Lucio Crinò, MD Silvestrini Hospital Perugia, Italy ALK INHIBITORS IN LUNG CANCER THERAPY

2 Identification of Aberrant Forms of the Anaplastic Lymphoma Kinase Expressed in ALCL with t(2;5) chromosome rearrangement resulting in a fusion protein of two genes: the novel tyrosine kinase gene (ALK) and NPM 1 Other chromosome translocations involving the ALK locus have also been identified in several different human cancers 2,3,4 Detection of phosphoprotein in an ALCL cell line in SCID mice compared with controls kDa 1 Shiota M & Mori S. Leuk Lymphoma. 1996;23:25  Pulford K, et al. J Cell Physiol. 2004;199:330  Palmer, et al. Biochem J. 2009;420:345–61. 4 Mano. Cancer Sci. 2008;99:2349–55. ALCL, anaplastic large-cell lymphoma; NPM, nucleophosmin; SCID, severe combined immunodeficiency.

3 Identification of the EML4-ALK Fusion in NSCLC Soda M, et al. Nature. 2007;448:561 – 67. ~3.6 kb EML4 EML4–ALK variant 1 HELP WDBasic TM EML4-ALK variant 1 Exon 13 ALKEML4 297 bp Exon 21 Kinase ALK EML, echinoderm microtubule-associated protein-like 4; HELP, hydrophobic echinoderm mizroxbule-associated protein-like protein

4 ALK Pathway InversionTranslocation Or ALK Partner gene product ALK fusion protein* Tumour cell proliferation Cell survival PI3K BAD AKT STAT3/5 mTOR S6K RAS MEK ErK PLC- Y PIP 2 IP 3 1 Inamura K, et al. J Thorac Oncol. 2008;3:13–17. 2 Soda M, et al. Proc Natl Acad Sci. U S A. 2008;105:19893–97. Figure based on: Chiarle R, et al. Nat Rev Cancer. 2008;8(1):11–23. Mossé YP, et al. Clin Cancer Res. 2009;15(18):5609–14; and Pfizer Inc, data on file. *Subcellular localisation of the ALK fusion gene, while likely to occur in the cytoplasm, is not confirmed. 1,2 BAD, BCL2-associated agonist of death; STAT3, signal transducer and activator of transcription 3; S6K, ribosome protein S6 kinase; ERK, extracellular signal-regulated kinase. See alternative slide in back-up (slide 28)

5 1 Soda M, et al. Nature. 2007;448:561–67. 2 Zhang, et al. Mol Cancer. 2010;9:188. EML4–ALK Is a Potent Oncogenic Driver 1 Other fusion partners for ALK have also been identified, including NPM, EML4, TPM3, ATIC, TFG, CARS, and CLTC 2 3T3 Nude mice tumour/ injection0/80/80/88/80/88/82/2 VectorEML4ALK EML4–ALKK589MNPM–ALKv-Ras TPM3, tropomyosin 3; ATIC, aminoimidazole-4-carboxamide ribonucleotide formyltransferase 11MP cyclohydrolase; TFG, TRK-fused gene; CARS, cysteinyl-tRNA synthetase; CLTC, clathrin, heavy chain.

6 ALK Fusion Prevalence in NSCLC Retrospective Data Prospective Data Lung Cancer Mutation Consortium 9 Adenocarcinoma 1 Takahashi, et al Wong, et al Perner, et al Paik, et al Boland, et al Paik, et al Takahashi, et al Rodig, et al Kris, et al. Presented at ASCO Abstract CRA7506. RT-PCRFISHIHC % ALK+ patients Unselected 1.6% % 2 2.7% % 4 1.7% % 6 % ALK+ patients Adenocarcinoma 2.4% % 2 5.6% 8 2.7% 5 RT-PCR, reverse transcription-polymerase chain reaction; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry.

