Academia Pharma Intersect: Lung Cancer. A. JOHN IAFRATE c

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1 The Oncologist Acdemi Phrm Intersect: Lung Cncer Crizotinib for the Tretment of ALK-Rerrnged Non-Smll Cell Lung Cncer: A Success Story to Usher in the Second Decde of Moleculr Trgeted Therpy in Oncology SAI-HONG IGNATIUS OU, CYNTHIA HUANG BARTLETT, b MARI MINO-KENUDSON, c JEAN CUI, d A. JOHN IAFRATE c Cho Fmily Comprehensive Cncer Center, University of Cliforni, Irvine, Cliforni, USA; b Pfizer Oncology, New York, New York, USA; c Deprtment of Pthology, Msschusetts Generl Hospitl, Boston, Msschusetts, USA; d Pfizer Globl Reserch, L Joll, Cliforni, USA Key Words. Crizotinib ALK inhibitor MET inhibitor ROS1 inhibitor Chromosoml rerrngements Receptor tyrosine kinse fusion positive mlignncies Non-smll cell lung cncer (NSCLC) Disclosures: Si-Hong Igntius Ou: Pfizer, Genentech, Amgen (C/A); Pfizer, Genentech, Lilly (H); Pfizer (RF); Cynthi Hung Brtlett: Pfizer (E); Jen Cui: Pfizer (E, OI); Crizotinib (IP); A. John Ifrte: Pfizer (C/A). The other uthor indicted no finncil reltionships. (C/A) Consulting/dvisory reltionship; (RF) Reserch funding; (E) Employment; (H) Honorri received; (OI) Ownership interests; (IP) Intellectul property rights/inventor/ptent holder; (SAB) Scientific dvisory bord ABSTRACT Crizotinib, n ALK/MET/ROS1 inhibitor, ws pproved by the U.S. Food nd Drug Administrtion for the tretment of nplstic lymphom kinse (ALK)-rerrnged non-smll cell lung cncer (NSCLC) in August 2011, merely 4 yers fter the first publiction of ALK-rerrnged NSCLC. The crizotinib pprovl ws ccompnied by the simultneous pprovl of n ALK compnion dignostic fluorescent in situ hybridiztion ssy for the detection of ALK-rerrnged NSCLC. Crizotinib continued to be developed s n ALK nd MET inhibitor in other tumor types driven by ltertion in ALK nd MET. Crizotinib hs recently been shown to be n effective ROS1 inhibitor in ROS1-rerrnged NSCLC, with potentil future clinicl pplictions in ROS1-rerrnged tumors. Here we summrize the heterogeneity within the ALK- nd ROS1-rerrnged moleculr subtypes of NSCLC. We review the pst nd future clinicl development of crizotinib for ALKrerrnged NSCLC nd the dignostic ssys to detect ALK-rerrnged NSCLC. We highlight how the success of crizotinib hs chnged the prdigm of future drug development for trgeted therpies by trgeting moleculrdefined subtype of NSCLC despite its rrity nd ffected the prctice of personlized medicine in oncology, emphsizing close collbortion between clinicl oncologists, pthologists, nd trnsltionl scientists. The Oncologist 2012; 17: INTRODUCTION The er of moleculrly trgeted therpy in lung cncer begn in 2004 with the discovery tht ctivting epiderml growth fctor receptor (EGFR) muttions in non-smll cell lung cncer (NSCLC) correlted with clinicl response to EGFR tyrosine kinse inhibitors (TKIs) [1 3], lthough EGFR TKIs were developed nd pproved for clinicl use prior to the knowledge of these ctivting EGFR muttions [4, 5]. Subsequently, five rndomized trils in ptients with NSCLC with ctivting EGFR muttions hve demonstrted sttisticlly significnt superior response rtes nd progression-free survivl for EGFR TKIs compred with stndrd doublet chemotherpy [6 10]. Activting EGFR muttions provide prtil moleculr explntion for the observtion tht NSCLC in neversmokers is considered distinct disese with high proportion of Asin femle denocrcinom ptients who hve better Correspondence: Si-Hong Igntius Ou, M.D., Ph.D., Cho Fmily Comprehensive Cncer Center, University of Cliforni Irvine Medicl Center, 101 City Drive, Bldg 56, RT81, Ornge, Cliforni 92868, USA. Telephone: ; Fx: ; e-mil: igntius.ou@uci.edu Received July 20, 2012; ccepted for publiction September 10, 2012; first published online in The Oncologist Express on September 18, AlphMed Press /2012/$20.00/0 The Oncologist 2012;17:

2 1352 Crizotinib: The Second Decde of Trgeted Therpy survivl outcome thn tht of smokers [11, 12]. This observtion lso provided the impetus for the identifiction of other driver muttions in NSCLC. In the Iress Pn-Asi Study (IPASS) [13, 14] nd First-SIGNAL [15] trils, despite the enrichment of Asin femle never-smokers with denocrcinom nd the employment of sophisticted sequencing techniques, only pproximtely 60% nd 44% of ptients in ech study, respectively, were found to crry ctivting EGFR muttions, indicting tht other potentil driver muttions remin to be discovered [16]. DISCOVERY OF ANAPLASTIC LYMPHOMA KINASE REARRANGEMENT IN THE PATHOGENESIS OF NSCLC ALK rerrngements were identified in NSCLC in 2007 by two independent groups. Sod et l. developed retrovirl-bsed cdna expression librries to screen for novel oncogenes [17, 18]. They trnsfected cdna librry derived from lung denocrcinom from 62-yer-old Jpnese mle smoker who ws prescreened to be negtive for KRAS nd EGFR muttions [17]. They identified n echinoderm microtubule ssocited protein-like 4 (EML4)-nplstic lymphom kinse (EML4- ALK) fusion trnscript tht possessed trnsforming ctivity in 3T3 cells [17]. 3T3 cells trnsfected with EML4-ALK nd implnted in nude mice resulted in rpid tumor growth [17]; n ALK inhibitor inhibited the growth of BA/F3 cells trnsfected with EML4-ALK. Finlly, preliminry survey of pnel of 33 NSCLC tumors reveled tht EML4-ALK rerrngement occurs independently of EGFR nd KRAS muttions [17]. To further demonstrte the role of EML4-ALK in the pthogenesis of NSCLC, Sod et l. generted trnsgenic mice engineered to specificlly express EML4-ALK in lung lveolr cells, which resulted in hundreds of lung denocrcinom nodules. Tretment of these trnsgenic mice with n ALK inhibitor resulted in reduced tumor burden compred with untreted mice. Intrvenous injection of EML4-ALK/3T3 cells resulted in mssive infiltrtion of EML4-ALK/3T3 cells in the lungs of nude mice nd rpid deth of the mjority of the mice within 1 month [18]. Tretment with the sme ALK inhibitor resulted in the bsence of EML4-ALK/3T3 cells in the lung nd prolonged survivl [18]. In summry, Sod et l. convincingly demonstrted tht EML4-ALK is unique driver muttion in NSCLC nd tht inhibition of EML4-ALK ctivity in vivo led to the reduction of lung cncer burden. Contemporneously, Rikov et l. chrcterized the phosphotyrosine profile in 191 NSCLC cell lines nd tumor smples using phosphoproteomic pproch to identify driver kinses in NSCLC [19]. They identified high level of ALK phosphoryltion in severl NSCLC tumor smples nd H2228 NSCLC cells. Rpid mplifiction of the 5 complementry DNA ends (5 RACE) of the RNA trnscripts isolted from these smples with highly phosphorylted ALK reveled the EML4-ALK fusion trnscript [19]. No muttions were found in the ALK kinse domin [19]. Thus, two groups using two different pproches independently identified ALK trnsloction, the first of its kind in common solid mlignncy. THE NORMAL PHYSIOLOGICAL ROLE OF ALK AND SIGNAL TRANSDUCTION PATHWAYS ACTIVATED BY EML4-ALK The full-length ALK cdna contins 29 exons. The full-length ALK protein contins 1,620 mino cids, with predicted moleculr weight of 177 kilodltons (kd) [20]. The 254-mino cid kinse domin comprises mino cid residues preceded by short trnsmembrne region of mino cids [20]. ALK is temporlly nd sptilly expressed during development of the murine neontl centrl nervous system with its expression highest in the neontl brin, nd is not expressed in ny non-neurl tissues during ny stge of development of the mouse [20, 21]. ALK ws so nmed when it ws discovered to be trnslocted in nplstic lrge cell lymphom (ALCL) [22]. Subsequently, ALK rerrngement with vrious fusion prtners hs been discovered in diffuse lrge B- cell lymphom nd inflmmtory myofibroblstic tumor (IMT) prior to the discovery of ALK rerrngement in NSCLC. All three of the mjor signling pthwys (PI3K-AKT, RAS- ERK, JAK-STAT3) hve been identified s being engged by the vrious ALK fusion proteins [23, 24]. Much less is known bout the norml function of the ntive ALK protein. Bsed on its expression pttern [21, 22], ALK is believed to be involved in erly neurogenesis. In Drosophil, the lignd for ALK is jelly belly, but no humn homolog of jelly belly hs been identified [25]. Insted, there re two known ALK lignds: pleiotrophin [26] nd midkine [27]. Both re polypeptide nerve growth fctors tht cn bind to other receptors besides ALK. Binding of pleiotrophin to ALK led to both MAPK- [28] nd phosphtidylinositol 3-kinse (PI3K) medited cell growth nd MAPK-medited ntipoptotic signls [28, 29]. The MAPK signling pthwy, especilly MEK, is importnt for the promotion of cell growth nd neurite outgrowth of the SK-N-SH neuroblstom cell line [30]. ALK is lso negtively regulted by receptor protein tyrosine phosphtse (RPTP) /, which dephosphoryltes ALK. RPTP / is lso receptor for pleiotrophin nd is inctivted by pleiotrophin upon binding to RPTP /, llowing the continul utophosphoryltion of ALK [30]. Thus, pleiotrophin cn ctivte ALK both directly nd indirectly. Expression of midkine during centrl nervous development correltes with the expression of ALK [31]. Midkine ctivtes ALK, resulting in incresed N-myc nd trkb expression nd the prolifertion of immture sympthetic neurons. Midkine lso (through different pthwy) increses the expression of trnscription fctor, Hnd2, tht results in the upregultion of ALK expression, resulting in positive utoregultory pthwy [31]. ALK hs recently been recognized s dependence receptor, where in its inctive form (without lignd binding) it is propoptotic, wheres ALK is ntipoptotic in its ctive form (with lignd binding or berrntly trnslocted in mlignncies) [32, 33]. ALK cn enhnce poptosis following cspse-medited clevge t position D1160 in the ALK juxtmembrne region, disrupting the ALK kinse domin nd exposing propoptotic region encompssing mino cids in the intrcellulr juxtmembrne domin [33].

