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Targeting ALK Signaling in NSCLC

Tianhong (Tina) Li, MD/PhD August 14, 2011

UC Davis Cancer Center Sacramento, CA

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Disclosure

• No conflict of interest in this presentation.

Acknowledgement

• Drs. D. Ross Camidge, Fred Hirsch, Marileila

Varella Carcia, Suresh Ramalingam, Robert Sweetman for providing their slides.

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Outlines: ALK Inhibitors in NSCLC

• A case study
• Update on the results from PROFILE 1001

and 1005 studies

• Unmet needs in clinical use of ALK inhibitors
• New insights in understanding the ALK

signaling pathway and resistance mechanisms

• Strategies to overcome the resistances to

first-generation ALK inhibitor crizotinib

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A 56-year-old female, never-smoker

• A 56-year-old Caucasian female, never-smoker, who initially

presented with persistent cough in August 2006.

• Workups revealed multiple lung masses with liver and bony
• metastases. Core needle biopsy of a liver mass revealed lung

adenocarcinoma.

• Summary of prior cancer treatment: Carbo/pac x 4, PD;

erlotinib x <1 mo, intolerability; bevacizumab and vinorelbine x 3, PD; nab paclitaxel x2, PD; pac weekly x 20 (5 mos.), PD; Pem 16 mos, PD; gem/docetaxel x 5 mos, PD; erlotinib 50 mg x 2 mos, PD; RT to right iliac bone (wheel chair-bound); Pem/Cis/Bev x3 mos then Pem/Bev x 5 mos, PD.

• July 15, 2010: Liver biopsy (initial diagnosis): EML4-ALK

Break-Apart FISH positive (90% of cells).

• Enrolled in PROFILE 1005, which is a phase II study of

crizotinib monotherapy in refractory, metastatic NSCLC

• patients. She started the treatment on 12/1/2010.

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Crizotinib monotherapy: 250 mg po bid

• Patient reports significant decrease in

nonproductive cough within one week after starting crizotinib. Right hip pain has also decreased and controlled.

• One level dose reduction of crizotinib at Cycle 3

for grade 2 peripheral neuropathy, which was resolved with dose reduction; no worsening symptoms since resuming original level dose of crizotinib since cycle 10. Grade 1 vision changes has been stable.

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Baseline CT Scan Crizotinib: Post 2 cycles

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Baseline CT Scan Crizotinib: Post 2 cycles

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Echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase (EML4-ALK) fusion oncogenes in NSCLC

Soda et al., Nature 448: 561-566, 2007 3T3 Nude mice Tumor/ injection 0/8 0/8 0/8 8/8 0/8 8/8 2/2 Vector EML4 ALK EML4–ALK K589M NPM–ALK v-Ras

ALK

Y Y P P Y Y P P TM TM Y Y P P Y Y P P Y Y P P Y Y P P TM TM

Extracellular Intracellular

Y Y P P Y Y P P

Kinase

Y Y P P Y Y P P Y Y P P Y Y P P Y Y P P Y Y P P Y Y P P Y Y P P

Kinase

EML4-ALK

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Cellular selectivity on 10 of 13 relevant hits Upstate 102 kinase 13 kinase “hits” <100X selective for c-MET

Kinase % Inhibition Met(h) 94 Tie2(h) 103 TrkA(h) 102 ALK(h) 100 TrkB(h) 100 Abl(T315I)(h) 98 Yes(h) 96 Lck(h) 95 Rse(h) [SKY] 94 Axl(h) 93 Fes(h) 93 Lyn(h) 93 Arg(m) 91 Ros(h) 90 CDK2/cyclinE(h) 87 Fms(h) 84 EphB4(h) 80 Bmx(h) 79 EphB2(h) 77 Fgr(h) 73 Fyn(h) 68 IR(h) 64 CDK7/cyclinH/MAT1(h) 58 cSRC(h) 58 IGF-1R(h) 56 Aurora-A(h) 54 Syk(h) 52 FGFR3(h) 50 PKCµ(h) 50 BTK(h) 35 CDK1/cyclinB(h) 25 p70S6K(h) 24 PRK2(h) 22 PAR-1Bα(h) 21 PKBß(h) 21 Ret(h) 21 GSK3ß(h) 18 Flt3(h) 17 MAPK1(h) 17 ZAP-70(h) 17 Abl(h) 16 c-RAF(h) 16 PKD2(h) 15 ROCK-II(h) 14 Rsk3(h) 14 GSK3α(h) 11 CDK5/p35(h) 10 PDGFRα(h) 10 Rsk1(h) 7 SGK(h) 6 CHK1(h) 5 ErbB4(h) 5 Rsk2(h) 5 JNK1α1(h) 4 PKBα(h) 4 Blk(m) 3 CDK3/cyclinE(h) 3 PKCι(h) 3 PKCθ(h) 3 CDK2/cyclinA(h) 2 PAK2(h) 2 PKCßI(h) 2 Pim-1(h) 1 PKCη(h) 1 SAPK4(h) 1 CaMKII(r) MKK7ß(h) CaMKIV(h)