UNIVERSTY OF TORINO – DEPT. OF CLINICAL & BIOLOGICAL SCIENCES Status of Actionable Driver Mutations in Lung Adenocarcinoma Tumor Specimens Johnson D, et al. ECCO ESMO Abstract No mutation detected KRAS (22%) EGFR (18%) EML4-ALK (7%) Double mutants (2%) BRAF (2%) AKT1 NRAS<1% MEK1<1% MET AMP<1% HER2 1% PIK3CA 1% RT-PCRFISHIHC % ALK+ patients Unselected 1.6% % 2 2.7% % 4 1.7% % 6 % ALK+ patients Adenocarcinoma 2.4% % 2 5.6% 8 2.7% 5 1 Takahashi, et al Wong, et al Perner, et al Paik, et al Boland, et al Paik, et al Takahashi, et al Rodig, et al Alk Fusion Prevalence in NSCLC: Retrospective Data

8 Patients With ALK Fusion Represent a Specific Molecular Subset of Adenocarcinoma Patients with the ALK fusion gene may not benefit from agents such as EGFR TKIs ALK-positive patients display similar sensitivity to platinum-based chemotherapy compared with ALK- negative patients 1 1 Shaw AT, et al. J Clin Oncol. 2009;27:4247 ‒ 53. TTP on EGFR-TKI monotherapy ALK may predict sensitivity to EML4–ALK inhibition but resistance to EGFR TKIs Patients with ALK-positive disease (n=15): 5 months Patients with EGFR-positive disease (n=25): 16 months Patients with EGFR WT/WT disease (n=49): 6 months Time (months) Percent progression free EGFR WT/WT EML4-ALK Percent progression free EGFR WT/WT EML4-ALK P =.004 (ALK vs EGFR) Time (months) TTP on platinum-based chemotherapy

9 Clinical Impact of ALK Rearrangement: A Single Center, Retrospective, Case-match Study 1100 patient cases with lung cancer of non-squamous histology were collected in the NSCLC database of Seoul National University Hospital 257 samples which were EGFR wild type or unresponsive to prior EGFR TKI therapy underwent FISH for ALK testing Each patient with an ALK-positive tumour was matched to:  Two patients with EGFR-mutation positive tumours and  Two patients with ALK WT/EGFR WT tumours (WT/WT) Matching variables included: age at diagnosis, sex, and stage of disease (IIIB or IV) Kim DW, et al. Presented at ASCO 2011; Abstract 7515.

10 Overall Survival and Progression-Free Survival by Genetic Characteristics Kim DW, et al. Presented at ASCO 2011; Abstract Early investigations do not support ALK rearrangement as a favourable prognostic factor ALK-positive patients might be more resistant to EGFR TKI treatment compared with WT/WT patients Patients who had received an ALK inhibitor were not included in this study ALK+ (N=22) EGFR mut+ (N=44) WT/WT (N=44) ALK+ (N=10) EGFR mut+ (N=40) WT/WT (N=24) *Excludes ALK-positive patients enrolled due to previous non- response to EGFR TKIs Months Months OS (%) PFS (%)* PFS of EGFR-TKI treated patientsOS

11 Status of ALK Inhibitors in Clinical Development

12 Kinase IC50 (nM) mean* Selectivity ratio c-Met8– ALK202X RON 29834X 18922X Axl 29434X 32237X Tie244852X Trk A58067X Trk B39946X Abl1,159166X IRK2,887334X Lck2,741283X Sky>10,000>1000X VEGFR2>10,000>1000X PDGFRβ>10,000>1000X Findings for crizotinib High probability of ALK and c-Met inhibition at clinically relevant doses Low probability of relevant inhibition of RON, Axl, Tie2, or Trk at clinical dose levels Cellular selectivity on 10 of 13 relevant hits Upstate 102 kinase panel *Measured using ELISA capture method Crizotinib: A Selective Inhibitor of ALK and c-MET 13 ‘hits’ <100X selective for c-Met Pfizer Inc, data on file. Crizotinib (PF )

13 Part 2: Molecularly defined cohorts Part 1: Dose escalation (n=37) Crizotinib: First-in-human/Patient Trial (A ) Modified from: Tan, et al. J Clin Oncol. 2010;28:15S. Abstract Cohort 6 (n=9) 250 mg BID MTD/RP2D BID, twice daily; QD, once daily; MTD, maximum tolerated dose; RP2D, randomised phase 2 dose. Cohort 1 (n=3) 50 mg QD Cohort 2 (n=4) 100 mg QD Cohort 3 (n=8) 200 mg QD Cohort 4 (n=7) 200 mg BID Cohort 5 (n=6) 300 mg BID