3 Ou, Brtlett, Mino-Kenudson et l Figure 1. Signling pthwys trnsduced by EML4 ALK. Abbrevitions: ALK, nplstic lymphom kinse; EGFR, epiderml growth fctor receptor. Activtion/phosphoryltion prevents cspse-medited clevge, resulting in n utoregultory cycle. Overexpression of ALK led to poptosis of primry corticl neurl cultures nd neuroblsts [33]. ALK knockout mice re vible nd exhibited no phenotypic bnormlities. They do exhibit n ge-dependent increse in bsl hippocmpl neurogenesis consistent with its norml role in propoptotic enhncement nd the restriction of ALK expression lrgely to the centrl nervous system [34]. Consistent with the role of ALK in neurogenesis, ctivting muttions in ALK led to both fmilil [35] nd spordic neuroblstom [36 38], nd provide the rtionle for n ALK inhibitor in the tretment of neuroblstom [37]. One of the mjor differences between the signling pthwys engged by ntive ALK nd EML4-ALK is the difference in the subcellulr loction. Ntive ALK is trnsmembrne nd generlly not ctivted, wheres EML4-ALK is cytoplsmic in loction nd in constitutively ctivted form. EML4- ALK hd been shown to engge ll three mjor signling pthwys involved in receptor tyrosine kinses (RTKs): MAPK/MEK/ERK, PI3K/AKT, nd RAS/STAT3 [39 41], but it remins reltively uncler which pthwy(s) re criticl to pthogenesis of NSCLC by EML4-ALK. Tkezw et l. recently demonstrted tht both the MAP kinse (MEK/ERK) nd JAK/STAT3 pthwys but not the PI3K/AKT pthwy re engged by EML4-ALK to induce poptosis. Cell lines with stbly trnsfected EML4-ALK vrints 1 nd 3 hd mrkedly incresed phosphoryltion of MEK, ERK, nd STAT3 but not AKT [42]. Inhibition of EML4-ALK with TAE684, smll-molecule ALK inhibitor, led to decresed phosphoryltion of ERK nd incresed levels of BIM, pro-poptotic protein. BIM is member of the BCL-2 fmily of proteins nd is degrded vi phosphoryltion by ERK. It hs been recently shown tht the level of BIM corresponds to the bility of tyrosine kinse inhibitors to induce poptosis in vrious cell lines hrboring driver muttions with the higher levels BIM ssocited with higher levels of poptosis. Simultneously nd by n independent mechnism, TAE684 led to decresed phosphoryltion of STAT3 nd reduced levels of survivin [42]. Survivin directly nd indirectly inhibits cspses, leding to poptosis. Thus, EML4-ALK in NSCLC results in ctivtion of ERK nd STAT3, which leds to decresed BIM nd incresed survivin, llowing for the synergistic effects of ntipoptosis nd promotion of tumor growth (Fig. 1). CLINICOPATHOLOGIC CHARACTERISTICS OF IN PATIENTS WITH ALK-REARRANGED NSCLC Mny retrospective tissue bnk studies hve demonstrted the incidence of ALK-rerrnged NSCLC to be pproximtely 3% 5% in NSCLC, with no pprent difference in incidence by ethnicity. Ptients with ALK-rerrnged NSCLC tend to be young (pproximtely 50 yers of ge t dignosis) nd neversmokers ( 70% 75%) or light smokers. The vst mjority present with denocrcinom nd there is no sex preference for mles or femles. Compred to ptients with EGFR muttions, ALK-positive ptients hd younger medin ge of dignosis

4 1354 Crizotinib: The Second Decde of Trgeted Therpy Tble 1. Ptients with simultneous ALK nd EGFR rerrngements reported in the literture Reference n of ptients Type of EGFR muttion Rimkuns et l. [56] 2 L858R (detected by specific EGFR L858R ntibody) Frequency of EGFR muttions in ALK-positive NSCLC Ptient chrcteristics Method of ALK detection 2/22 NR Biopsy, FISH, nd IHC Response to EGFR TKI Lee et l. [55] 3 Exon 19 del/a750p NA Mn, 73 yrs, former Biopsy, FISH PD PR smoker, denocrcinom Exon 19 del Mn, 43 yrs, neversmoker Biopsy, FISH NA NA L718P Womn, 46 yrs Biopsy, FISH NA NA Yng et l. [53] 2 4/42 Never-smokers, denocrcinom RT-PCR vrint 1 (3 pts) nd vrint 6 (1 pt) Response to crizotinib (61 vs. 57 yers old, respectively) nd higher proportion of never-smokers or light smokers ( 100 cigrettes per lifetime; 48% vs. 67%) [43]. The vst mjority of ALK-rerrnged NSCLCs possess wild-type EGFR nd KRAS genes [44, 45], lthough coexisting ALK rerrngements nd other driver muttions hve been described (Tble 1) [46 56]. Severl lrge prospective moleculr profiling epidemiology studies (e.g., the Lung Cncer Muttion Consortium in the USA [47], the Institut Ntionl Du Cncer in Frnce, nd LUNGSCAPE in Europe [57]) will provide dt on the incidence nd clinicl chrcteristics of ALK rerrngement s groups begin to report their findings. Most recently, the Lung Cncer Muttion Consortium reported tht, mong 901 ptients using ALK brekprt ssy, ALK positivity ws 8.3% in denocrinom in cliniclly enriched popultion (younger, with more women nd never smokers) [58]. In ddition, Li et l. reported rte of 3.2% of ALK-positivity fter lrge-scle screening 4,500 ptients with NSCLC formlin-fixed prffinembedded (FFPE) tissue using multiplex reverse trnscriptsepolymerse chin rection (RT-PCR) ssys tht included nine known EML4-ALK fusion trnscripts nd ALK RNA levels [59]. Mny histologic fetures hve been scribed to ALK-rerrnged NSCLC, including cinr [60, 61], ppillry [60, 62 63], microppillry [63], bronchiolveolr [60, 62 63], nd the presence of signet ring cells [64 66]. Yoshid et l. performed n extensive histology review of 54 ALK-rerrnged NSCLC resections nd identified two mjor nd different histologic ptterns: solid growth pttern with focl signet ring cell component nd mucinous cribriform pttern ssocited with significnt extrcellulr mucus mterils [67]. Severl studies hve shown tht signet ring cell differentition is common specific feture of ALK-positive NSCLC within up to two-thirds of tumors exhibiting this morphology [64 67]. Interestingly, mucinous cribiform component hs recently been shown to be much more common in receptor tyrosine kinse fusion-positive NSCLC nd my be used s nother feture to drw ttention to the potentil presence of ALK NR 2 pts hd PR (L858R, exon 19) Doebele et l. [46] 1 S768I 1/42 Womn, 53 yrs, Biopsy, FISH NR NA never-smoker, denocrcinom with focl spindle differentition Sski et l. [51] 3 L868R 3/50 Adenocrcinom Biopsy, FISH Erlotinib, PR NA Exon 19 del Adenocrcinom Biopsy, FISH 9 months: NA erlotinib, PR A767_V769dup ASV Adenocrcinom Biopsy, FISH 5 months: NA NA Kris et l. [47] 2 NR 2/43 Adenocrcinom Biopsy, FISH NR NA Popt et l. [50] 1 Exon 19 del NA Womn, 65 yrs, white, never-smoker, denocrcinom, TTF- 1 positive Kuo et l. [48] 1 Exon 19 del NA Womn, 72 yrs, Asin, never-smoker, denocrcinom, TTF- 1 positive Tiseo et l. [52] 1 Exon 19 del NA Mn, 48 yrs, white, never-smoker, poorly differentited denosqumous Zhng et l. [54] 1 Exon 19 del 1/12 Womn, Chinese, never-smoker, denocrcinom Biopsy, FISH RT-PCR (vrint 1) Biopsy, FISH RT-PCR (vrint 3b) Erlotinib, 25 months Gefitinib, clinicl response, 232 dys Erlotinib, no response Abbrevitions: FISH, fluorescent in situ hybridiztion; IHC, immunohistochemistry; NA, not pplicble; NR, not reported; NSCLC, non-smll cell lung cncer; PD, progressive disese; PR, prtil response; pt, ptient; RT-PCR, reversetrnscriptse polymerse chin rection; TKI, tyrosine kinse inhibitor. NR NA NA NA NA NA NA

