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

EML4–ALK Is an Oncogenic Driver Vector EML4 ALK EML4–ALK K589M NPM–ALK v-Ras 3T3 Nude mice tumour/ injection 0/8 0/8 0/8 8/8 0/8 8/8 2/2 As shown in nude mice experiments EML4 ALK is a potent oncogenic driver In addition transgenic mice expressing EML4 Alk in lung epithelial cells numerous bilateral lung adenocarcinomas develop shortly after birth supporting the oncogenic nature of the fusion protein and again the adminstration of a specific inhibitor of ALK tyorsine kinase activity resulted in a rapid eradication of these nodules 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 1Soda M, et al. Nature. 2007;448:561–67. 8

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 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 Incidence ALCL NPM-ALK 60-80% TPM3-ALK 12-18% TGF-ALK rare CLTC1-ALK ATIC-ALK TPM4-ALK MSN-ALK ALO17-ALK MYH9-ALK IMT 50-60% CARS-ALK RANBP2-ALK CTLC1-ALK SEC 31L1-ALK ALK fusion Incidence Lung EML4-ALK 3-5% KIF5B-ALK rare TGF-ALK Breast 0-2.4% Colorectal DLBCL CTLC1-ALK NPM-ALK Esophageal TPM4-ALK - Renal VCL-ALK NPC TBD 14/51 (27.5%) Atypical myeloproliferative leukemia RANBP2-ALK Grande et al., Mol Cancer Ther 2011; 10:569-579 Barreca et al., J Molec Endocrinol 2011; 47:R11-R23 Garber, J Natl Cancer Inst 2010; 102:672-675 Röttgers et al., Leukemia 2010; 24:1197-1200

Other ALK alterations (mutations, gene amplification) Incidence Thyroid Mutations (L1198F, G1201E) 11% Neuroblastoma (F1174L, R1275Q) 6-8% Amplification 4% Glioblastoma ALK protein expression Growth factor PTN protein, mRNA expression - Murugan et al., Cancer Res 2011; 71:4403–4411 Grande 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 IC50 (nM) mean* Selectivity ratio c-MET 8 – ALK 40-60 5-8X ROS 60 7X RON 80 10X 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 PDGFR 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) 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%) N Engl J Med 2010;363:1693-703.

Updated of the phase I study Common treatment-related grade 1/2 AE Lancet Oncol 2012; 13: 1011–19

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)

Primary endpoint = PFS met PROFILE 1005 – phase II Key entry criteria Positive for ALK by central laboratory Progressive disease in Arm B of study A8081007 >1 prior chemotherapy Crizotinib 250 mg BID (N=250) administered on a continuous dosing schedule Primary endpoint = ORR PROFILE 1007 – phase III Crizotinib 250 mg BID (n=159) administered on a continuous dosing schedule Key entry criteria Positive for ALK by central laboratory 1 prior chemotherapy (platinum-based) Pemetrexed 500 mg/m2 or docetaxel 75 mg/m2 (n=159) infused on day 1 of a 21-day cycle PROFILE 1007: NCT00932893; PROFILE 1005: NCT00932451 Primary endpoint = PFS met

Decrease or increase from baseline (%) Marked Activity of Crizotinib in ALK+ NSCLC Update of the Phase 2 Study 100 PD SD PR CR 80 60 40 20 Decrease or increase from baseline (%) –20 –40 –60 + + –80 –100 + + –120 *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 Kim et al., ASCO 2012

Probability of survival without progression Marked Activity of Crizotinib in ALK+ NSCLC Update of the Phase 2 Study 1.0 Median PFS 8.1 months (95% CI: 6.8–9.7) 28% patients in follow-up for progression 0.8 + Censored 95% Hall-Wellner Band 0.6 Probability of survival without progression 0.4 0.2 0 5 10 15 20 Time (months) n at risk 261 175 95 26 2 Kim et al., ASCO 2012

PROFILE 1005: Any-Grade Treatment-Related AEs in ≥10% of Patients Mature 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 diplopia Rare instances of fatal pneumonitis and fatal hepatotoxicity were reported in crizotinib clinical trial program

Result of PROFILE 1007 N Engl J Med 2013. DOI: 10.1056/NEJMoa1214886

1st line setting (PROFILE 1014): Crizotinib 250 mg BID administered on a continuous dosing schedule R A N D O M I Z E Key entry criteria Positive for ALK by central laboratory No prior systemic therapy Pemetrexed 500 mg/m2 + Cisplatin 75mg/m2 OR Pemetrexed 500mg/m2 + Carboplatin AUC 5 or 6 infused on day 1 of a 21-day cycle Study Start Date: January 2011 Estimated Study Completion Date: December 2013 Primary endpoint = PFS

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. As an example, in 5% to 10% of EGFR TKI–resistant cases, resistance is mediated by focal amplification of c-MET. c-MET activates downstream signaling independently of EGFR, allowing resistant cells to grow despite EGFR inhibition. Nat Rev Clin Oncol. 2012 Apr 3 Current Opinion in Pharmacology 2013

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

Acquired Crizotinib Resistance Alternative 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 2012 Cancer 2011 Sci Transl Med 2012 Of note, EGFR mutations have not been identified in any resistant, ALK-positive tumor specimens, so mutational activation is unlikely to account for the increase in phospho-EGFR.

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 1/3 ALK amplification-> new ALK Hsp 90 inhibitor

Novel Agents to Overcome Crizotinib-resistance ALK+ NSCLC 2nd generation ALK inhibitors HSP90 inhibitors 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

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)

ALK-Positive Timeline EML4-ALK defines a molecular subset of NSCLC with distinct clinical characteristics[4] FDA approves crizotinib for treatment of ALK+ NSCLC[6] EML4-ALK chromosomal rearrangements reported in NSCLC[1] 2007 2009 2011 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] ? 2012 ? 2nd generation ALK inhibitor TKIs and hsp inhibitors ALK, anaplastic lymphoma kinase; EML4, echinoderm microtubule-associated protein-like 4; NSCLC, non-small-cell lung cancer; PFS, progression-free survival. 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.

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

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