ALK IN LUNG CANCER: PAST, PRESENT, AND FUTURE

Gene Mutations in Lung Adenocarcinomas

17 years ago……

• 2;5 chromosomal translocation in most anaplastic large-cell non-Hodgkin's lymphomas , which fused the NPM gene on chromosome 5q35 to ALK, on chromosome 2p23.

• Expressed in the small intestine, testis, and brain but not in normal lymphoid cells, ALK shows greatest sequence similarity to the insulin receptor subfamily of kinases.

• Unscheduled expression of the truncated ALK may contribute to malignant transformation in these lymphomas.

The Mechanism of Carcinogenesis

Discovery of the EML4-ALK fusion in NSCLC

Initially reported in 2007 as a result of an inversion in chromosome 2p, which results in the fusion of the N-terminal portion of the echinoderm microtubule-associated protein-like 4 (EML4) with the kinase domain of ALK.

Soda et al., Nature 2007; 448:561-567

1Soda M, et al. Nature. 2007;448:561–67.

EML4–ALK Is an Oncogenic Driver

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

•Expression plasmids for WT, EML4, ALK, EML4-ALK, EML4-ALK K589M, and NPM-ALK were introduced into 3T3 fibroblasts.•Subcutaneous injection of the transfected 3T3 cells into nude mice revealed those that formed tumors

Evidence for EML4-ALK as a Lung Cancer Oncogene

• Insertion of EML4-ALK into NIH 3T3 fibroblasts was tumorigenic when implanted subcutaneously into nude mice

• Engineered the specific expression of EML4-ALK fusion gene in lung progenitor cells using a surfactant protein C gene promoter

• 100% of EML4-ALK transgenic mice developed lung adenocarcinoma that were + for ALK by IHC. No other primary cancers were observed.

• Following IV injection of EML4-ALK/3T3 cells into nude mice, all developed lung cancer. Ten animals were treated with an ALK-specific TKI and 10 were observed:

PNAS December 16, 2008 vol. 105 no. 50 19893–19897

Frequency of ALK Rearrangements

Author Total Number

Pos % Notes

Shaw ASCO 2009

141 19 13% More likely in adenocarcinoma, light or never smokers, didn’t overlap with EGFR or KRAS, younger patients

Inamura, JTO 2008

149 5 3% No overlap with EGFR or KRAS

Takeuchi, CCR 2008

253 11 4%

Koivuner, CCR 2008

305 8 3% More common in never or light smokers

Wong, Cancer 2009

266 13 5% Mostly adenocarcinoma, never smokers, younger

Rodig, CCR 2009

358 20 6% More common in younger, never smokers, adenocarcinoma with signet ring features, no overlap with EGFR mutations

Kris, ASCO 2011

598 43 7% Rare overlap with EGFR, BRAF, KRAS

ALK fusions occur in numerous tumors

ALK fusion Incidence

ALCL

NPM-ALK 60-80%

TPM3-ALK 12-18%

TGF-ALK rare

CLTC1-ALK rare

ATIC-ALK rare

TPM4-ALK rare

MSN-ALK rare

ALO17-ALK rare

MYH9-ALK rare

IMT

TPM3-ALK 50-60%

TPM4-ALK 50-60%

CARS-ALK rare

RANBP2-ALK rare

CTLC1-ALK rare

SEC 31L1-ALK rare

ALK fusion Incidence

Lung

EML4-ALK 3-5%

KIF5B-ALK rare

TGF-ALK rare

Breast EML4-ALK 0-2.4%

Colorectal EML4-ALK 0-2.4%

DLBCLCTLC1-ALK rare

NPM-ALK rare

Esophageal TPM4-ALK -

Renal VCL-ALK -

NPCTBD 14/51

(27.5%)

Atypical myeloproliferative

leukemia

RANBP2-ALK -

Grande et al., Mol Cancer Ther 2011; 10:569-579Barreca et al., J Molec Endocrinol 2011; 47:R11-R23

Garber, J Natl Cancer Inst 2010; 102:672-675Röttgers et al., Leukemia 2010; 24:1197-1200

Other ALK alterations (mutations, gene amplification)

ALK alteration Incidence

ThyroidMutations

(L1198F, G1201E)11%

Neuroblastoma

Mutations(F1174L, R1275Q)

6-8%

Amplification 4%

Glioblastoma

ALK protein expression

Growth factor PTN protein, mRNA

expression

-

Murugan et al., Cancer Res 2011; 71:4403–4411Grande et al., Mol Cancer Ther 2011; 10:569-579

Powers et al., J Biol Chem 2002; 277:14153-14158 Lu et al., J Biol Chem 2005; 280:26953-26964

How do we test for ALK rearrangments?

