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 NPM1

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 controls1

1 2 3 4 5 6 7

1 2 3 4 5 6 7

80 kDa80 kDa

1Shiota M & Mori S. Leuk Lymphoma. 1996;23:2532. 2Pulford K, et al. J Cell Physiol. 2004;199:33058. 3Palmer, et al. Biochem J. 2009;420:345–61. 4Mano. Cancer Sci. 2008;99:2349–55.

1Shiota M & Mori S. Leuk Lymphoma. 1996;23:2532. 2Pulford K, et al. J Cell Physiol. 2004;199:33058. 3Palmer, et al. Biochem J. 2009;420:345–61. 4Mano. 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

EML4EML4

EML4–ALK variant 1EML4–ALK variant 1

HELPHELP11 496496 981981

WDWDBasicBasic

11 496496 10591059

11 10581058 16201620

TMTM

EML4-ALK variant 1EML4-ALK variant 1

Exon 13Exon 13

ALKALK EML4EML4

297 bpExon 21Exon 21

KinaseKinaseALKALK

EML, echinoderm microtubule-associated protein-like 4; HELP, hydrophobic echinoderm mizroxbule-associated protein-like protein

4

ALK Pathway

Inversion TranslocationOr

ALK

Partner gene product

ALK fusion protein*

Tumour cellproliferation

Cellsurvival

PI3KPI3K

BADBAD

AKTAKT

STAT3/5STAT3/5

mTORmTOR

S6KS6K

RASRAS

MEKMEK

ErKErK

PLC-PLC-YY

PIPPIP22

IPIP33

1Inamura K, et al. J Thorac Oncol. 2008;3:13–17. 2Soda 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)

51Soda M, et al. Nature. 2007;448:561–67.

2Zhang, et al. Mol Cancer. 2010;9:188.

1Soda M, et al. Nature. 2007;448:561–67.2Zhang, et al. Mol Cancer. 2010;9:188.

EML4–ALK Is a Potent Oncogenic Driver1

Other fusion partners for ALK have also been identified, including NPM, EML4, TPM3, ATIC, TFG, CARS, and CLTC2

3T3

Nudemice

tumour/ 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

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 NSCLCRetrospective Data Prospective Data

Lung Cancer Mutation Consortium9

Adenocarcinoma

1Takahashi, et al. 2010. 2Wong, et al. 2009. 3Perner, et al. 2008. 4Paik, et al. 2011. 5Boland, et al. 2009. 6Paik, et al 2011. 7Takahashi, et al. 2010.

8Rodig, et al. 2009. 9Kris, et al. Presented at ASCO 2011. Abstract CRA7506.

1Takahashi, et al. 2010. 2Wong, et al. 2009. 3Perner, et al. 2008. 4Paik, et al. 2011. 5Boland, et al. 2009. 6Paik, et al 2011. 7Takahashi, et al. 2010.

8Rodig, et al. 2009. 9Kris, et al. Presented at ASCO 2011. Abstract CRA7506.

RT-PCR FISH IHC

% ALK+ patients

Unselected 1.6%1 - 4.9%2 2.7%3 - 4.2%4 1.7%5 - 8.6%6

% ALK+ patients

Adenocarcinoma2.4%7 - 4.9%2 5.6%8 2.7%5

RT-PCR, reverse transcription-polymerase chain reaction; FISH, fluorescence in situ hybridization; IHC, immunohistochemistry.

Status of Actionable Driver Mutations in Lung Adenocarcinoma Tumor Specimens

Johnson D, et al. ECCO ESMO 2011. Abstract 9018.

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-PCR FISH IHC

% ALK+ patients

Unselected 1.6%1 - 4.9%2 2.7%3 - 4.2%4 1.7%5 - 8.6%6

% ALK+ patients

Adenocarcinoma2.4%7 - 4.9%2 5.6%8 2.7%5

1Takahashi, et al. 2010. 2Wong, et al. 2009. 3Perner, et al. 2008. 4Paik, et al. 2011. 5Boland, et al. 2009. 6Paik, et al 2011. 7Takahashi, et al. 2010.

8Rodig, et al. 2009.

1Takahashi, et al. 2010. 2Wong, et al. 2009. 3Perner, et al. 2008. 4Paik, et al. 2011. 5Boland, et al. 2009. 6Paik, et al 2011. 7Takahashi, et al. 2010.

8Rodig, et al. 2009.

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 patients1

1Shaw AT, et al. J Clin Oncol. 2009;27:4247‒53.1Shaw AT, et al. J Clin Oncol. 2009;27:4247‒53.

TTP on EGFR-TKI monotherapyALK 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

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)0 12 24 36 48 60

100

80

60

40

20

Per

cen

t pr

ogre

ssio

n fr

ee

EGFRWT/WTEML4-ALK

Per

cen

t pr

ogre

ssio

n fr

ee EGFRWT/WTEML4-ALK

P =.004(ALK vs EGFR)

Time (months)

100

80

60

40

20

0 12 24 36 48 60

TTP on platinum-based chemotherapyTTP 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 7515.