5 Ou, Brtlett, Mino-Kenudson et l Figure 2. List of ALK fusion vrints in NSCLC nd proportion of vrious of EML4-ALK fusion vrints. (A): Schemtic of vrious forms of ALK fusions in ALK-rerrnged NSCLC [17, 19, 39, 63, 66, 68, 70 78, 75]. Adpted from Sski T, Rodig SJ, Chiriec LR et l. The biology nd tretment of EML4-ALK non-smll cell lung cncer. Eur J Cncer 2010;46: , with permission. (B): Pie chrt of distribution of EML4 ALK vrious fusions [17, 39, 41, 49, 61, 63, 66, 71 75, 75, 79, 80, 90 94]. rerrngement [68]. However, none of the documented histologic fetures were sufficiently sensitive or specific to predict for ALK rerrngement [69]. Likewise, lthough lymphovsculr invsion nd necrosis, TTF-1 expression, nd p63 expression (generlly considered squmous cell mrker but found to be expressed in ALK-positive denocrcinom) were ll found significntly more often in ALK-positive thn in ALK-negtive smples, such fetures should not be used to replce current dignostic tests for ALK rerrngement [67]. There re now t lest 27 different ALK fusion vrints in NSCLC reported in the literture: 21 EML4-ALK isoforms (including one with fusion to exon 19 of ALK) [17, 39, 63, 66, 70 75, 75, 75b], 3 KIF5B-ALK isoforms [68, 75, 75, 76], 1 KCL1-ALK isoform [77], 1 TFG-ALK isoform [19] nd 1 ALK- PTPN3 isoform [17, 19, 39, 63, 66, 68, 70 75, 75, 76 78] (Fig. 2A, Tble 2). The distribution of EML4-ALK vrints hs been previously reported [89] nd we hve performed n updted nlysis bsed on totl of 371 published EML4-ALK vrints. Vrint 1 (49.6%) mde up the most common type of the EML4-ALK fusion trnscripts, followed by vrint 3/b (25.6%) nd vrint 2 (10%) [17, 39, 41, 49, 61, 63, 66, 71 75, 75, 79, 80, 90 94] (Fig. 2B).

6 1356 Crizotinib: The Second Decde of Trgeted Therpy Tble 2. List of ALK fusion vrints in NSCLC reported in the literture Vrint Symbol References c EML4-ALK 1 E13; A20 Sod et l. [17] E10del54 E13;A20 Wng et l. [75] 2 E20; A20 Sod et l. [17] E20ins18; A20 Tkhshi et l. [73] 3 E6; A20 Choi et l. [70] 3b E6ins33; A20 Choi et l. [70] E6ins18; A20 Wng et l. [75] 4 E14; ins11del49a20 Tkeuchi et l. [74] 5 E2; A20 Tkeuchi et l. [74] 5b E2; ins117a20 Tkeuchi et l. [74] 6 E3; ins69a20 Tkeuchi et l. [75] 7 E14; del12a20 Tkeuchi et l. [75] E14; del36a20 Yoshid et l. [66] 8 b E17; ins30a20 Snders et l. [72] 8b E17ins61; ins34a20 Snders et l. [72] E17del58ins39; A20 Wng et l. [75] E17ins65; A20 Wng et l. [75] E17ins68; A20 Tkhshi et l. [73] V4 E15del19; del20a20 Koivunen et l. [39] V5 E18; A20 Wong et l. [63] E6; A19 Doebele et l. [71] KIF5B-ALK KI24; A20 Tkeuchi et l. [75] KI15; A20 Wong et l. [76] KI17; A20 Tkeuchi et l. [68] KLC1-ALK KL9; A20 Togshi et l. [77] TFG-ALK T3; A20 Rikov et l. [19] ALK-PTPN3 Exons 10 nd 11 of ALK inserted between exons 2 nd 3 of PTPN3 Jung et l. [78] Only the reference tht first reported the vrint is cited here. These vrints hve not been officilly nmed. b Vrint 8 contins n internl stop codon within the 30 bp insertion. Vrint 8 ws discovered in the sme smple s vrint 8b. c Only reference tht first reported the vrint is cited here. Despite these vrious EML4-ALK fusion trnscripts in NSCLC, Li et l. did not find ny significnt difference in the medin ge of dignosis nd gender composition mong vrints V1 5 in 152 ptients with ALK-rerrnged NSCLC [59]. However, there is in vitro evidence tht vrious ALK fusions hve different sensivitivites to ALK inhibitors nd my hve different intrinsic protein stbility within the cell [78], lthough EML4-ALK vrints 1 nd 3 hd similr clinicl outcome treted with first-line pltinum-bsed chemotherpy [78b]. EML4-ALK fusion trnscripts hve been found in solid tumors other thn NSCLC [79, 80]. Using tumor repository, Lin et l. reported EML4-ALK in 2.4% of brest cncer (5/209) nd colorectl cncer (2/83) [80]. Recently, EML4-ALK hs lso been detected in non-cler cell renl cell crcinom using n immunohistochemicl screening method [81]. The frequency of KIF5B-ALK in denocrcinom of the lung estimted from the three reports ws 4 of 1,331 ptients or pproximtely 0.3% [68, 75, 76]. It is importnt to note tht the

7 Ou, Brtlett, Mino-Kenudson et l Tble 3. Comprison of overll survivl of ptients with ALK-rerrnged NSCLC treted with crizotinib s second-line or third-line tretment versus crizotinib-nïve ptients with ALK-rerrnged NSCLC [95] Crizotinib-treted ptients Crizotinib-nïve ptients p vlue n of ptients Medin overll survivl, mos (95% CI) NR (14 NR) 6 (4 17) 1-yer survivl (95% CI) 70% (50 83) 44% (23 64) 2-yer survivl (95% CI) 55% (33 72) 12% (2 30).004 Abbrevitions: CI, confidence intervl; NR, not reched. Tble 4. Comprison of overll survivl of crizotinib-treted ptients with ALK-rerrnged NSCLC nd tyrosine kinse inhibitor-treted ptients with EGFR-mutted NSCLC [95] Crizotinib-treted ptients TKI-treted ptients p vlue Overll n of ptients Medin overll survivl, mos (95% CI) NR (17 NR) 24 (15 34) 1-yer survivl (95% CI) 71% (58 81) 74% (61 83) 2-yer survivl (95% CI) 57% (40 71) 52% (38 65).786 As second-line or third-line tretment n of ptients Medin overll survivl, mos (95% CI) NR (14 NR) 15 (12 32) 1-yer survivl (95% CI) 70% (50 83) 72% (46 87) 2-yer survivl (95% CI) 55% (33 72) 43% (20 64).578 Abbrevitions: CI, confidence intervl; NR, not reched; TKI, tyrosine kinse inhibitor. three fusion prtners to ALK EML4, KIF5B, nd KLC1 ll interct with the microtubule pprtus [82, 86]. Therefore, gents tht interrupt the ssembly of microtubules such s txnes or vinc lkloids my be useful in the tretment of ALKrerrnged NSCLC lone nd/or in combintion with n ALK inhibitor. Finlly, n ALK-PTPN3 vrint hs been observed from the insertion of genomic DNA encompssing exons 10 nd 11 of ALK into the intronic region between exons 2 nd 3 of protein tyrosine phosphtse, nonreceptor type 3 gene (PTPN3) [78]. Of note, ALK-PTPN3 cn theoreticlly generte splitprt fluorescent in situ hybridiztion (FISH) signl but will not respond to crizotinib s the ALK kinse domin is bsent. The clinicl presenttion nd course of ptients with ALKrerrnged NSCLC hve not been extensively described. One study reported tht ptients with ALK-rerrnged NSCLC tended to present with higher frequency of pericrdil nd pleurl-bsed disese, n incresed propensity for liver but not brin metstses, nd n incresed number of sites of metstsis compred with EGFR nd ALK /EGFR ptients [46]. Similrly of the 901 ptients with stge IIIB/IV denocrcinom profiled to dte for ALK rerrngement by the Lung Cncer Muttion Consortium, ALK-positive disese ws significntly ssocited with liver metstsis compred with ALK-negtive disese (23% vs. 10%; p.004), but no difference ws detected in bone, brin, or drenl glnd metstses [58]. Ptients with ALK-rerrnged NSCLC my lso present with uncommon sites of metstsis such s retinl metstsis [46]. In retrospective study, Shw et l. hve demonstrted mong contemporneously dignosed ptients with ALK-rerrnged NSCLC tht those treted with crizotinib hd significnt survivl dvntge over those who did not receive crizotinib (Tble 3) [95]. In fct, crizotinib-treted ptients with ALK-rerrnged NSCLC chieved excellent survivl outcomes comprble with EGFR muttion-positive ptients who received EGFR TKIs (Tble 4) [95]. Tken together, these studies indicte tht it is importnt to dignose ptients with ALK-rerrnged NSCLC erly in the tretment course to initite effective ntitumor tretment. Recently, severl retrospective studies hve shown tht pemetrexed-bsed chemotherpy my confer high tumor response either s first-line combintion with pltinum [96], s single gent in second-line or beyond tretment [97, 98] or longer progression-free survivl when first-line pltinum/pemetrexed combintion is used in ptients with ALK-rerrnged NSCLC [98]. However, these nlyses my hve confounded by some of the clinicl demogrphics of the ALK-positive popultion such s younger ge nd/or never-smoking sttus [98]. On the other hnd, lrger retrospective nlysis of tretment