Histology/IHC FISH/Cytogenetics PCR Sequencing

What can we do in ALK+ NSCLC patient?

Crizotinib: A Dual MET/ALK Tyrosine Kinase Inhibitor

KinaseIC50 (nM)mean*

Selectivity ratio

c-MET 8 –

ALK 40-60 5-8X

ROS 60 7X

RON 80 10X

Axl294 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

Co-crystal structure of crizotinib (PF-02341066) bound to c-MET

Cui et al. J. Med. Chem. 2011;54:6342-63 and Pfizer data on file

Early-phase clinical trial of crizotinib (PF-02341066)

N Engl J Med 2010;363:1693-703.

Key entry criteria

• Positive for ALK by central laboratory

• Expanded from phase I dose escalation trial

• Most were previously treated

• N=82

Crizotinib 250mg bid for 28-day cycle

6-month PFS among crizotinib users was estimated at 72% (95% CI, 61–83%)

Lancet Oncol 2012; 13: 1011–19

Updated of the phase I study

Common treatment-related grade 1/2 AE

Overview of ongoing trials

● ALK inhibition, NSCLC

– PROFILE 1007 – Ph III 2nd line (NCT00932893)

– PROFILE 1014 – Ph III frontline (NCT01154140)

– PROFILE 1005 – Ph II pretreated (NCT00932451)

– PROFILE 1001 – Ph II expansion cohort (NCT00585195)

● ALK inhibition, other tumor types

– PROFILE 1013 – Ph I in non-NSCLC (NCT01121588)

● Met inhibition

– Study 1002 – Ph I/II with erlotinib (NCT00965731)

– Study 1006 – Ph I with PF-0299804 (dacomitinib), NSCLC (NCT01121575)

Key entry criteria

• Positive for ALK by central laboratory

• 1 prior chemotherapy (platinum-based)

PROFILE 1007 – phase III

Key entry criteria

• Positive for ALK by central laboratory

• Progressive disease in Arm B of study A8081007

• >1 prior chemotherapy

PROFILE 1005 – phase II

Crizotinib 250 mg BID (n=159)administered on a continuous dosing schedule

Pemetrexed 500 mg/m2 ordocetaxel 75 mg/m2 (n=159)infused on day 1 of a 21-day cycle

Primary endpoint = PFS met

Crizotinib 250 mg BID (N=250)administered on a continuous dosing schedule

Primary endpoint = ORR

PROFILE 1007: NCT00932893; PROFILE 1005: NCT00932451

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*n=240 response-evaluable patients from the mature population, and excludes patients with early death, indeterminate response and non-measurable disease+Per RECIST 1.1, percent change from baseline for subjects with best overall response of CR can be less than 100% when lymph nodes are included as target lesions

PD

++

++

Marked Activity of Crizotinib in ALK+ NSCLCUpdate of the Phase 2 Study

SD PR CR

Kim et al., ASCO 2012

Median PFS 8.1 months (95% CI: 6.8–9.7)28% patients in follow-up for progression

1.0

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0 5 10 15 20Time (months)

+ Censored 95% Hall-Wellner Band

n at risk 261 175 95 26 2

Marked Activity of Crizotinib in ALK+ NSCLCUpdate of the Phase 2 Study

Kim et al., ASCO 2012

PROFILE 1005: Any-Grade Treatment-Related AEs in ≥10% of Patients

AEMature population, n=261

n (%)Overall population, n=901

n (%)

Any AE 245 (93.9) 827 (91.8)

Vision disorder* 154 (59) 468 (51.9)

Nausea 148 (56.7) 423 (46.9)

Vomiting 116 (44.4) 352 (39.1)

Diarrhea 106 (40.6) 369 (41.0)

Constipation 86 (33.0) 249 (27.6)

Peripheral edema 72 (27.6) 211 (23.4)

Fatigue 64 (24.5) 163 (18.1)

Decreased appetite 59 (22.6) 167 (18.5)

Increased alanine aminotransferase 45 (17.2) 146 (16.2)

Dysguesia 43 (16.5) 149 (16.5)

Dizziness 40 (15.3) 95 (10.5)

Neutropenia 36 (13.8) 84 (9.3)

Increased aspartate aminotransferase 33 (12.6) 106 (11.8)

*Includes visual impairment, photopsia, vision blurred, vitreous floaters, photophobia and diplopiaRare instances of fatal pneumonitis and fatal hepatotoxicity were reported in crizotinib clinical trial program

N Engl J Med 2013. DOI: 10.1056/NEJMoa1214886

Result of PROFILE 1007

1st line setting (PROFILE 1014):

