.0 Probability of survival 12 24 36 4860 72 Months Δ=3.7 months 0 Bevacizumab + FOLFIRI (n=295) HR=0.77 p=0.017 28.7 25.0 Cetuximab + FOLFIRI (n=297) 0.0 0.75 1.0 0.50 0.25 Δ = 7.5 months 12 24 36 48 60 72 Months 0 Probability of survival Bevacizumab + FOLFIRI (n=171) HR=0.70 p=0.011 33.1 25.6 Cetuximab + FOLFIRI (n=171) 15% Fire3 – Expanded RAS analysis

14. Ciardiello F et al. Annals of Oncology 2014

18. Comparison of methodologies StudyMethodSensitivity* FIRE-3 1 Pyrosequencing≤5% 2 OPUS 3 Inostics BEAMing technology (detection cut-off 0.1%) 0.01% 4 CAPRI 5 Next-generation sequencing: Ion AmpliSeq™ Colon and Lung Cancer Panel 2% 5 PRIME 6 Bidirectional Sanger sequencing and WAVE-based SURVEYOR ® Scan Kits (Transgenomic) 10−20% (Sanger sequencing) 8 1% (WAVE-based SURVEYOR®) 9 PEAK 7 20020408 8 Next-generation sequencing, Sanger sequencing, and independently conducted WAVE-based SURVEYOR ® Scan Kits (Transgenomic) 10–20% (Sanger sequencing) 8 De Roock et al 10 Sequenom MALDI-TOF MassARRAY multiplex PCR and genotyping 5–15% 10 1. Stintzing S, et al. ECC 2013 (Abstract No. LBA17); 2. Anderson SM. Expert Rev Mol Diagn 2011;11:635–642; 3. Data on file; 4. Aung KL, et al. Hugo J 2010;4:11–21; 5. Ciardiello F, et al. ECC 2013 (Abstract No. LBA31); 6. Douillard J-Y, et al. N Engl J Med 2013;369:1023–1034; 7. Karthaus M, et al. ECC 2013 (Abstract No. 2262); 8. Peeters M, et al. WCGC 2013 (Abstract No. PD-0008); 9. Jänne PA et al. Clin Cancer Res 2006;12:751–758; 10. De Roock W, et al. Lancet Oncol 2010;11:753–762 *Values refer to the lowest percentage of mt sequence that is detectable

20. Multiple gene mutations in KRAS mutated tumors Multiple gene mutations in PIK3CA mutated tumors Type of mutationsn n KRAS only (no other mutation detected)15PIK3CA only (no other mutation detected)5 KRAS + TP538PIK3CA + KRAS5 KRAS + PIK3CA ex93PIK3CA + BRAF1 KRAS + FBXW72PIK3CA + TP532 KRAS + PIK3CA ex202PIK3CA + NRAS1 KRAS + FGFR31PIK3CA + MET1 KRAS + PTEN1PIK3CA + KRAS + TP533 KRAS + MET1PIK3CA + FBXW7 + TP531 KRAS + SMAD41PIK3CA + KRAS + ERBB2 + TP531 KRAS + PIK3CA ex9 + BRAF + TP531PIK3CA + KRAS + BRAF + TP532 KRAS + PIK3CA ex9 + TP532PIK3CA + KRAS + EGFR + TP531 KRAS + FBXW7 + TP532PIK3CA + KRAS + BRAF + FBXW71 KRAS + EGFR + PIK3CA ex9 + TP531Total PIK3CA mutations24 KRAS + PIK3CA ex20 + BRAF + TP531 Two tumors had two concomitant PIK3CA mutations in both exon 9 and exon 20 KRAS + PIK3CA ex20 + TP531 KRAS + PIK3CA ex20 + BRAF + FBXW71 KRAS + BRAF + TP531 KRAS + PIK3CA ex9 + PIK3CA ex20 + ERBB2 + TP531 Total KRAS mutations45 Gene mutations are not mutually exclusive in mCRC treated with FOLFIRI + cetuximab One tumor had two concomitant KRAS mutations