• 1

CHK2(h)

• 1

CK2(h)

• 1

JNK2α2(h)

• 1

MKK6(h)

• 1

CK1δ(h)

• 2

PKCα(h)

• 2

MAPK2(h)

• 3

MEK1(h)

• 3

PKCδ(h)

• 3

PKCε(h)

• 3

Plk3(h)

• 3

PKCßII(h)

• 5

MSK1(h)

• 6

PDGFRß(h)

• 6

PKCζ(h)

• 6

SAPK3(h)

• 6

MAPKAP-K2(h)

• 7

PKA(h)

• 7

AMPK(r)

• 9

CDK6/cyclinD3(h)

• 9

CSK(h)

• 9

SAPK2a(h)

• 9

JNK3(h)

• 10

PKBγ(h)

• 10

IKKα(h)

• 11

NEK2(h)

• 11

*The cellular kinase activities were measured using ELISA capture method Kinase IC50 (nM) mean* Selectivity ratio c-MET 8 – ALK 20 2X RON 298 34X 189 22X Axl 294 34X 322 37X Tie-2 448 52X Trk A 580 67X Trk B 399 46X Abl 1,159 166X IRK 2,887 334X Lck 2,741 283X Sky >10,000 >1,000X VEGFR2 >10,000 >1,000X PDGFR >10,000 >1,000X

Pfizer Inc. Data on file

Crizotinib – a dual c-MET and ALK inhibitor at clinically relevant dose levels

Crizotinib (PF-02341066)

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Crizotinib: cell growth inhibition and apoptosis induction in H3122 cells

0.0001 0.01 0.1 1 10 Crizotinib concentration (mM) 125 100 75 50 25 –25 –50 % control

IC50 = 96 nM

Crizotinib: Induces Potent Growth Inhibition and Apoptosis in either c-Met amplified or EML4-ALK fusion oncogene-positive NSCLC cells

Crizotinib: cell growth inhibition in NSCLC cell lines Pfizer Inc. Data on file IC50 = 50% inhibitory concentration *MET amplification

1,000 800 600 400 200

H1993 H3122 H23 H1734 H82 H520 H526 SKMES1 H2347 H1975 H1838 H226 H1703 H647 H1755 CALU6 CALU1 H358 H1581 H2087 H1651 HCC827 HCC2935 H1355 H1573 H1650 H1666 H2405 A549

IC50 value (nM) *

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Milestones for Crizotinib (PF-02341066) Development

Online access to Pfizer’s submission to ODAC NPM, nucleophosmin; ALK, anaplastic lymphoma kinase; ALCL, anaplastic large cell lymphoma; IMT, inflammatory myofibroblastic tumor; NSCLC, non-small cell lung cancer; NBL, neuroblastoma lymphoma

May

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Part 2: Molecularly defined cohorts (ALK and c-MET) Prescreening for patients with ALK (non-lung) or MET activation Part 1: Dose escalation (n=37) Modified from Tan et al. J Clin Oncol 2010;28:15S abstract 2596 Soda, Nature 2007;448:561-6 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 Cohort 6 (n=9) 250 mg BID MTD/RP2D

BID, twice daily; QD, once daily; MTD, maximum tolerated dose; RP2D, randomized phase 2 dose

ALK-positive NSCLC cohort added 2008 Discovery of ALK gene rearrange- ments in NSCLC in 2007

PROFILE 1001: Phase 1 Safety, Pharmacokinetic And Pharmacodynamic Study Of Crizotinib, A c-Met/HGFR Selective Tyrosine Kinase Inhibitor, Administered Orally To Patients With Advanced Cancer (Began in 2006) N=175

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PROFILE 1001: Crizotinib-Treated ALK-Positive NSCLC Cohort

• ALK FISH-positive NSCLC

– No limit on prior lines of therapy

• Brain metastases allowed if treated and stable
• Crizotinib 250 mg po BID continuously in 28-day cycles

– Tumor response evaluated every other cycle (RECIST v1.0) – Safety evaluated every 2 wks for first 8 wks then every 4 wks

• Evolving data set, reports at ASCO annual meetings:
• 2009: In 19 ALK-positive NSCLC patients1
• 2010: In 82 ALK-positive NSCLC patients2,3
• 2011: In 119 ALK-positive NSCLC patients with a median

duration of follow-up of 11 mos (data cutoff: 10/29/2010)4

1Kwak et al. J Clin Oncol 2009;27:15S abstract 3509 2Bang et al. J Clin Oncol 2010;28:18S abstract 3 3Kwak et al. New Engl J Med 2010;363:1693–1703 4Camidge et al. J Clin Oncol 2011;29:15S abstract 2501