8 1358 Crizotinib: The Second Decde of Trgeted Therpy histories from ptients enrolled in the PROFILE 1005 study, which is globl phse II single rm study of crizotinib in ptients with ALK-rerrnged NSCLC, showed tht mong the 711 ptients who hd received pemetrexed s combintion therpy or s single gent, overll response rte (ORR) nd time-to-progression outcomes were similr to unselected ptients with NSCLC who were enrolled in phse III rndomized trils compring the use of first-line pltinum/pemetrexed or second-line pemetrexed nd treted with pemetrexed [99]. It will be importnt to complete the enrollment nd wit the results of the rndomized trils compring crizotinib to pltinum pemetrexed-bsed chemotherpy in the first-line tretment setting (PROFILE 1014) or to stndrd second-line chemotherpy (pemetrexed or docetxel; PROFILE 1007), which ws recently reported to meet its primry endpoint [100]. The prognostic significnce of ALK rerrngement in NSCLC hs not been settled. In two seprte reports [65, 95], Shw et l. did not demonstrte ny significnt differences in overll survivl (OS) for ptients with NSCLC by EML4-ALK sttus in the er before crizotinib. Similrly, Zhng et l. reported no survivl difference ccording to ALK sttus fter djusting for disese stge, histology, nd EGFR/KRAS mutnt sttus [101]. Lee et l. showed tht ptients with ALK-rerrnged NSCLC hd the shortest overll survivl compred to wild-type ptients, but the difference ws not significnt [102]. On the other hnd, Kim et l. were ble to demonstrte tht ptients with ALK-rerrnged NSCLC hd significnt worse OS outcomes fter fctoring in ge, sex, histology, stge, nd performnce sttus [103]. Similrly, Yng et l. found tht EML4-ALK sttus is poor prognostic fctor for relpse-free survivl fter fctoring in stge, sex, ge, nd tretment [104]. Conversely, Wu et l. found tht ptients with EML4-ALK NSCLC identified from pleurl effusion cytology hd significntly improved survivl outcome compred with ptients without EML4-ALK NSCLC [94]. Furthermore, Tkeuchi et l. found tht receptor kinse fusion-positive NSCLC is n independent fvorble prognostic fctor fter tking into considertion ge, sex, stge, nd smoking sttus [68]. All of these studies re limited by the smll number of ALK-positive ptients, different comprison group of ptients, the heterogeneous tretment ptients received, nd differences in the blnce of prognostic fctors (e.g., smoking sttus, surgicl tretment, ge) compred with ALK-rerrnged ptients. ORIGINAL DESIGN AND SYNTHESIS OF CRIZOTINIB Crizotinib Development nd Preclinicl Activity Crizotinib is competitive ATP inhibitor ginst both wildtype MET nd ginst MET ATP-binding site mutnts (V1092I, H1094R), P-loop mutnt (M1250T), nd juxtmembrne domins mutnts (R988C, T1010I), but is not ctive ginst ctivtion loop mutnts (Y1230C, Y1235D) [105]. The cellulr effects from MET inhibition by crizotinib re multifold: induction of poptosis, decrese in prolifertion, nd decrese in ngiogenesis [106]. Crizotinib inhibited the growth of the MET-dependent GTL-16 gstric crcinom cell line, nd inhibited cell migrtion nd invsion of the HGF-stimulted NCI-H441 lung cncer cell line nd the growth of multiple humn crcinom xenogrft models including gstric crcinom (GTL-16), renl cell (Cki-1), glioblstom (U87MG), prostte (PC-3), nd NSCLC (NCI-H441) [106]. After its synthesis s MET inhibitor [ ] (Figures 3A nd 3B [ ]), crizotinib ws evluted ginst pnel of 120 kinses in biochemicl ssys nd 12 cell-bsed phosphoryltion ssys reveled tht it lso inhibited phosphoryltion of NPM-ALK in both Krps 299 nd SU-DHL-1 ALCL cells with men IC 50 of 24 nmol/l [110]. Since both Krps 299 nd SU-DHL-1 cell lines express detectble MET levels, the lck of inhibitory ctivity of two specific MET inhibitors (PHA nd PF ) indicted tht the inhibitory effect of crizotinib on these two cell lines ws not due to its nti-met ctivity. Crizotinib lso resulted in dose-dependent growth inhibition of the Krps299 xenogrft in SCID-Beige mice. This inhibition ws gin ccomplished by inhibition of NPM-ALK phosphoryltion nd the phosphoryltion of NPM-ALK s downstrem signling meditors: PLC- nd STAT3 over wide rnge of crizotinib concentrtions, nd AKT nd ERK kinses t reltively high concentrtion of crizotinib [110]. CLINICAL DATA FOR CRIZOTINIB First In-Humn Phse I Tril of Crizotinib The first in-humn crizotinib tril (PROFILE 1001, NCT ) ws written s n open-lbel, multicenter tril tht ws ctivted in 2006 with n initil stndrd dose-escltion phrmcokinetic (PK) portion followed by clinicl efficcy portion tht imed to enroll smll number of moleculrly enriched ptients to ssess the ntitumor ctivity of crizotinib. The initil PK portion used the stndrd doseescltion finding schem to determine the mximl tolerted dose (MTD) nd the recommended phse II dose (RP2D). There were severl substudies to investigte the effect of food on crizotinib PK, midzolm substudy to ssess the effect of CYP3A isozyme inhibition by crizotinib, nd study to ssess the fesibility of using fluoro-l-thymidine positron emission tomogrphy to monitor response to crizotinib s n lterntive to fluorodeoxyglucose positron emission tomogrphy [111]. Phrmcokinectics of Crizotinib Crizotinib ws first dosed t 50 mg orlly once dily nd eventully esclted to 300 mg orlly b.i.d., t which dose two ptients experienced grde 3 ftigue [112]. The dose of crizotinib ws reduced to 250 mg orlly b.i.d. nd ws found to be tolerble; this dose ws determined to be the MTD nd RP2D dose [112]. Eleven ptients were enrolled onto the food effect substudy. The bsorption of single dose of 250 mg of crizotinib fter high-ft, high-clorie mel ws 14% lower thn the concentrtion of single dose of 250 mg of crizotinib tken on n empty stomch [112, 113]. Thus crizotinib could be tken with or without food. The pek plsm concentrtion ws reched

9 Ou, Brtlett, Mino-Kenudson et l Figure 3. Synthesis of crizotinib. (A): Design nd synthesis of crizotinib. Figure 3A provided by Jen Cui. Plese refer to reference 107 for detiled synthesis of crizotinib. (B): Crystllogrphy of crizotinib in unphosphorylted MET nd ALK [108, 109]. PDB ID 2wgj for PF /c-MET complex; PDB ID 2xp2 for PF /ALK complex. Figure 3B provided Jen Cui. Abbrevition: MW, moleculr weight. 4 6 hours fter single dose of crizotinib [113]. There ws liner PK from 100 mg of crizotinib once dily to 300 mg of crizotinib b.i.d. [114]. Stedy-stte concentrtion of crizotinib ws reched within 15 dys of repeted dministrtion of crizotinib t 250 mg orlly b.i.d., with hlf-life of pproximtely hours [114]. The men stedy-stte trough plsm level for 250 mg crizotinib b.i.d. (the recommended phse II dose) is 274 ng/ml or 57 nm of free drug, which exceeded the trget efficcy levels predicted for the inhibition of MET ( 13 nm) nd ALK ( 26 nm) bsed on preclinicl mouse models [114]. Metbolism nd Drug Interctions Thirteen ptients were enrolled in the midzolm substudy. The PK of single 2 mg midzolm dose ws evluted before nd 28 dys fter 250 mg of crizotinib orlly b.i.d.. Midzolm is metbolized by CYP3A isozymes nd there ws 3.6-fold increse in the midzolm level (90% confidence intervl [CI]: 2.7