21. Multiple gene mutations in BRAF mutated tumors Multiple gene mutations in NRAS mutated tumors Type of mutationsn n BRAF only (no other mutation detected)3NRAS only (no other mutation detected)8 BRAF + TP535NRAS + TP533 BRAF + PIK3CA (ex20)1NRAS + MET1 BRAF + FBXW71NRAS + PIK3CA ex91 BRAF + KRAS + TP531Total NRAS mutations13 BRAF + KRAS + PIK3CA (ex9) + TP531 BRAF + KRAS + PIK3CA (ex20) + TP531 BRAF + SMAD4 + FGFR2 + TP531 BRAF + KRAS + PIK3CA (ex20) + FBXW71 Total BRAF mutations15 Gene mutations are not mutually exclusive in mCRC treated with FOLFIRI + cetuximab

22. Mutated cases (N=182 analyzed) KRASNRAS BRAF PIK3CA ex9 PIK3CA ex20 MET EGFR SMAD4 CTNNB1 FGFR3 PTEN ERBB2 FGFR2 FBXW7 TP53 KRAS (30/45)* 495111111518 NRAS (5/13)* 113 BRAF (12/15)* 4 1311129 PIK3CA ex9 (14/16)* 911 21118 PIK3CA ex20 (7/10)* 532 123 MET (4/7)* 111 1 EGFR (1/2)* 111 SMAD4 (2/2)* 11 11 CTNNB1 (2/2)* 1 FGFR3 (2/2)* 11 1 PTEN (1/1)* 1 ERBB2 (1/1)* 111 1 FGFR2 (1/1)* 11 FBXW7 (9/9)* 522 5 TP53 (36/72)* 1839831111115 *cases with multiple mutations/total mutated cases 22 multiple gene mutation analysis in mCRC treated with FOLFIRI + cetuximab

25. Clinical activity of FOLFIRI + cetuximab Total patients (KRAS exon 2 wt, n=340) Patients with 22 multiple gene mutation analysis (n=182) Complete response, %26/340 (7.6%)12/182 (6.6%) Partial response, %166/340 (48.8%)92/182 (50.5%) Stable disease, %115/340 (33.8%)61/182 (33.5%) Progressive disease, %33/340 (9.7%)17/182 (9.3%) ORR, %192/340 (56.4%)104/182 (57.1%) Median PFS, months (95% CI) 9.9 (8.8–11.3) 9.8 (8.7–11.5) Clinical activity of FOLFIRI + cetuximab KRAS/NRAS wt (n=124)KRAS/NRAS mt (n=58) Complete response, % 8/124 (6.4%)4/58 (6.9%) Partial response, % 69/124 (55.6%)23/58 (39.7%) Stable disease, % 35/124 (28.2%)26/58 (44.8%) Progressive disease, % 12/124 (9.7%)5/58 (8.6%) ORR, % 77/124 (62%)27/58 (46.6%) Median PFS, months (95% CI) 11.1 (9.2–12.8) 8.9 (7.4–9.6) Clinical activity of FOLFIRI + cetuximab KRAS/RAS/ BRAF/PIK3CA wt (n=104) KRAS/NRAS/ BRAF/PIK3CA mt (n=78) Complete response, % 8/104 (7.7%)4/78 (5.1%) Partial response, % 59/104 (56.7%)33/78 (42.3%) Stable disease, % 28/104 (26.9%)33/78 (42.3%) Progressive disease, % 9/104 (8.6%)8/78 (10.3%) ORR, % 67/104 (64.4%)37/78 (47.4%) Median PFS, months (95% CI) 11.3 (9.4–13.2) 7.7 (5.4–9.4) A B C

26. Conclusions Results confirm the activity of cetuximab + FOLFIRI in 1 st line treatment of mCRC: ORR, 56.4%; mPFS, 9.9 months. A 22 multiple gene mutation analysis using the next generation sequencing Ion AmpliSeq™ Colon and Lung Cancer Panel was feasible on FFPE tumor tissues Increased clinical activity of FOLFIRI + cetuximab was observed in mCRC patients whose tumors were wild type for both KRAS and NRAS genes: –KRAS, NRAS wild type:ORR, 62.0%; mPFS, 11.1 months. –KRAS or NRAS mutant:ORR, 46.6%; mPFS, 8.9 months. Similar differences in clinical activity were observed in mCRC patients whose tumors were wild type for KRAS, NRAS, BRAF and PIK3CA genes: –KRAS, NRAS, BRAF, and PIK3CA wild type: ORR 64.4%; mPFS 11.3 months. –KRAS, NRAS, BRAF or PIK3CA mutant: ORR, 47.4%; mPFS, 7.7 months CAPRI GOIM trial