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Baseline Characteristics (N=119)

Characteristics N=119 Age, yrs Median 51 Range 21–79 Gender, n (%) Male / Female 59 (50) / 60 (50) Race, n (%) White 74 (62) Asian 34 (29) Other 11 (9) Smoking status, n (%) Never smoker 86 (72) Former/current smoker 32 (27) / 1 (1) Histology, n (%) Adenocarcinoma 116 (97) Other 3 (3) ECOG PS, n (%) 41 (34) 1 63 (53) ≥2 15 (13) Prior systemic regimens, n (%) 16 (13) (advanced/metastatic disease) 1 36 (30) 2 24 (20) 3 17 (14) ≥4 26 (22) Camidge et al. J Clin Oncol 2011;29:15S abstract 2501.

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Best Tumor Response Baseline in Target Lesions*

*excludes patients with early death and indeterminate response (n=106) **includes patients with early death and indeterminate response (n=116) % Decrease or increase from baseline Progressive disease Stable disease Partial response Complete response 100 80 60 40 20 –20 –40 –60 –80 –100 Objective response details (all evaluable patients) N=116** ORR (95% CI) 61% (52, 70) Median response duration 48 weeks Median time to response 8 weeks Disease control rate at 8, 16 weeks 79%, 67% Camidge et al. J Clin Oncol 2011;29:15S abstract 2501.

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Objective Response in Evaluable Patients (n=116)

Best Response N (%) Complete response 2 (1.7%) Partial response 69 (59.5%) Stable disease 31 (26.7%) Progressive disease 6 (5.2%) Other* 8 (6.9%) ORR (95% CI) 61% (52, 70) Median response duration 48 weeks Median time to response 8 weeks DCR at 8 weeks 79% DCR at 16 weeks 67%

*includes patients with early death and indeterminate response. DCR, disease control rate. Camidge et al. J Clin Oncol 2011;29:15S abstract 2501.

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Rapid Responses Seen In Some Patients

Ou et al. J Thoracic Oncol 2010;5:2044–2046

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Objective Responses by Patient Characteristics

Patient group n/N ORR, % Age: <65 years old 60/100 60 ≥65 years old 11/16 69 Sex: Male 36/59 61 Female 35/57 61 ECOG PS: 21/39 54 1 39/62 63 ≥2 11/15 79

• No. prior systemic therapies:

(metastatic disease) 12/15 80 1 21/35 60 ≥2 38/66 58 Race: Asian 28/34 82 Non-Asian 43/82 52

Camidge et al. J Clin Oncol 2011;29:15S abstract 2501.

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Progression-Free Survival (N=119)

1.0 0.8 0.6 0.4 0.2 Survival distribution function 5 10 15 20 Months

119 73 29 8 1 n at risk

Censored 95% Hall-Wellner Band 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 et al. J Clin Oncol 2011;29:15S abstract 2501.

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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 1st dose of crizotinib:

– 6 months: 90% (95% CI 82.7, 94.4) – 12 months: 81% (95% CI 70.9, 87.2)

Camidge et al. J Clin Oncol 2011;29:15S abstract 2501.

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• Overall, 114 patients (96%) reported a treatment-related AE
• The majority (n=95; 80%) of these patients experienced Grade 1/2

AEs; 19 patients (16%) experienced Grade 3/4 events

• 3 discontinuations due to treatment-related AEs: 2 cases of pneumonitis,

1 case of increased ALT (G4)

• Common treatment-related AEs (≥20%):

Treatment-Related Adverse Events

Event (N=119) All grades, n (%) Grade 3/4 Median time to first

• nset, days

Visual effects* 74 (62) – 13 Nausea 58 (49) – 2 Diarrhea 51 (43) – 2 Vomiting 42 (35) – 2 Edema** 33 (28) – 74 Constipation 32 (27) 1 (1)

• *Cluster term including visual impairment, photopsia, vision blurred, vitreous floaters and diplopia.

**Cluster term including localized edema, edema and edema peripheral.

Camidge et al. J Clin Oncol 2011;29:15S abstract 2501.

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• „Trails‟ from lights in peripheral vision in low light conditions

(e.g. dawn and dusk)

Characteristic Visual Effects With Crizotinib

• At edges of vision in low light conditions:
• Image persistence
• Flashes of light, which do not appear to be connected to a real

light source

• Flipped registration from high contrast images (e.g. stripes)

Camidge et al. J Clin Oncol 2011;29:15S abstract 2501.

• No patients required dosing interruption, dose reduction, or

permanent discontinuation of crizotinib due to visual effects.