10 1360 Crizotinib: The Second Decde of Trgeted Therpy Figure 4. Stedy-stte crizotinib levels in Asins nd non-asins receiving repeted doses of crizotinib 250 mg twice dily [115]. 4.9) fter 28 dys of crizotinib dministrtion, indicting tht crizotinib is moderte CYP3A inhibitor [114]. Codministrtion of strong CYP3A inhibitors or inducers should be voided. Ethnicity The phse I study reveled higher exposure to crizotinib in Asin ptients t stedy stte fter crizotinib 250 mg b.i.d. thn in non-asin ptients (Fig. 4) [115]. Exposure to crizotinib remined higher in Asin ptients even fter correction for idel body weight, body surfce re, or body mss index [115]. Recently, exposure-response nlyses from both PROFILE 1001 nd 1005 showed tht Asin ptients hd higher ORR nd longer progression-free survivl (PFS) times, ssocited with higher crizotinib exposure thn non-asin ptients [116]. CLINICAL EFFICACY OF CRIZOTINIB IN PATIENTS WITH ALK-REARRANGED NSCLC Following the discovery of the EML4-ALK trnsloction in NSCLC in 2007 [17, 19] nd in the context of crizotinib s ALK inhibitor ctivity [110], commercilly vilble brek-prt FISH ssy for detecting ALK rerrngement in ALCL ws modified to detect ALK rerrngement in NSCLC. The first ptient with EML4-ALK NSCLC ws treted with crizotinib on the phse I dose-esclting cohort (300 mg b.i.d.) t Msschusetts Generl Hospitl on December 26, 2007 with rpid clinicl benefit, but ws tken off tril due to liver enzyme elevtions despite dose reduction. Six months lter, the second ptient with EML4-ALK NSCLC ws treted with crizotinib, with clinicl benefit nd stble disese. Bsed on the clinicl benefit from crizotinib for these two ptients, there ws concerted shift mong the phse I clinicl sites to screen for NSCLC ptients with ALK-rerrngement. The clinicl efficcy dt for crizotinib in ALK-rerrnged NSCLC ptients hve been remrkbly consistent t multiple dt cutoff dtes, with n ORR of pproximtely 60% over the 4 yers since the first report from 19 ptients in 2009 (Tble 5) [90, 112, ]. The updted estimted PFS is stble t pproximtely 9.7 months [118]. The clinicopthologic chrcteristics of ptients with ALK-rerrnged NSCLC enrolled onto the study remined remrkbly constnt with medin ge of 51, lthough ptients s young s 21 nd s old s 86 were enrolled gin underlying the heterogeneous popultion of ptients with NSCLC hrboring ALK rerrngement. Only 70% of these ptients were never- or light-smokers. The vst mjority of ptients with ALK-rerrnged NSCLC presented with denocrcinom (Tble 5). An ongoing single-rm, globl phse II study of crizotinib ws lunched in 2009 (PROFILE 1005, NCT ) bsed on the initil dt from the phse I study, with centrl dignosis of ALK rerrngement using the Vysis FISH test (Abbott Moleculr, Abbott Prk, IL; t the time lso under evlution by the U.S. Food nd Drug Administrtion [FDA]) for the first 250 ptients enrolled. (The protocol ws subsequently mended to llow NSCLC ptients with ny ALK-positive test performed outside the centrl lbortory to be enrolled on cse-by-cse bsis.) This tril lso provides n venue for ptients with ALK-rerrnged NSCLC rndomized to the control chemotherpy rm of the second-line phse III PROFILE 1007 (NCT ) tril to ccess crizotinib if they experience progression. Dt from PROFILE 1005 showed tht ptients hd very similr clinicopthologic chrcteristics (Tble 6) to those enrolled in PROFILE 1001, nd the first 261 ptients enrolled (whose ALK positivity ws centrlly confirmed by FISH) chieved response rte of 60% (95% CI: 54% 66%) with medin PFS of 8.1 months (95% CI: ) nd medin durtion of response of 46 weeks [119]. In ddition, best overll response ppered to be independent of the percentge of cells positive for ALK rerrngement by FISH in the dignosis smple [120]. ORR ws independent of ge, sex, nd number of prior metsttic tretment regimens. Responses were chieved rpidly, with pproximtely hlf of the observed responses occurring t the time of first follow-up scn: within 8 weeks (PROFILE 1001) nd 6 weeks (PROFILE 1005) [118, 119]). Adverse Events with Crizotinib As of Jnury 2012, more thn 1,000 ptients hd been treted in PROFILE 1001 nd PROFILE 1005, nd the sfety profile for crizotinib hs been consistent between these studies [118, 119]. Crizotinib ws generlly well tolerted with the mjority of dverse events being grde 1 or 2, nd low rtes of with-

11 Ou, Brtlett, Mino-Kenudson et l Tble 5. Summry of clinicopthologic chrcteristics of ptients with ALK-rerrnged NSCLC nd clinicl ctivity of crizotinib in ptients enrolled in the first in-humn tril (PROFILE 1001) over period of 4 yers Clendr yer reported 2009 [112] 2010 [90] 2011 [117] 2012 [118] Dt cutoff dte Mrch 9, 2009 April 7, 2010 October 10, 2010 June 1, 2011 n of ptients (totl/evluble) 19/19 82/82 119/ /143 Medin ge, yrs (rnge) 50 (28 73) 51 (25 78) 51 (21 79) 52 (21 86) Never-smokers (%) Adenocrcinom (%) Men (%) Ethnicity (%) White NR Asin Other No prior tretment regimen (%) ECOG PS 2 (%) ORR (95% CI) ( ) 60.8 ( ) SD (%) DCR t 8 weeks (95% CI) ( ) 82.5 ( ) Medin time on tretment (95% CI) NR 6.4 mos 31.9 wks ( ) 43.1 wks ( ) Medin follow-up time (mos) NR NR 9.9 mos 16.3 mos Estimted PFS, mos (rnge) NR NR 10.0 ( ) 9.7 ( ) Medin durtion of response (wks) NR NR Men. Abbrevitions: CI, confidence intervl; DCR, disese control rte; ECOG PS, Estern Coopertive Oncology Group performnce sttus; NR, not reported; PFS, progression-free survivl; ORR, overll response rte; SD, stble disese. drwl due to dverse events. The most commonly reported tretment-emergent, ll-cuslity dverse events were nuse, dirrhe, vomiting, constiption, nd visul effects [113]. Other events reported t frequencies 20% in one or both studies included peripherl edem, dizziness, ftigue, nd decresed ppetite [113]. Visul effects with crizotinib re prticulrly noteworthy s such events re not commonly ssocited with cncer therpies; however, no ptient required dosing interruption, dose reduction, or permnent discontinution of crizotinib tretment becuse of visul effects. Ptientreported symptom dt reported from ptients enrolled on PROFILE 1005 showed tht visul events were short-lived (the mjority of events lsted 1 minute), occurred less frequently s tretment continues, nd were not bothersome, with no effect on ptient s ctivities of dily living [119]. Further detil will be elucidted s these dt mture nd more ptients re included. Bsed on dt from ophthlmologic exmintions performed t bseline nd post-tretment in clinicl trils, no cliniclly meningful chnges in visul cuity, biomicroscopy, or ophthlmoscopy in ptients experiencing visul disturbnces occurred [121]. Heptic nd Pulmonry Adverse Events with Crizotinib Trnsminse elevtions hve been noted with crizotinib, generlly occurring within the first 2 months of tretment, with medin onset of 40 dys for ny grde ALT elevtion. Among 1,054 ptients treted with crizotinib in both PROFILE 1001 nd PROFILE 1005, ALT elevtions of greter thn 3, 5, nd 10 times the upper limit of norml occurred in 15%, 7.4%, nd 3% of ptients, respectively. The incidences of lnine minotrnsferse (ALT), sprtte minotrnsferse, nd lkline phosphtse (AP) elevtion were comprble for grde 1 nd grde 2 events, but grde 3 nd grde 4 events were more frequent for ALT elevtions. Totl bilirubin elevtion ws less common, suggesting tht heptic function is less ffected in most cses of liver injury. Temporry discontinution or dose reduction occurred in 5.3% of ll ptients, nd permnent discontinution rte due to heptic dverse events ws 1.3%. Trnsminse elevtions were usully reversible nd ptients were ble to resume the tretment t the sme or t lower dose. Ftl drug-induced heptotoxicity occurred in 1% of ptients in these studies [122]. Concurrent elevtions in ALT greter thn 3 times the upper limit of norml nd totl bilirubin greter thn 2 times the upper limit of norml, with norml AP, occurred in less thn 1% of ptients in clinicl trils. Liver function test monitoring is recommended, including ALT nd totl bilirubin once month nd s cliniclly indicted, with more frequent repet testing for incresed liver trnsminses, AP, or totl bilirubin in ptients who develop trnsminse elevtions [113]. Crizotinib hs been ssocited with severe, life-threten-

12 1362 Crizotinib: The Second Decde of Trgeted Therpy Tble 6. Clinicopthologic chrcteristics of ptients with ALK-rerrnged NSCLC enrolled in the PROFILE 1005 tril [119] Ptient chrcteristics First ptients enrolled, with ll confirmed to be ALK-positive vi centrlized FISH (mture cohort) Enrolment to dte, with ALK-positivity confirmed centrlly or loclly n of ptients Age, medin yrs (rnge) 52 ( ) 53 ( ) Women (%) 142 (54.4) 514 (57) Ethnicity (%) White 154 (59) 485 (53.8) Blck 8 (3.1) 18 (2.0) Asin 94 (36) 379 (42.1) Other 5 (1.9) 19 (2.1) Bseline ECOG PS (%) 0 68 (26.1) 225 (25.0) (56.7) 511 (56.7) 2 42 (16.1) 134 (14.9) 3 3 (1.1) 31 (3.4) Adenocrcinom (%) 245 (93.9) 826 (91.7) Smoking clssifiction (%) Never smoker 176 (67.4) 592 (65.7) Former smoker 73 (28.0) 271 (30.1) Smoker 12 (4.6) 38 (4.2) Prior therpies for loclly dvnced/ metsttic disese (%) 0 0 (0) 3 ( 1.0) 1 32 (12.3) 248 (27.5) 2 91 (34.9) 299 (33.2) (52.8) 351 (39.0) Dt re n (%) unless noted. Three ptients were not eligible due to prior djuvnt tretment only. Abbrevitions: ECOG PS, Estern Coopertive Oncology Group performnce sttus; FISH, fluorescense in situ hybridiztion. ing, or ftl tretment-relted pneumonitis/interstitil lung disese (ILD). Four cses mong 255 ptients (1.6%) in PROFILE 1005 nd PROFILE 1001 hve been reported [113]. Ptients should therefore be monitored for pulmonry symptoms indictive of pneumonitis/ild in ddition to NSCLC progression. Other cuses of pulmonry symptoms, such s other pulmonry disese, infection, or rdition effects, should be excluded [113]. Endocrine Adverse Events Recently, the University of Colordo reported tht 13 men with NSCLC experienced rpid decrese in totl testosterone levels fter inititing crizotinib potentilly vi hypothlmic or pituitry effect, which my ccount for some of the ftigue experience by some ptients [123]. Testosterone promptly returned to the norml level fter crizotinib discontinution. However, hypogondism is multifctoril in its etiology in dvnced lung cncer. Further investigtion in n pproprite control setting will provide more comprehensive description nd understnding of this phenomenon. Specific Clinicl Situtions (Tretment Beyond Progression/Brin Metstsis) Both the PROFILE 1001 nd 1005 protocols llow crizotinib to be continued if the investigtors deem the ptient will continue to derive continul clinicl benefit. Of 138 ptients who continued on crizotinib for 2 weeks postprogression on PROFILE 1001 nd PROFILE 1005, totl of 53 ptients (46%) hd progression in the brin s new lesion or nontrget lesion [125]. Among the 42 ptients who received more thn 6 months tretment postprogression, hlf hd brin metstses nd some ptients hd received 40 weeks of postprogression tretment, with tretment still ongoing [125]. The level of crizotinib chieved in cerebrospinl fluid (CSF) hs been re-