27. Cetuximab beyond progression in RAS wild type (WT) metastatic colorectal cancer (mCRC): The CAPRI-GOIM randomized Phase II study of FOLFOX versus FOLFOX plus cetuximab Fortunato Ciardiello*, Nicola Normanno, Erika Martinelli, Teresa Troiani, Claudia Cardone, Anna Nappi, Anna Maria Rachiglio, Matilde Lambiase, Salvatore Pisconti, Francesco Giuliani, Carlo Barone, Maria Biglietto, Vincenzo Montesarchio, Giuseppe Tonini, Daniele Rizzi, Saverio Cinieri, Roberto Bordonaro, Antonio Febbraro, Ferdinando De Vita, Michele Orditura, Giuseppe Colucci, and Evaristo Maiello on the behalf of the CAPRI-GOIM Investigators * Second University of Naples, Italy

28. 153 patients randomized to 154 patients a) no second line for R0 operated LLD (20) b) lost to follow-up (32) c) refusal (19) d) other 2nd line therapies (68) e) KRAS or NRAS mutant after November 2013 (15) 340 patients (KRAS exon 2 wt according to local pathology lab assessment) FOLFIRI + cetuximab 307 patients CR/PR/SD at progression FOLFOX (Arm B) 79 patients FOLFOX + cetuximab (Arm A) 74 patients 33 patients PD

29. Progression free survival* (n=153 [ITT]) (KRAS exon 2 wild type patients by local laboratory assessment) 3232 N at Risk: Arm A Arm B 3232 0000 2222 74 79 7575 42 36 20 14 0101 Months Progression-Free Survival (PFS) Arm B (FOLFOX) Arm A (FOLFOX + cetuximab) FOLFOX + cetuximab (Arm A) (n=74) FOLFOX (Arm B) (n=79) Median PFS, months6.44.5 HR (95% CI)0.81 (0.58–1.12) p=0.19 *From randomization at start of 2nd line treatment

30. Tumor responses: FOLFOX + cetuximab (Arm A) (n=74) FOLFOX (Arm B) (n=79) Complete response, n (%)2 (2.7)1 (1.3) Partial response, n (%)14 (18.9)9 (11.4) Stable disease, n (%)32 (43.2)37 (46.8) Progressive disease, n (%)22 (29.7)27 (34.2) Not evaluable, n (%)4 (5.4)5 (6.3) Response rate (n=153 [ITT]) (KRAS exon 2 wild type patients by local laboratory assessment)

31. Overall survival* (n=153 [ITT]) (KRAS exon 2 wild type patients by local laboratory assessment) 3232 N at Risk: Arm A Arm B 7676 0000 3535 2222 47 74 79 19 14 67 64 34 27 1010 Overall Survival (OS) FOLFOX + cetuximab (Arm A) (n=74) FOLFOX (Arm B) (n=79) Median OS, months17.614.0 HR (95% CI)0.86 (0.61–1.2) p=0.41 Arm B (FOLFOX) Arm A (FOLFOX + cetuximab) Months *From randomization at start of 2nd line treatment

32. Mutations in KRAS, NRAS, BRAF and PIK3CA genes as detected by next generation sequencing 1,2 (n=117) 1. Ciardiello F et al., Annals of Oncology 25:1756-61, 2014; 2. Normanno N et al., Annals of Oncology, 2015 in press *Analysis of single tumor multiple gene mutations, as detected by NGS, has been previously reported 1, 2. **19/117 (16.2%) cases had a KRAS exon 2 mutation, but were originally classified as KRAS exon 2 wild type upon local laboratory assessment and, therefore, were treated with FOLFIRI plus cetuximab in 1st line Gene: Number of cases with at least one mutation*, n (%) (n=51/117, 43.6%) KRAS32 (27.4)19 at exon 2 (16.2)**;13 at exons 3 or 4 (11.1) PIK3CA2 (1.8)1 at exon 9 (0.9); 1 at exon 20 (0.9) BRAF7 (6.0)6 at codon 600 (5.1); 1 at other (0.9) NRAS10 (8.5)10 at exons 2 or 3 (8.5)