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PROFILE 1007 (N=318)

• ALK-positive by central laboratory
• 1 prior chemotherapy

(platinum-based)

R A N D O M I Z E

Crizotinib 250 mg BID (n=159) [continuous] Pemetrexed 500 mg/m2 or docetaxel 75 mg/m2 (n=159) infused on day 1 of a 21-day cycle

Other Ongoing Crizotinib PROFILE Program

Crizotinib 250 mg BID (n=400) [continuous] PROFILE 1005 (N=400)

• ALK-positive by central laboratory
• ≥1 prior chemotherapy and not

eligible for 1007 PROFILE 1014 (N=334)

• ALK-positive locally advanced/

metastatic non-squamous NSCLC

• No prior treatment for advanced

disease

R A N D O M I Z E

Crizotinib 250 mg BID (n=167) [continuous] Pemetrexed/cisplatin or pemetrexed/carboplatin (n=167) infused on day 1 of a 21-day cycle Crossover on PD Crossover on PD

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N=400 (planned) Crizotinib 250 mg BID administered continuously (21-day cycle) Key entry criteria:

• ALK+ NSCLC with measureable

lesions

• ECOG PS: 0–2
• Not eligible for Phase 3 study

(A8081007)

• PD in chemo arm of study

A8081007

• ≥1 prior chemotherapy
• Stable/controlled brain metastases

allowed

PROFILE 1005: Initial Phase 2 Results with Crizotinib in Advanced ALK+ NSCLC

Crino et al. J Clin Oncol 2011;29:15S abstract 7501. Primary endpoints: ORR, safety, and tolerability Secondary endpoints: OS, TTR, duration of response, disease control rate, PK, biomarkers, PRO/HRQoL (EORTC QLQ-C30 and LC-13)

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Baseline Characteristics

Characteristic N=136 Male/ female, n (%) 64/72 (47/53) Median age, years (range) 52 (29–82) Ethnicity, n (%) White 86 (63) Asian 43 (32) Other 7 (5) Smoking status, n (%) Never/ former/ current 92 (68) / 39 (29) / 5 (4) Adenocarcinoma/ Other histology, n (%) 130 (96) / 6 (4) ECOG performance status, n (%) 0/ 1/ 2 37 (27) / 74 (54) / 24 (18)

• No. prior treatment regimens, n (%)

1 9 (7) 2 39 (29) 3 42 (31) 4 21 (15) ≥5 25 (18) Data cut off date: Feb 1, 2011. Crino et al. J Clin Oncol 2011;29:15S abstract 7501.

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Crizotinib (n=133) Overall response rate, % (95% CI) 51.1 (42.3, 59.9) Best response, n (%) Complete response Partial response Stable disease Progression Stable disease n (%) 0–<3 months 3–<6 months 6–<9 months 9–<12 months Disease control rate, % (95% CI) Week 6 Week 12 Duration of response, range in weeks 1 (<1.0) 67 (50.4) 45 (33.8) 10 (7.5) 9 (20) 29 (64.4) 5 (11.1) 2 (4.4) 85 (77.7, 90.6) 73.7 (65.3, 80.9) 1.0–15.0

Best Overall Response to Crizotinib (Response-Evaluable Patients)

Crino et al. J Clin Oncol 2011;29:15S abstract 7501.

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Response Evaluable population, N=123 (excludes patients with early death and indeterminate response). CR = complete response; PD = progressive disease; PR = partial response; SD = stable disease. 100 80 60 40 20 –20 –40 –60 –80 –100 % Decrease or Increase From Baseline BOR PD SD PR CR Best Overall Response (all evaluable patients) N=133 ORR, % (95% CI) 51.1% (42.3, 59.9) CR, n (%) 1 (<1.0) PR, n (%) 67 (50.4) SD, n (%) 45 (33.8) Progression, n (%) 10 (7.5) Duration of response, range 1.0-15.0 weeks Disease control rate at 6, 12 wks 85%, 73.7%

PROFILE 1005: Tumor Response to Crizotinib

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Patients Experienced Clinically Meaningful Benefits* in Key Domains of Pain, Dyspnea, Cough, and Fatigue

BL=baseline; N=number of subjects that completed the scale at the respective cycle; *Clinically meaningful difference defined as ≥10 points at time points shown. Mean change from baseline N 122 114 110 112 103 97 70 Cycle 2 3 4 5 6 7 8 Fatigue (QLQ-C30) 5 –5 –10 –15 –20 Mean change from baseline N 122 114 110 112 103 97 70 Cycle 2 3 4 5 6 7 8 5 –5 –10 –15 –20 Mean change from baseline N 122 114 110 112 103 97 70 Cycle 2 3 4 5 6 7 8 5 –5 –10 –15 –20 Mean change from baseline N 119 112 107 111 101 96 69 Cycle 2 3 4 5 6 7 8 5 –5 –10 –15 –20 Pain (QLQ-C30) Cough (QLQ-LC13) Dyspnea (QLQ-C30) * * * * * * * * * * * * * * * * * * * * * * * * *