13 Ou, Brtlett, Mino-Kenudson et l ported in one ptient who received crizotinib 250 mg orlly b.i.d. [124]. The CSF level ws only 0.26% of the plsm level (0.616 ng/ml; mol/l), level expected to be insufficient to inhibit ALK. Thus, progression in the brin lone likely reflect sntury site for crizotinib rther thn true resistnce to crizotinib given mny ptients continued to be on crizotinib fter progression in the brin. Additionlly, PROFILE 1001 nd PROFILE 1005 protocols empiriclly specify tht crizotinib be withheld the dy before, during, nd the dy fter ny rdition tretment, llowing ptients who received definitive rdition to the brin to continue crizotinib without undue interruption. This strtey hs been successfully employed by the University of Colordo to provide dditionl 6 months of PFS for selected ptients with ALK-rerrnged NSCLC who presented with oligoprogressive disese [126]. FDA APPROVAL OF CRIZOTINIB AND THE BREAK-APART FISH ASSAY The FDA greed to ccept clinicl efficcy nd sfety dt of crizotinib from the PROFILE 1001 nd PROFILE 1005 trils s the bsis for the new drug ppliction (NDA) filing in April Crizotinib ws grnted orphn drug designtion for ALK-positive NSCLC in September Fst-trck designtion for crizotinib ws grnted in December 2010 nd review of the NDA on rolling bsis begn in Jnury The brek-prt FISH ssy kit mnufctured by Abbott Moleculr ws required to stisfy two criteri: n nlyticl component nd clinicl utility. The exclusive use of the Abbott kit in PROFILE 1005 stisfied the clinicl utility requirement bsed on response rtes in tht study [115]. For the nlyticl component, Abbott Moleculr nd the Deprtment of Pthology t Msschusetts Generl Hospitl (which served s the centrl lbortory for PROFILE 1001) simultneously nd independently re-evluted the ptients enrolled in PROFILE 1001 using the Abbott Vysis probes with excellent concordnce ( 90%). The Abbott Moleculr brek-prt FISH testing regultory pckge ws submitted in My 2011, completing the regultory submission pckge to the FDA. Crizotinib ws pproved in the USA on August 26, 2011, primrily bsed on response rtes of 50% from the first 136 ptients with ALK-rerrnged NSCLC enrolled on PROFILE 1005 [127] nd secondrily on response rte of 61% from the first 119 ptients with ALK-rerrnged NSCLC enrolled on PROFILE 1001 [117]. The pprovl lnguge sttes: Xlkori is kinse inhibitor indicted for the tretment of ptients with loclly dvnced or metsttic non-smll cell lung cncer (NSCLC) tht is nplstic lymphom kinse (ALK)-positive s detected by n FDA-pproved test. This indiction is bsed on response rte. There re no dt vilble demonstrting improvement in ptient reported outcomes or survivl with Xlkori [113], with no restriction s to which line of tretment crizotinib could be used to tret ptients with ALK-positive NSCLC. Crizotinib is now lso pproved in mny countries, including Argentin, Cnd, Isrel, Jpn, Kore, Mcu, Mexico, nd Switzerlnd nd imminently by the Europen Union. WHAT IS THE BEST COMPANION DIAGNOSTIC TEST FOR DETECTING ALK REARRANGEMENTS? Brek-prt FISH Assy s the Gold Stndrd Moleculrly trgeted therpy in oncology is criticlly dependent on vlidted test to detect the moleculr ltertion in question, especilly when moleculr ltertions constitute smll subgroup of ptients. Idelly, the test should be both highly sensitive nd highly specific, reltively inexpensive, nd fesible in most dignostic lbortories to fcilitte worldwide doption. FISH is routinely used in oncology to detect chromosoml trnsloctions which ply n importnt role in soft tissue nd hemtologic mlignncies, including the dignosis of ALK-positive ALCL. Until recently, chromosoml trnsloctions were considered uncommon in epithelil tumors [128]. The sme ALK brek-prt FISH ssy used for ALCL ws the bsis for the ALK dignostic test eventully pproved by the FDA to detect ALK-rerrnged NSCLC in conjunction with the pprovl of crizotinib in the USA [113]. The performnce of the FISH ssy hs been rigorously evluted nd shown to be effective in nlyzing FFPE NSCLC specimens. Techniclly, ALK FISH cn be more chllenging thn most brek-prt ssys, becuse ALK nd its most common fusion prtner EML4 re situted very ner to ech other on chromosome 2 (pproximtely 12 megbses prt). EML4-ALK rerrngement is generted most commonly from n intrchromosoml inversion, which results in split signls tht my not be very fr prt in positive cses nd thus occsionlly difficult to interpret. FISH positivity is defined by the seprtion between the 5 nd 3 signls of greter thn two signl dimeters (Fig. 5A, 5B) [129] nd t lest 50 cells to be counted to ensure 100% sensitivity nd specificity [130]. A second common pttern in positive cses is the presence of isolted ALK 3 probes (red signls only; Fig. 5C), likely reflecting the presence of n ALK rerrngement with loss of the nonfunctionl derivtive chromosome. A creful nlysis of known ALK-negtive cses reveled cutoff of 15% of tumors cells to hve split signls, bove which cse will be clled positive (15% is 2 stndrd devitions bove the men cell count in negtive cses). Some cells my be flsely positive becuse the tumor cells counted my hve rtificil split signls due to sectioning rtifct or poor nucleus morphology. The men proportion of cells with split signls in ALK-positive NSCLC (n 13) ws 53.8% (rnge: 22.25% 86.62%) nd 5.98% (rnge: ) in ALK-negtive NSCLC (n 56; p.0001; Tble 7, Fig. 5D) [ ]. With the inclusion of more ALK-positive NSCLC smples (n 90), the men percentge positive cells remined similr t 56% (rnge: 18% 100%) [133]. The reson the percentge of positive cells in ALK-positive smples is not lwys 100% is due to the inherent technicl limittions of the ssy to detect rerrngement on cell-bycell bsis nd not becuse of tumor heterogeneity. Furthermore, 5 nd 3 split signls re more common thn isolted 3 signls in FISH-positive smples (Tble 8) [131, ]. Interestingly, NSCLC with isolted 3 signls hs on verge more percentge cells positive thn NSCLC with split signls,

14 1364 Crizotinib: The Second Decde of Trgeted Therpy Figure 5. ALK brekprt FISH ssy. (A): Greter thn 2 signl dimeter seprtion s criterion for nplstic lymphom kinse (ALK) brek-prt fluorescense in situ hybridiztion (FISH) positivity. (B): 5 3 brek-prt ALK FISH. Figure provided by John Ifrte. (C): Isolted 3 brek-prt ALK FISH. (D): Using the 15% criterion for ALK brek-prt, FISH resulted in cler seprtion of two groups of NSCLC ptients. Adpted nd modified from figure 2 of reference [130], with permission. likely due to the difficulty in clling split signl with the inversion pttern (Tble 9) [133]. Importntly, there ws no correltion between the percentge of cells positive by FISH nd response to crizotinib from nlyses of both PROFILE 1001 [133] nd PROFILE 1005 [120]. There re dvntges nd disdvntges to the brek-prt FISH ssy. As mentioned, the brek-prt FISH ssy is currently the only ALK ssy tht hs been cliniclly vlidted. This ssy cn be performed on FFPE tissue, which is how the vst mjority of lung cncer tissue is processed. Furthermore, it is not necessry to know the specific fusion prtner to perform brek-prt FISH, n importnt fctor considering tht more fusion prtners my be discovered in NSCLC nd other solid tumors, such s VCL-ALK in sickle cell trit-ssocited renl medullry crcinom [135, 136] nd C2orf44-ALK in colorectl cncer [84]. A stndrdized brek-prt FISH protocol will therefore llow detection of ALK rerrngements beyond NSCLC. The mjor chllenge with the ALK FISH ssy is tht interprettion of the ALK brek-prt FISH ssy requires experience, ptience, nd technicl expertise. For exmple, there is potentil to misinterpret ALK mplifiction (copy number gin) s positive for ALK rerrngement due to the existence of multiple single red signls; however, this is not n indiction for crizotinib use bsed on current evidence. The close involvement or supervision by n ntomic pthologist fmilir with NSCLC morphology nd FISH techniques will llow optiml FISH ssessment. The College of Americn Pthologists (CAP) hs recommended tht brek-prt FISH nlysis be performed by two independent observers nd confirmed by cytogeneticist or