33. Progression free survival* according to KRAS/NRAS (n=117) 2121 N at Risk: Arm A Arm B 2121 0000 2121 39 36 5353 24 20 11 8 0101 Months Progression-Free Survival (PFS) 0101 N at Risk: Arm A Arm B 0101 0000 0101 14 28 0202 4 11 1515 0000 Months Progression-Free Survival (PFS) KRAS/NRAS mutated (n=42) Arm A (n=14)Arm B (n=28) Median PFS, months2.74.1 HR (95% CI)1.53 (0.78–2.96) p=0.2 KRAS/NRAS wild type (n=75) Arm A (n=39)Arm B (n=36) Median PFS, months6.85.5 HR (95% CI)0.80 (0.50–1.29) p=0.4 Arm B (FOLFOX) Arm A (FOLFOX + cetuximab) Arm B (FOLFOX) Arm A (FOLFOX + cetuximab) *From randomization at start of 2nd line treatment

34. Tumor responses in KRAS/NRAS wt patients: FOLFOX+cetuximab (Arm A) (n=39) FOLFOX (Arm B) (n=36) Complete response, n (%)1 (2.6)1 (2.8) Partial response, n (%)9 (23.1)5 (13.9) Stable disease, n (%)18 (46.2)19 (52.8) Progressive disease, n (%)9 (23.1)10 (27.8) Not evaluable, n (%)2 (5.1)1 (2.8) Response rates according to KRAS/NRAS (n=117) Tumor responses in KRAS/NRAS mt patients: FOLFOX+cetuximab (Arm A) (n=14) FOLFOX (Arm B) (n=28) Complete response, n (%)00 Partial response, n (%)1 (7.4)4 (14.3) Stable disease, n (%)4 (28.6)11 (39.3) Progressive disease, n (%)7 (50.0)11 (39.3) Not evaluable, n (%)2 (14.2)2 (7.1)

35. Overall survival* according to KRAS/NRAS (n=117) 5454 N at Risk: Arm A Arm B 16 8 1010 2222 39 36 23 18 37 28 24 1010 Months Overall Survival (OS) 0202 N at Risk: Arm A Arm B 1414 0000 0202 14 28 4646 11 23 5 16 0000 Months Overall Survival (OS) 2121 2121 KRAS/NRAS mt (n=42) Arm A (n=14)Arm B (n=28) Median OS, months11.612.7 HR (95% CI)1.45 (0.74–2.83) p=0.27 KRAS/NRAS wt (n=75) Arm A (n=39)Arm B (n=36) Median OS, months21.419.8 HR (95% CI)0.78 (0.46–1.32) p=0.35 Arm B (FOLFOX) Arm A (FOLFOX + cetuximab) Arm B (FOLFOX) Arm A (FOLFOX + cetuximab) *From randomization at start of 2nd line treatment

36. Progression free survival* according to KRAS/NRAS/BRAF/PIK3CA (n=117) 2020 N at Risk: Arm A Arm B 2020 0000 2020 34 32 4141 23 17 10 5 0000 Months Progression-Free Survival (PFS) 0202 N at Risk: Arm A Arm B 0202 0000 0202 19 32 1414 6 14 2828 0101 Months Progression-Free Survival (PFS) KRAS/NRAS/BRAF/PIK3CA mt (n=51) Arm A (n=19)Arm B (n=32) Median PFS, months2.74.4 HR (95% CI)1.70 (0.94–3.05) p=0.07 KRAS/NRAS/BRAF/PIK3CA wt (n=66) Arm A (n=34)Arm B (n=32) Median PFS, months6.95.3 HR (95% CI)0.56 (0.33–0.94) p=0.025 *From randomization at start of 2nd line treatment