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AE, n (%) Grade 1 Grade 2 Grade 3 Grade 4/5 Nausea 68 (50) 10 (7) – – Vomiting 53 (39) 6 (4) – – Diarrhea 54 (40) 4 (3) – – Visual effects 58 (43) – – Constipation 28 (21) 9 (7) – – Peripheral edema 26 (19) 9 (7) – – Fatigue 17 (13) 15 (11) 2 (2) – Decreased appetite 20 (15) 9 (7) – Dysgeusia 17 (13) 3 (2) – – ↑ Alanine aminotransferase 4 (3) 3 (2) 9 (7) 1 (1)* Dizziness 14 (10) 3 (2) – –

Treatment-Related Adverse Events (≥10%)

*Mistakenly reported as Grade 5, but actually Grade 1.

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Treatment-Related Adverse Events: Summary

• Most common (≥10%) treatment-related Grade 3 and above

AEs were reported in 25.7% of patients including:

• fatigue (n=2 grade 3)
• increased alanine aminotransferase (n=9 grade 3; n=1

grade 5)

• Discontinuation due to AEs included:
• raised alanine aminotransferase (n=3)
• pneumonitis (n=2)
• Two deaths were considered treatment-related (pneumonitis

& unknown cause) by the investigators

• Pneumonitis confounded by prior radiation therapy and

history of pulmonary embolism, pleural effusion, and pleural catheter treatment

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• Preliminary data from this phase 2 single arm study suggest crizotinib

was well-tolerated in patients with pre-treated ALK-rearranged NSCLC

• There was evidence of clinically meaningful antitumor activity

– ORR: 51% (1 CR; 67 PR) – Disease control rate was 85% at 6 weeks and 73.7% at 12 weeks

• Patients reported clinically meaningful reductions in pain, dyspnea,

cough, and fatigue and quality of life was maintained with therapy

• These data support earlier efficacy and safety findings reported from

PROFILE 1001 study, a Phase I expanded cohort of crizotinib in patients with ALK-positive advanced NSCLC, which reported an

• bjective response rate of 61% with a similar AE profile

Conclusions

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Vysis LSI ALK Dual Color, Break Apart Rearrangement Probe (Abbott Molecular) ALK rearrangement positive in ≥15% of tumor cells: split 3’/5’ (gap >2 signal diameters

• single 3’ ALK (red)

ALK rearrangement negative: 3’/5’ signal fusion 3’ 5’ Garcia 2011 WCLC

FISH Assay for ALK Rearrangement

Failure rate: 11%

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Applications for Regulatory Approval

• Crizotinib (PF-02341066) is a first-in-class, oral, and

selective small-molecule TKI targeting ALK fusion

• ncogenes. It is well tolerated and has demonstrated

consistent ORR of 51-61% and durable clinical benefit rate in advanced NSCLC patients with EML4-ALK fusion

• ncogenes.
• Pfizer Oncology has filed for accelerated approval of

crizotinib to the US FDA based on the results from phase I (PROFILE-1001) and available 1005 studies in conjunction with the ongoing confirmatory phase III program (PROFILE-1007) studies in May of 2011.

• Abbott Molecular Diagnostics has also sought approval

from the US FDA and the Japanese Ministry of Health, Labour, and Welfare for ALK FISH test in conjunction to identify candidate patients for crizotinib.

www.abbott.com/pressrelease

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Unmet Clinical Needs for ALK Inhibitors (Crizotinib) in ALK-positive Advanced NSCLC Patients

• It is critical to identify the rare (3-5%) subset of

NSCLC patients who have tumors harboring ALK fusion oncogenes correctly because of the dramatic response that many patients have with ALK targeted therapy.

• Only a few, large US academic centers have

clinical experience with crizotinib.

• Highly needed treatment strategies for those ALK-

positive patients who have progressed to crizotinib monotherapy.