15 Ou, Brtlett, Mino-Kenudson et l Tble 7. Men percentge of FISH-positive cells in ALK-positive nd ALK-negtive non-smll cell lung cncer University of Colordo Cncer Center (USA) [130] Ntionl Cncer Center (Tokyo, Jpn) [132] Grenoble University Hospitl (Frnce) [131] ALK ALK ALK ALK ALK ALK n of ptients Men percentge of positive cells (rnge) 56 (18 100) 6 ( ) 41 (17 b ) 5(3 b ) 74.5 NR Positive FISH signl ws defined s 1 signl dimeter prt. b Stndrd devition. Abbrevitions: FISH, fluorescense in situ hybridiztion; NR, not reported. Tble 8. Distribution of ptterns of brek-prt FISH-positivity in ptients with ALK-rerrnged non-smll cell lung cncer University of Colordo Cncer Center (USA) [133] pthologist with trining in FISH [137]. The Internl System of Humn Cytogenetic Nomenclture (ISCN) my lso be recommended for use with ll ALK brek-prt reports (exmples of ISCN reports re given in Tble 10) [137]. The brek-prt FISH ssy is generlly more expensive thn some other ssys, nd so its primry role in the detection of ALK rerrngement will need to be crefully evluted for both performnce nd cost-effectiveness ginst other techniques tht re currently being introduced. ALK FISH hs been vlidted in pthologic specimens but not on cytologic specimens s prt of the PROFILE 1005 tril, lthough the pckge insert of the Abbott ALK FISH Vysis test kit does not mke this fine distinction. If the FISH test is to be performed on cell blocks from tumor cells spun down nd collected from pleurl, pericrdil, or scitic fluids nd not from tumor cells, it is recommended to use cell blocks mde of spun down tumor cells or to spred the specimens isolted from fine needle biopsy on glss slides to prevent cells from overlpping one nother. Grenoble University Hospitl (Frnce) [131] n of ptients Split signls pttern (%) 49 (54) 19 (90.5) 14 (73.7) Single red (3 ALK) pttern (%) 33 (37) 2 (9.5) 5 (26.3) Both split signl nd single red pttern (%) 8 (9) 0 0 Abbrevition: FISH, fluorescense in situ hybridiztion. Tble 9. Correltion of FISH pttern positivity nd percentge of ALK-positive cells (n 90) [133] Men percentge of ALK cells Pttern of FISH positivity (rnge) p vlue Overll positivity 56 Split signls pttern 48 (18 82) Single red (3 ALK) pttern 74 (26 100).0001 Abbrevition: FISH, fluorescense in situ hybridiztion. Seoul Ntionl University Bundng Hospitl (South Kore) [134] Dul-Color Brek-prt Chromogenic In Situ Hybridiztion Chromogenic in-situ hybridiztion (CISH) hs been developed in recent yers to simplify FISH s detection ssy for HER2 overexpression in brest cncer; it hs shown high concordnce with FISH in clinicl studies [138]. Insted of using fluorescent probes s in FISH, CISH uses probes tht cn be detected by routine cytochemicl techniques. The dvntge of CISH is tht it hs been utomted, cn be ssessed under light microscope (thus enbling simultneous cytomorphologic exmintion), nd the signls re stble in room temperture, mening slides cn be stored s permnent records. Using the dpted stndrd criteri of FISH positivity, Kim et l. demonstrted 94% sensitivity (17/18) nd 100% specificity (425/425) for dul-color brek-prt CISH compred with brek-prt FISH [139]. Seprtely, Yoshid et l. lso compred CISH with FISH using RT-PCR s the criterion stndrd. In 15 ALK-positive NSCLC nd 30 ALK-negtive NSCLC smples identified by RT-PCR, CISH nd FISH were ble to detect the sme 14 ALK-positive smples nd ll of the 30 ALK-negtive smples were FISH- nd CISH-negtive [132]. However, one of the criteri for FISH/CISH positivity in the study ws seprtion of the probes by greter thn only one signl dimeter rther thn the stndrd criterion of greter thn two signl dimeter seprtions. Bsed on these two studies, dul-color brek-prt CISH hs the potentil to be n lterntive to FISH s the dignostic choice for ALKrerrngement in NSCLC, but the criteri for positivity will likely hve to be different from FISH nd will need to be vlidted to rech consensus.

16 1366 Crizotinib: The Second Decde of Trgeted Therpy Tble 10. Exmples of Internl System of Humn Cytogenetic Nomenclture (ISCN) for ALK FISH results ISCN nomenclture ALK rerrngement Notes nuc ish (ALK 2) 50/50 Negtive 2 copies of norml ALK copies in ll 50 cells exmined nuc ish (5 ALK, 3 ALK) 2(5 ALK con 3 ALK 2) 50/50 Negtive 2 copies of 5 nd 3 ALK probes (fused) in ll 50 cells exmined nuc ish (ALK 2) (5 ALK sep 3 ALK 1) 30/50 Positive Reverse-Trnscriptse Polymerse Chin Rection ALK rerrngement in NSCLC my lso be detected by RT- PCR. RT-PCR is esy to perform, nd the mjority of current ALK fusion vrints were detected by RT-PCR in fresh frozen tumor tissue [17, 19, 39, 63, 66, 71, 73 75]. However, RT- PCR requires ALK fusion vrints to be known so tht primers to ll vrints re included in the rection. With n ever-expnding list of ALK fusion vrints (Fig. 2A), primers for RT- PCR must be constntly updted to keep pce with published literture. Further, in dily clinicl prctice, most of the tumor tissue vilble for moleculr profiling is from FFPE tissue, where the integrity of RNAs is likely to be gretly compromised compred with fresh/frozen tissue. Although FISH nd immunohistochemistry (IHC) cn be performed on single FFPE slide, RT-PCR requires multiple slides in order to extrct sufficient RNA for successful rection. Li et l. demonstrted tht RT-PCR cn be successfully performed to detect the common EML4-ALK fusion vrints from 4,750 rchived FFPE NSCLC smples t commercil dignostic lbortory, but the flse-negtive rte is unknown [59]. Therefore, hed-to-hed comprison with FISH nd/or IHC is still needed before RT-PCR cn become useful mss screening test. Thus, lthough RT-PCR remins criticl lbortory technique to investigte the vrious ALK fusion vrints, to identify new fusion vrints nd to ultimtely multiplex other mrkers, it remins to be optimized s definite method to detect ALK rerrngement in NSCLC. 2 copies of ALK, but 30 out of 50 cells show seprtion of the 5 ALK nd 3 ALK probes, indicting the presence of ALK trnsloction nuc ish (5 ALK 1, 3 ALK 2) 30/50 Positive 30 out of 50 cells show isolted 3 ALK signls, indicting the presence of ALK trnsloction nuc ish (5 ALK 2, 3 ALK 4)(5 ALK con 3 ALK 2) 30/50 nuc ish (ALK 2) (5 ALK sep 3 ALK 1) 25/50 /(5 ALK 1, 3 ALK 2) 5/50 Positive Positive 30 out of 50 cells show incresed 3 ALK signls, indicting the presence of ALK trnsloction 25 out of 50 cells show seprtion of 5 nd 3 ALK signls, with n dditionl 5 out of 50 cells showing isolted 3 ALK signls, indicting the presence of ALK trnsloction nuc ish (5 ALK 2, 3 ALK 1) 30/50 Negtive 30 out of 50 cells show isolted 5 ALK signls nuc ish (5 ALK, 3 ALK) /50 Negtive 30 out of 50 cells show 4 5 copies of 5 ALK nd 3 ALK probes, indicting ALK polysomy nuc ish (ALK 3) 30/50 Negtive 30 out of 50 cells show 3 copies of ALK signls, indicting ALK polysomy nuc ish (ALK 1) 30/50 Negtive 30 out of 50 cells show one copy of ALK signl, indicting monosomy of ALK signl Abbrevitions: ISCN, Interntionl System of Humn Cytogenetic Nomenclture; nuc ish, interphse in situ hybridiztion; 5 ALK con 3 ALK, fusion of ALK probes; 5 ALK sep 3 ALK, seprtion of ALK probes. Immunohistochemistry Although ALK FISH is currently considered the criterion stndrd for dignosing ALK-positive NSCLC, ALK IHC holds promise s rpid nd ffordble method tht could be preferred for routine screening nd dignosis by pthology lbortories worldwide. Similr to ALK FISH, IHC requires one unstined slide cut from n FFPE block s long s there re t lest few clusters of vible tumor cells. IHC cn be performed successfully on vriety of different tumor specimens, including FNA cell blocks. The mjor chllenge for ALK IHC is the low level of ALK fusion protein expression in ALK-rerrnged NSCLC (t lest fivefold lower thn ALK fusion protein expression in ALCL; Fig. 6A D) [140]. It is most likely due to the weker trnscription ctivity of the EML4 promoter compred with the trnscriptionl ctivity of the nucleophosmin (NPM) promoter in the genertion of NPM-ALK protein in ALCL. There re three ALK ntibodies (ALK1, 5A4, nd D5F3) tht hve been studied in depth in NSCLC (Tble 11) [131, 134]. D5F3 is promising rbbit monoclonl ALK ntibody (developed by Cell Signling Technology, Dnvers, MA) for the detection of ALK rerrngement in NSCLC tht hs shown high sensitivity nd specificity in recent report [140]. However, the current impediments to ALK IHC being widely dopted in screening for ALK rerrngement include the lck of rigorous lrgescle performnce comprison with FISH nd the lck of vlidtion of clinicl responses to crizotinib with ALK IHC. IHC is more likely to yield flse-negtive results, especilly with low- ALK-expressing vrints [59] or when specimens re smll nd/or poorly preserved. Unsuccessful FISH will be reported s