37. Tumor responses in KRAS/NRAS/BRAF/PIK3CA wt patients: FOLFOX+cetuximab (Arm A) (n=34)FOLFOX (Arm B) (n=32) Complete response, n (%)1 (2.9)0 Partial response, n (%)9 (26.5)3 (9.4) Stable disease, n (%)16 (47.1)19 (59.4) Progressive disease, n (%)6 (17.6)9 (28.1) Not evaluable, n (%)2 (5.9)1 (3.1) Response rates according to KRAS/NRAS/BRAF/PIK3CA (n=117) Tumor responses in KRAS/NRAS/BRAF/PIK3CA mutant patients: FOLFOX+cetuximab (Arm A) (n=19)FOLFOX (Arm B) (n=32) Complete response, n (%)01 (3.1) Partial response, n (%)06 (18.8) Stable disease, n (%)7 (36.8)12 (37.5) Progressive disease, n (%)10 (52.6)11 (34.4) Not evaluable, n (%)2 (10.5)2 (6.2)

38. Overall survival* according to KRAS/NRAS/BRAF/PIK3CA (n=117) 5151 N at Risk: Arm A Arm B 15 6 0000 2121 34 32 22 15 32 25 26 21 1010 Months Overall Survival (OS) 0303 N at Risk: Arm A Arm B 0404 0000 0101 19 32 5959 16 27 7 19 0000 Months Overall Survival (OS) 2020 2020 1616 0000 KRAS/NRAS/BRAF/PIK3CA mt (n=51) Arm A (n=19)Arm B (n=32) Median OS, months11.614.0 HR (95% CI)1.60 (0.89–2.96) p=0.10 KRAS/NRAS/BRAF/PIK3CA wt (n=66) Arm A (n=34)Arm B (n=32) Median OS, months23.719.8 HR (95% CI)0.57 (0.32–1.02) p=0.056 Arm B (FOLFOX) Arm A (FOLFOX + cetuximab) Arm B (FOLFOX) Arm A (FOLFOX + cetuximab) *From randomization at start of 2nd line treatment

39. ●CAPRI-GOIM is an academic non-profit, multicenter, randomized Phase II study in which 153 patients with KRAS exon 2 wt mCRC (by local pathology lab assessment), who initially responded to 1st line therapy with FOLFIRI plus cetuximab, were treated in 2nd line with FOLFOX plus cetuximab (Arm A; n=74) or FOLFOX (Arm B; n=79) ●In the ITT patient population a non-significant difference was observed for both PFS and OS: Median PFS Arm A: 6.4 months Arm B: 4.5 months Median OS Arm A: 17.6 months Arm B: 14.0 months Conclusions (1) HR=0.81 (95% CI 0.58–1.12); p=0.19 HR=0.86 (95% CI 0.61–1.22); p=0.41

40. Conclusions (2) ●In 117/153 (76.5%) cases, KRAS, NRAS, BRAF and PIK3CA mutations were centrally assessed by NGS ●In 66/117 cases, tumors had no mutations in KRAS, NRAS, BRAF and PIK3CA genes, whereas in 51/117 cases a mutation in at least one of these genes was found ●19/117 (16.2%) tumors, that were originally classified as wt, had a KRAS exon 2 mutation

41. Median PFS Arm A: 2.7 months Arm B: 4.4 months Median OS Arm A: 11.6 months Arm B: 14.0 months Median PFS Arm A: 6.9 months Arm B: 5.3 months Median OS Arm A: 23.7 months Arm B: 19.8 months ●A significant improvement in PFS with a similar trend in OS was observed in the FOLFOX plus cetuximab arm in patients with KRAS, NRAS, BRAF and PIK3CA wild type tumors, whereas a detrimental effect was observed in patients whose tumors were mutated for at least one of these genes ●KRAS, NRAS, BRAF, PIK3CA wild type ●KRAS, NRAS, BRAF, PIK3CA mutated Conclusions (3) HR=0.56 (95% CI 0.33–0.94), p=0.025 HR=0.57 (95% CI 0.32–1.02), p=0.056 HR=1.70 (95% CI 0.94–3.05), p=0.07 HR=1.60 (95% CI 0.89–2.96), p=0.10 Continuing EGFR inhibition with cetuximab and switching chemotherapy backbone is a potential therapeutic strategy that deserves further evaluation in a randomized Phase III study in mCRC patients with an EGFR-dependent cancer

42. Dubbink HJ et al, Neuro-Oncology 2015

44. Molecular Pathological

45. Dubbink HJ et al, Neuro-Oncology 2015

46. NGS “FINESTRA SULLA COMPLESSITA’ “