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Frequency of ALK-positive NSCLC by different screening tests

EML4-ALK positive NSCLC: ~ 4% overall frequency ~ 8-10% in lung adenocarcinoma

First Author Method of Pollulalion of ALK+ Case Tolal No. of NSCLC ALK+ Case (Year) Testing Origin identified Tumors Tested Percentage Soda (2007) RT.PCR Japan 5 75 6.7 Rikova (2007)

• RT. PCR

China 4 103 3.9 Pemer (2008) FISH Switzerl and, USA 16 603 2.7 Inamurn (2008) RT· PCR Japan 5 200 2.5 Koivunen (2008) FISH USA (138), Korea (167) 8 305 2.6 Takeuchi (2008) RT·PCR Japan I I 253 4.3 Takeuchi (2008) mc, RT·PCR Japan 8 130 6.2 Shinmurn (2009) RT·PCR Japan 2 77 2.6 Wong (2009) RT·PCR China 13 266 4.9 Boland (2009) IHC, FISH USA 6 335 1.9 Martelli (2009) RT·PCR Italy, Spain 9 120 7.5 Rodig (2009) IHC, FISH USA I 227 0.4 Shaw (2009) FISH USA 19 141 13.5 Takahashi (2010) RT-PCR Japan 5 313 1.6 Zhang (2010) RACE PRC China 12 103 11.6 Li (2011) RT-PCR USA 75 1889 4.0 Total 187 5140 3.6

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Methods for Detection of ALK Translocation

Shaw, A. T. et al. J Clin Oncol; 27:4247-4253 2009 Hirsch F et al. Clin Cancer Res 2010;16:4909–4911

1. FISH 2. IHC 3. RT-PCR 4. Sequencing

Potential ALK Fusion Partners:

• EML4
• KIF5B
• TFG

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IHC may also be a good screening tool to identify actual ALK protein expression but one needs the right antibody and assay conditions.

• IHC is a routine procedure in many pathology settings
• Less labor intensive and cost-effective
• Can visualize cell morphology
• Good initial screening test

FISH may be a good screening tool to identify patients with ALK break-apart but

• Break-apart signal can be subtle and missed
• Technically sophisticated- significant expertise for accurate assessment
• Difficult to identify cell morphology in dark field microscopy
• Labor intensive and costly for thousands of patients

EML4-ALK Detection

RT-PCR may be a good screening tool to identify different variants of ALK fusion genes

• RNA could be used for other genetic and genomic analyses
• Very sensitive and fast turnaround time, routinely performed in most of the

labs

• requires knowledge of breakpoints and could identify different variants of ALK

fusion genes.

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Recent published reports describing sensitivity and specificity of IHC for ALK+ lung cancer

Author Journal Year Antibody Supplier Detection method Standard Sensitivity Specificity

Wong Cancer 2009 Polyclona l Invitrogen Dako HRP complex, RT-PCR 100%) N/A Shaw JCO 2009 ALK1 Dako N/A FISH 100% N/A Inamura Mod Pathol 2009 ALK1 Dako EnVision + RT-PCR 100% N/A Takeuchi CCR 2009 5A4 Abcam iAEP method RT-PCR 100% 100% EnVision+ DAB RT-PCR 18% 100% ALK1 Dako iAEP method RT-PCR 100% 100% EnVision+ DAB RT-PCR 9% 100% SP8 Abcam iAEP method RT-PCR 100% 30% EnVision+ DAB RT-PCR 18% 100% Boland Hum Pathol 2009 ALK1 Dako ADVANCE FISH, RT-PCR 100%(tested in 40 of 335 cases) 100% Rodig CCR 2009 ALK1 Dako tyramide amplification or EnVision+ FISH 80% 100% Mino- Kenudson CCR 2010 ALK1 Dako EnVision+ FISH 67% 97% D5F3 Cell Signaling Technology EnVision+ FISH 100% 99% Yi JTO 2011 ALK1 Dako ADVANCE FISH 90.0% 97.8% Paik JTO 2011 5A4 Novocastra i-view detection kit (Ventana) FISH 100% 95.8% Mitsudomi ASCO 2011 5A4 Abcam EnVision FLEX+ FISH 100% 100%

Courtesy of Dr. Hirsch, 2011 WCLC

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• Immunohistochemistry staining patterns:
• IHC score 0 (A), 1+ (B), 2+ (C), and 3+ (D)
• Current available data suggest:

– Those patients having tumors with ALK IHC score of 0 and 3+ correlate well with FISH negative and positive, respectively. – Those patients having tumors with ALK IHC score of 1+ or 2+ should be considered to test ALK FISH in order to confirm ALK translocation.

ALK IHC Assay

A B C D Courtesy of Dr. Hirsch, 2011 WCLC

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Many EML4 and Other 5‟ Partners

Sasaki T, Rodig SJ, Chirieac LR, Janne PA. The biology and treatment of EML4-ALK non-small cell lung cancer. Eur J Cancer 2010; 46:1773-80.

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RT-PCR Assay for EML4-ALK fusion variants

Primer sets designed for each fusion gene variant amplified the cognate synthetic fragment with 100% specificity within its validated range.