Key entry criteria

• Positive for ALK by central laboratory

• No prior systemic therapy

RANDOMIZE

Crizotinib 250 mg BIDadministered on a continuous dosing schedule

Pemetrexed 500 mg/m2 + Cisplatin 75mg/m2 OR Pemetrexed 500mg/m2 + Carboplatin AUC 5 or 6infused on day 1 of a 21-day cycle

Primary endpoint = PFS

Study Start Date: January 2011Estimated Study Completion Date: December 2013

The Future: Overcoming Crizotinib Resistance

Resistance develops on average within the first year or two of TKI therapy…

Acquired Crizotinib Resistance

The target gene can be altered by mutation or by amplification, limiting the ability of the drug to inhibit the kinase. (like T790M in EGFR and T315I in BCR-ABL)

Alternative signaling pathways (bypass tracks) can be activated in resistant cells, bypassing the need for signaling from the target.

Nat Rev Clin Oncol. 2012 Apr 3 Current Opinion in Pharmacology 2013

Acquired Crizotinib ResistanceMutation

Up to 1/3 of relapsing patients, crizotinib resistance is mediated by secondary resistance mutations located in the ALK TK domain.Mutation Mechanism

L1196M gatekeeper mutation, hinder TKI binding through steric hindrance

Most common

G1269A lies directly in the ATP-binding Pocket

G1202R and S1206Y Locate in solvent-exposed region of the kinase domain, decrease binding affinity of Crizotinib

Acquired Crizotinib ResistanceAmplification

Amplification of the ALK fusion gene has also been reported in a small number of crizotinib-resistant tumors

Acquired Crizotinib ResistanceAlternative Pathway

In crizotinib-resistant tumors, several distinct bypass tracks mediating resistance have been reported. EGFR

½ cases with crizotinib resistance showed increased EGFR activity

c-KIT Confirmed by IHC, FISH, and c-Kit

ligand Stem cell factor (SCF) Can be overcome by Imatinib

combine with crizotinib There may be more than 1

bypass pathway in 1 individual

Sci Transl Med 2012Cancer 2011

Sci Transl Med 2012

Mechanisms of resistance to crizotinib in ALK-positive NSCLC

Camidge, D. R. Nat Rev Clin Oncol. 2012 Apr 3

What Can We Do?

Summary of Crizotinib Resistance

Novel Agents to Overcome Crizotinib-resistance ALK+ NSCLC

LDK378 (Novartis, Basel, Switzerland)

AP26113(ARIAD Pharmaceuticals, Cambridge, MA),

AF802(Chugai Pharmaceutical, Tokyo, Japan)

ASP3026 (Astellas Pharma, Tokyo, Japan)

STA-9090(Ganetespib)

AUY922 IPI-504 AT 13387 DS-2248

2nd generation ALK inhibitors HSP90 inhibitors

The Possible Difficulties

There may be more than 1 bypass mechanisms in one patient, therefore combination therapy may be needed.

The mutation may be different in different tumor sites in the same patient. Biopsy in different site may be indicated

There may be other unknown bypass pathway

Frontline 2nd ALK inhibitor, sequential use, or combine with other agent/CT (cocktail use)

1. Soda M, et al. Nature 2007; 448: 561-566. 2. McDermott U, et al. Cancer Res 2008; 68: 3389-3395.3. Koivunen JP, et al. Clin Cancer Res 2008; 14: 4275-4283.4. Shaw AT, et al. JCO 2009; 27: 4247-4253.5. Kwak EL, et al. N Engl J Med. 2010; 363: 1693-1703. 6. US Food and Drug Administration.

ALK-Positive Timeline

2007 2009

EML4-ALK chromosomal rearrangements reported in NSCLC[1]

2011

EML4-ALK defines a molecular subset of NSCLC with distinct clinical characteristics[4]

Preclinical studies document antitumor activity of ALK inhibitors in lung cancer cell lines and xenografts[2,3]

2008 2010

Crizotinib produces a response in 47/82 ALK+ patients and a 6-month PFS of 72%[5]

FDA approves crizotinib for treatment of ALK+ NSCLC[6]

? 2012 ?2nd generation ALK inhibitor TKIs and hsp inhibitors

Take Home Message

EML4-ALK defines a new molecular subset of NSCLC

Patients are more likely to be young, never/light smokers with adenocarcinoma

Crizotinib results in a 6-month PFS of 72% and overall response rate of 57% at 6.4 months

2nd generation ALK TKIs and HSP90 inhibitors offer promise in patients with crizotinib resistance

Thanks for Your Attention!!