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)

0 20 40 60 80 100

*Excludes ALK-positive patients

enrolled due to previous non-

response to EGFR TKIs

Months

100

80

60

40

20

0

Months

OS

(%

)

PF

S (

%)*

100

80

60

40

20

00 10 20 30 40

PFS of EGFR-TKI treated patientsPFS of EGFR-TKI treated patientsOSOS

11

Status of ALK Inhibitors in Clinical Development

12

KinaseIC50 (nM)

mean*Selectivity

ratio

c-Met 8 –

ALK 20 2X

RON298 34X

189 22X

Axl294 34X

322 37X

Tie2 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 >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

Kinase % InhibitionMet(h) 94Tie2(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) 90CDK2/cyclinE(h) 87

Fms(h) 84

EphB4(h) 80

Bmx(h) 79EphB2(h) 77Fgr(h) 73Fyn(h) 68IR(h) 64

CDK7/cyclinH/MAT1(h) 58cSRC(h) 58IGF-1R(h) 56

Aurora-A(h) 54Syk(h) 52

FGFR3(h) 50PKCµ(h) 50BTK(h) 35

CDK1/cyclinB(h) 25p70S6K(h) 24PRK2(h) 22

PAR-1Bα(h) 21PKBß(h) 21Ret(h) 21

GSK3ß(h) 18Flt3(h) 17

MAPK1(h) 17ZAP-70(h) 17

Abl(h) 16c-RAF(h) 16PKD2(h) 15

ROCK-II(h) 14Rsk3(h) 14

GSK3α(h) 11CDK5/p35(h) 10PDGFRα(h) 10

Rsk1(h) 7SGK(h) 6CHK1(h) 5ErbB4(h) 5Rsk2(h) 5

JNK1α1(h) 4PKBα(h) 4Blk(m) 3

CDK3/cyclinE(h) 3PKCι(h) 3PKCθ(h) 3

CDK2/cyclinA(h) 2PAK2(h) 2PKCßI(h) 2Pim-1(h) 1PKCη(h) 1SAPK4(h) 1CaMKII(r) 0MKK7ß(h) 0CaMKIV(h) -1CHK2(h) -1CK2(h) -1

JNK2α2(h) -1MKK6(h) -1CK1δ(h) -2PKCα(h) -2

MAPK2(h) -3MEK1(h) -3PKCδ(h) -3PKCε(h) -3Plk3(h) -3

PKCßII(h) -5MSK1(h) -6

PDGFRß(h) -6PKCζ(h) -6SAPK3(h) -6

MAPKAP-K2(h) -7PKA(h) -7AMPK(r) -9

CDK6/cyclinD3(h) -9CSK(h) -9

SAPK2a(h) -9JNK3(h) -10PKBγ(h) -10IKKα(h) -11NEK2(h) -11

Pfizer Inc, data on file.

Crizotinib (PF-02341066)

13

Part 2:Molecularly defined cohorts

Part 1:Dose escalation(n=37)

Crizotinib: First-in-human/Patient Trial (A8081001)

Modified from: Tan, et al. J Clin Oncol. 2010;28:15S. Abstract 2596.

Cohort 6 (n=9)250 mg BIDMTD/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

14

15

16

17

18

19

20

A8081001: Rapid Responses Reported

Day 7 Day 14

Ou, et al. J Thorac Oncol. 2010;5(12):2044–46.

Symptoms at study entry Improvement in symptoms

Cough Significant improvement at day 3, completely resolved by week 2

Daily low-grade fevers Resolved by day 3

Anorexia Gained 1.5 kg of weight by week 2

Right neck pain due to tumour invasion Resolved by day 3

21

A8081001: Progression-Free Survival (N=119)

Censored

95% Hall-Wellner Band

1.0

0.8

0.6

0.4

0.2

0

Su

rviv

al d

istr

ibu

tio

n f

un

ctio

n

0 5 10 15 20

Months119 73 29 8 1n 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

A8081001: 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/AustraliaN=36

2nd lineN=23

Never/lightsmoker

AdenoCAN=21

ALK-positiveCrizotinib-naïve

ALK CRIZOTINIB

ALK-positiveCrizotinib-treated

N=82

US/AustraliaN=56

2nd/3rd lineN=30

WT/WT CONTROLS

US (MGH)N=253

2nd lineN=125

ALK-negativeEGFR-wild type

Never/lightsmoker

AdenoCAN=28

Shaw AT, et al. Presented at ASCO 2011; Abstract 7507.

Never/lightsmoker

AdenoCAN=48

25

Overall Survival: 2nd Line Subset

ALK

Crizotinib(n=30)

ALK Control(n=23)

Median Survival, mo NR 6

1-yr Survival, % 70 44

WT/WT Control(n=125)

11

47

0

0%

20%

40%

60%

80%

100%

1 2 3 4

From 2nd/3rd line crizotinib

2-yr Survival, % 55 12 32

HR=0.49, P=.02

YearsShaw 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 2nd-/3rd- 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