V1 V2 V3A V3B V4 V5A V5B V6 V7 EML4- ALK.V1 +++

• EML4-

ALK.V2

• +++
• EML4-

ALK.V3A

• +++
• EML4-

ALK.V3B

• +++
• EML4-

ALK.V3AB

• +++

+++

• EML4-

ALK.V5A

• +++
• EML4-

ALK.V5B

• +++
• EML4-

ALK.V6

• +++
• EML4-

ALK.V5A/6

• +++
• +++
• EML4-

ALK.V4

• +++
• EML4-

ALK.V7

• +++

EML4- ALK.V4/7

• +++
• +++

Primer Set PCR Specificity for Fusion Variants of EML4-ALK

• Synthetic fragments representing

the nine EML4-ALK fusion genes variants 1, 2, 3a, 3b, 4, 5a, 5b, 6 and 7 were generated by recursive PCR technology.

• Primer probes were designed using

the synthetic fragments as templates to detect specific EML4- ALK fusion gene fragments with a maximum amplicon of 170 bases by RT-PCR.

• Beta-actin was used as the

reference gene to determine quantity of cDNA.

Danenberg et al., ASCO 2010

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Patient Characteristics

EML4-ALK Positive (N=75) Study Period: 07/2010 - 12/2010 Incidence (of 1889 NSCLCs tested): 4.0% Median Age (range): 53.3 (33-84) Gender (Female/Male): 35/40 (47%/ 53%) Histology: Adenocarcinomas: 75 (100%) Li et al. J Clin Oncol 2011;29:15S abstract 10520.

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SLIDE 15

Slide 43

Variable Inter-Tumor Expression Level of ALK Gene by RT-PCR Assay

Value ALK 0.0001 0.001

0.0004

0.01

0.004

0.1

0.04

1

0.4

10

4

1 EML4-ALK Pos or WT P< 0.0001 EML4-ALK Negative (N=1772) EML4-ALK Positive (N=67) ALK mRNA Level (ALK/β-Actin) Note: While EML4-ALK-negative NSCLC tumors could have high level of ALK gene expression, EML4-ALK-positive tumors could have low level of ALK gene expression.

Li et al. J Clin Oncol 2011;29:15S abstract 10520.

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EML4-ALK Variants: (N=75) V1 V2 V3 V4

• No. of Cases:

38 7 28 2 Age: Median (Range) 51.8 (33-64) 56.4 (36-74) 55.0 (33-84) 56.5 (50-63) Gender: Female (%) 20 (53%) 5 (71%) 11 (39%) 1 (50%) EGFR Mutations: K-Ras Mutations:

Genotyping

Expression Level of ALK Gene 1 2 3 4 5 6 V1 (N=38) V2 (N=7) V3 (N=28) V5a (N=2) ELM4-ALK Variants

2.3 2.3 0.4 0.2

• EML4-ALK-positive

NSCLC tumors could have low level of ALK gene expression.

• The clinical signicant of

this finding is unknown.

• These tumors might be

falsely negative by IHC or FISH. Li et al. J Clin Oncol 2011;29:15S abstract 10520.

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ALK Abnormalities

Mossé et al., Clin Cancer Res 2009;15(18):5609–14 Potential ALK Fusion Partners:

• EML4
• KIF5B
• TFG

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SLIDE 16

Slide 46

Resistance Mechanisms and Strategies to Overcome Resistance to Crizotinib

• -An Evolving Story

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Crizotinib: Post 8 cycles Post 10 cycles

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What would you do next?

1. Continue crizotinib till symptomatic progression 2. A second-generation of ALK inhibitor 3. A HSP-90 inhibitor 4. Continue crizotinib, add pemetrexed 5. Others ?

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SLIDE 17

Slide 49

PROFILE 1001: Ongoing Clinical Benefit Beyond Documented Disease Progression

• Patients with documented PD could continue to receive

crizotinib if deriving ongoing clinical benefit as judged by the investigator – Subjective – guidelines do not currently exist – Scans continued but data were not routinely collected centrally following PD, so data are limited

• 40 patients (34%) experienced RECIST-defined PD at

the data cut-off (29 October 2010) – 16 patients have received crizotinib for >2 weeks post-PD

Camidge et al. J Clin Oncol 2011;29:15S abstract 2501.

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Acquired Somatic Mutations in the TK Domain of ALK Gene in NSCLC Confer Resistance to ALK Inhibitors

Choi et al., N ENGL J MED 2010; 363:1734-1739.

Sputum Pleural Effusion Pt #1: Pt #2:

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Predicted Crystal Structure of the Kinase Domain of ALK

Choi et al., N ENGL J MED 2010; 363:1734-1739.

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SLIDE 18

Slide 52

ALK Inhibitors in Clinical Development

Drug Company Clinical Stage Crizotinib Pfizer Phase II-III for NSCLC Phase I-II in neuroblastoma and other solid tumors Phase I ALCL LDK378 Novartis phase I NSCLC (ALKi-naïve, resistant) and other tumors GSK2141795 GlaxoSmithKine Phase I, solid tumors or lymphomas CEP-28122 Cephalon Preclinical AP-26113 Ariad Pharmacetical Preclinical Xcovery X-277 Preclinical Others: Eli Lilly, Chugai, Nerviano, etc.