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20 A : Rapid Responses Reported Day 7Day 14 Ou, et al. J Thorac Oncol. 2010;5(12):2044–46. Symptoms at study entryImprovement in symptoms CoughSignificant improvement at day 3, completely resolved by week 2 Daily low-grade feversResolved by day 3 AnorexiaGained 1.5 kg of weight by week 2 Right neck pain due to tumour invasionResolved by day 3

21 A : Progression-Free Survival (N=119) Censored 95% Hall-Wellner Band Survival distribution function Months n at risk Median PFS=10.0 months (95% CI: 8.2, 14.7) 50 events (42%; 40 PD events) 69 patients (58%) censored, 59/69 (86%) in follow-up for PFS Camidge DR, et al. Presented at ASCO 2011; Abstract 2501.

22 A : Overall Survival Median OS had not been reached as of the data cut-off >23 deaths (19%) >2 patients (2%) censored (lost to follow-up) >94 patients (79%) remain in follow-up for OS  No deaths were related to study treatment Survival probabilities from first dose of crizotinib:  6 months: 90% (95% CI: 82.7, 94.4)  12 months: 81% (95% CI: 70.9, 87.2) Camidge DR, et al. Presented at ASCO 2011; Abstract 2501.

23 Impact of ALK Inhibition on Overall Survival of Patients With Advanced, ALK–Positive NSCLC Study background and rationale  The true impact of crizotinib on OS may be difficult to establish in ongoing randomised phase 3 studies due to crossover  In the absence of randomised data, determination of survival benefit requires a comparator population of ALK-positive, crizotinib-naïve patients Study objectives  Examine OS of crizotinib-treated ALK-positive NSCLC patients  Compare the survival outcomes of crizotinib-treated vs crizotinib-naïve, ALK-positive NSCLC patients  Explore the prognostic significance of ALK rearrangement by comparing the survival outcomes of crizotinib-naïve ALK-positive and ALK-negative NSCLC patients Shaw AT, et al. Presented at ASCO 2011; Abstract 7507.

24 Study Populations ALK CONTROLS US/Australia N=36 2 nd line N=23 Never/light smoker AdenoCA N=21 ALK-positive Crizotinib-naïve ALK CRIZOTINIB ALK-positive Crizotinib-treated N=82 US/Australia N=56 2 nd /3 rd line N=30 WT/WT CONTROLS US (MGH) N=253 2 nd line N=125 ALK-negative EGFR-wild type Never/light smoker AdenoCA N=28 Shaw AT, et al. Presented at ASCO 2011; Abstract Never/light smoker AdenoCA N=48

25 Overall Survival: 2 nd Line Subset ALK Crizotinib (n=30) ALK Control (n=23) Median Survival, mo NR 6 1-yr Survival, % WT/WT Control (n=125) % 20% 40% 60% 80% 100% 1234 From 2 nd /3rd line crizotinib 2-yr Survival, % HR=0.49, P=.02 Years Shaw AT, et al. Presented at ASCO 2011; Abstract 7507.

26 Conclusions Targeting the ALK fusion gene in NSCLC, a direct driver of oncogenesis, has resulted in promising clinical response rates and PFS in patients with advanced NSCLC treated with crizotinib  ORR: 61%  Median PFS: 10 months The most frequent adverse events observed with crizotinib were mild and moderate gastrointestinal events and mild visual disturbances Overall survival of patients with advanced, ALK-positive NSCLC treated with 2 nd -/3 rd - line crizotinib is significantly longer than that of clinically comparable, crizotinib-naïve controls Safety and efficacy of a molecularly targeted agent may be assessed in molecularly defined cohorts directly following traditional phase 1 dose escalation  US FDA has approved crizotinib for ALK-positive NSCLC based on data from this study  Crizotinib has also been successfully filed with the EMA These results are an example of rapid clinical development, from target identification to clinical validation, and support a personalised approach to NSCLC treatment