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Hsp90 Client Proteins Influence All Aspects

• f Oncogenic Transformation

1Banerji U. Clin Cancer Res, 2009 2Chen Z et al. Cancer Res 2010;70:9827- 9836

• > 200 Hsp90 client proteins

have been described, including EML4-ALK fusion gene1

• Many oncoproteins are relevant in

NSCLC1

• EML4-ALK associated proteins in

complex with Hsp90, Hsp70, HSC70, BIP/Grp78, Cdc23, cdc372 Courtesy of Dr. Ramalingam ASCO 2011

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HSP 90 Inhibitors in Clinical Development

Agent Sponsor Administration 17AAG (Tanespimycin) NCI/Kosan iv 17DMAG Kosan iv & oral IPI-504 (Retaspimycin) Infinity iv STA-9090 (Ganetespib) Synta iv AUY-922 Novartis iv DS-2248 Daiichi Oral XL-888 Exelixis Oral IPI-493 Infinity Oral MPC-3100 Myrexis Oral SNX-5422 Serenex Oral CNF-2024 Biogen Idec Oral Source: Clinicaltrials.gov, accessed 3/29/2011.

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SLIDE 19

Slide 55

Clinical Results: HSP90 Inhibition in ALK+ NSCLC

• 60
• 50
• 40
• 30
• 20
• 10

10 20 30

Response(% change from baseline) Patients

IPI-504 STA-9090 N=11 *simulated waterfall plot based on reported results *Durability of responses not reported Courtesy of Dr. Ramalingam, ASCO 2011

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Prognosis of ALK+ NSCLC Patients

Camidge J Thorac Oncol, 2011.

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PFS

Response to Pemetrexed in ALK+ NSCLC Patients

(N=19)

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SLIDE 20

Slide 58

Expression of Chemotherapy-Related Biomarkers in Available EML4-ALK Positive NSCLC Tumors (N=37)

RNA Level: Low High Not Available ERCC1 (N=37): 24 (65%) 13 (35%) TS (N=36): 23 (64%) 13 (36%) 1 RRM1 (N=32): 14 (44%) 18 (56%) 5 The clinical significance remains to be defined in large cohorts of patients with annotated clinical outcomes.

ERCC1, Excision repair cross-complementation group 1; TS, Thymidylate synthase; RRM1, Ribonucleoside-diphosphate reductase M1.

0.1 1 10 EML4-ALK V1 Variant (N=17)

2.33

0.1 1 10 EML4-ALK V1 Variant (N=17)

2.33

0.1 1 10 100 EML4-ALK V3 Variant (N=16)

2.33

0.1 1 10 100 EML4-ALK V3 Variant (N=16)

2.33 Li et al. J Clin Oncol 2011;29:15S abstract 10520.

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R A N D O M I Z E 1:1 Crizotinib 250 mg BID continuous oral daily dosing + Pemetrexed 500 mg/m2 IV D1 (1 cycle = 21 days) Pemetrexed 500 mg/m2 IV D1 (1 cycle = 21 days) Rebiopsy 1:1

• Non-squamous NSCLC

patients with ALK-positive tumors

• Systemic progression on

crizotinib monotherapy after clinical benefit, i.e., patients with either ORR or SD ≥ 3 mos

• Starting treatment within 60

days after discontinuing crizotinib

• Stable or treated brain

metastases

• Pemetrexed-naïve

Stratification Factors

• Number of prior therapy (0 vs ≥1)
• Zubrod Performance status (0-1 vs 2)

SWOG: A randomized, phase II trial of crizotinib plus pemetrexed versus pemetrexed alone in non-squamous NSCLC at acquired resistance to crizotinib monotherapy

Co-PIs: Li and Camidge Primary Endpoint: 3-month improvement in PFS Secondary Endpoints: ORR, time to treatment failure, OS, safety and tolerability

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Summary

• Crizotinib (PF-02341066) offers a new standard of care

for advanced NSCLC patients whose tumors harboring EML4-ALK fusion oncogenes.

• ORR 51-61%, median PFS 10 months
• The development of ALK inhibitor crizotinib is an

example of rapid clinical development from target identification to clinical validation, supporting a genetically defined, personalized treatment for NSCLC.

• Unmet clinical needs:

1) To establish a cost-effective strategy to select candidate patients in the community setting for the treatment of ALK inhibitor crizotinib. 2) To identify treatment strategies for those ALK-positive NSCLC patients who have progressed on crizotinib monotherapy.

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