17 Ou, Brtlett, Mino-Kenudson et l Figure 6. Comprison of immunohistochemistry (IHC) using nplstic lymphom kinse (ALK) 1 nd 5A4 in nplstic lrge cell lymphom nd ALK-rerrnged non-smll cell lung cncer. Both 5A4 IHCs were detected with Leic utomtion. Mgnifiction: 200. Figure provided by Mrie Mino-Kenudson. Abbrevitions: ALCL, nplstic lrge cell lymphom; NSCLC, non-smll cell lung cncer. Tble 11. Immunohistochemistry versus fluorescent in situ hybridiztion IHC 3 IHC 2 IHC 1 IHC 0 Seoul Ntionl University Bundng Hospitl Test set Totl ALK FISH-positive ALK FISH-negtive Vlidtion set Totl ALK FISH-positive ALK FISH-negtive Grenoble University Hospitl b Totl ALK FISH-positive ALK FISH-negtive Immunohistochemistry (5A4) versus ALK brek-prt FISH study ( 0.92) [134]. b Immunohistochemistry (5A4) versus ALK brek-prt FISH study [131]. A totl of 19 tumors for which FISH ws uninterpretble were not included in the tble. Abbrevitions: FISH, fluorescense in situ hybridiztion; IHC, immunohistochemistry. technicl filure, wheres unsuccessful IHC will be reported s negtive result. If IHC methods (with or without mplifiction or scoring systems) with 5A4 nd/or D5F3 re eventully proven to be ccurte in detecting ALK rerrngements in lrger-scle studies, IHC could become the primry screening modlity for ALK-rerrnged NSCLC, with only rre IHC-positive cses requiring confirmtion with the FDA-pproved ALK FISH test. In reserch project, Sugwr et l. utilized 5A4 with n interclted ntibody-enhnced method nd identified two ALK IHC-positive cses of non-cler cell renl cell crcinom tht were confirmed to hrbor ALK rerrngements (EML4-ALK, TPM3-ALK) [81]. AFTER FDA APPROVAL OF CRIZOTINIB The frontline PROFILE 1014 tht compres crizotinib to pltinum/pemetrexed is ongoing (Tble 12). Recently, the secondline PROFILE 1007 study ws reported s meeting its primry endpoint [100]. Both trils llow ptients to receive crizotinib on disese progression if they re rndomized to chemotherpy. Therefore, it is unlikely tht significnt improvement in OS with crizotinib will be shown in the first- or second-line setting bsed on these studies. Shw et l. hve performed retrospective study nd demonstrted tht OS times in contemporneously dignosed ptients with ALK-rerrnged NSCLC who enrolled onto PROFILE 1001 were significntly im-

18 1368 Crizotinib: The Second Decde of Trgeted Therpy Tble 12. List of mjor ongoing trils with crizotinib Tril number NCT number Brief description Primry endpoint Phse I PROFILE Originl crizotinib phse I tril, now ccruing RR ALK-negtive NSCLC (cmet mplified) nd ROS-rerrnged tumors A Combintion of crizotinib nd erlotinib MTD A Combintion of crizotinib nd dcomitinib (PF- MTD ) COG-ADVL Children s Oncology Group phse I study in MTD peditric popultion with relpsed/refrctory solid tumors nd ALCL Phse II PROFILE Clinicl efficcy nd sfety of crizotinib in RR ptients with ALK-rerrnged NSCLC who were previously treted for their dvnced NSCLC PROFILE Clinicl efficcy nd sfety of crizotinib in ALKrerrnged RR tumors except NSCLC CREATE (EORTC 91010) Cross tumorl study exploring crizotinib in RR ptients with dvnced tumors induced by cusl ltertions of ALK nd/or cmet Phse III PROFILE Rndomized tril compring pltinum/pemetrexed PFS versus crizotinib in tretment-nïve dvnced/ metsttic ALK-rerrnged NSCLC PROFILE Rndomized tril compring docetxel or PFS pemetrexed versus crizotinib s second-line tretment of dvnced/metsttic ALK-rerrnged NSCLC Closed to ccrul. Abbrevitions: ALCL, nplstic lrge cell lymphom; EORTC, Europen Orgniztion for Reserch nd Tretment of Cncer; MTD, mximum tolerted dose; NCT, Ntionl Clinicl Tril; NSCLC, non-smll cell lung cncer; PFS, progression-free survivl; RR, response rte. proved compred with ptients who did not enroll (Tbles 3,4) [95]. Although there re certin limittions to this nlysis, such retrospective nlyses re likely to be the only evidence tht crizotinib confers survivl dvntge in ptients with ALK-rerrnged NSCLC in the ner future. SCREENING FOR ALK-REARRANGED NSCLC The current drft version of the guidelines from CAP, the Interntionl Assocition for the Study of Lung Cncer, nd the Assocition for Moleculr Pthology recommends ALK testing in ll ptients with NSCLC exhibiting n denocrcinom component, regrdless of ge, ethnicity, sex, nd smoking history [137]. The current Ntionl Comprehensive Cncer Network guidelines, developed by n expert opinion pnel, recommend reflex testing of ll denocrcinom, lrge cell, nd NSCLC not otherwise specified for both EGFR muttions nd ALK rerrngement nd the use of crizotinib s first-line tretment of ALK-rerrnged NSCLC [141, 142]. Of the 24 ALK-rerrnged ptients who received crizotinib s first-line tretment of their metsttic disese in PROFILE1001, 14 of 22 response-evluble ptients chieved n objective response (ORR, 64% with 95% CI, 40% 83%). The medin PFS ws 18.3 months (95% CI, 8.3 -not reched) [118]. The French Ntionl Cncer Institute recommends brekprt ALK FISH ssy to screen ll EGFR/KRAS-negtive denocrcinoms of the lung mong the 28 French moleculr genetics tumor lbortories [131]. Prior to the pprovl of crizotinib nd the ALK compnion dignostic test in the USA nd prior to the pprovl of crizotinib in Cnd, Cndin consensus report did not recommend routine screening for ALK-rerrngement in NSCLC with the rgument tht there ws no pproved tretment for ALK-rerrnged NSCLC nd no proven dignostic test [143]. It is likely tht individul countries will need to set up screening guidelines, especilly in loctions where crizotinib is pproved. In regions (i.e., Est Asi) where the incidence of ctivting EGFR muttions is high, sequentil testing my be considered providing the overll turnround time is within 14 dys to llow the first-line use of crizotinib. However, doublepositive mutnt EGFR/ALK hs been reported with incresing frequency from Asin tumor smples (Tble 1), which could complicte formultion of screening strtegy.

19 Ou, Brtlett, Mino-Kenudson et l RESISTANCE TO CRIZOTINIB Resistnce to RTK inhibitors cn be divided into primry (intrinsic) or secondry (cquired). Accelerted in vitro mutgenesis hs identified muttions in six mino cid residues in ALK (C1156, I1171, F1174, G1269, L1196, S1206) tht confer resistnce t the highest level of crizotinib tested [41]. Muttions t three of these mino cid residues (L1196 in the gtekeeper region, S1206 ner the ribose binding pocket, nd G1269 in the DFG motif) confer the gretest resistnce to crizotinib in vitro [41]. To dte, multiple secondry point muttions in ALK tht confer cquired resistnce to crizotinib hve been identified in ptients with crizotinib-treted NSCLC (L1196M, C1156Y, L1152R, G1269A, S1206Y, G1202R, 1151Tins) [51, 71, ] nd inflmmtory fibroblstic tumor (IMT; F1174L) [147] nd they occurred in the sme mino cid residues identified in the ccelerted in vitro mutgenesis screen [41]. These secondry muttions ccounted for pproximtely one-qurter of the resistnce mechnisms. Furthermore, different ALK muttions cn confer differentil resistnce ccording to the specific type of ALK inhibitor [145, 148]. Mny second-genertion ALK inhibitors re being developed with stronger ALK inhibitor properties thn crizotinib, but lso with the cpbility to inhibit to vrying degree these secondry cquired muttions, including the L1196M gtekeeper muttion [145, 149]. L1196M is considered gtekeeper muttion similr to T351I, T670I, nd T790M tht developed in ABL, KIT, nd EGFR, respectively [150]. L1196M is lso muttion tht cn be generted fter exposing ALKdependent NSCLC cell lines to incresing concentrtions of crizotinib without the concomitnt use of mutgen [151]. Currently, t lest four second-genertion ALK inhibitors AP26113 [41, 151], CH [152], AS3062 [145], nd X396 [153] hve been reported to be ble to inhibit the gtekeeper L1196M muttion. Other resistnce mechnisms such s ALK gene copy number gin [71, 140], loss of the ALK fusion gene [71], nd EGFR pthwy ctivtion [51, 140] including EGFR muttion [71], KRAS muttion [71], nd KIT mplifiction [140] hve so fr been reported to confer resistnce to crizotinib. Finlly, in substntil number of crizotinib resistnce cses, the moleculr mechnisms tht underlie the resistnce remin to be determined [71, 140]. Two het shock protein (HSP) 90 inhibitors, retspimycin (IPI-504) [154] nd gnetespib (STA-9090) [155], hve demonstrted clinicl ctivity in ptients with ALK-rerrnged NSCLC. HSPs re fmily of chperone proteins tht shepherd the berrntly expressed EML4-ALK proteins to their subcellulr loction nd substrtes. In vitro, the ddition of IPI- 504 to n EML4-ALK-dependent cell line led to the rpid degrdtion of EML4-ALK [156], nd this inhibition of EML4- ALK kinse ctivity occurs even in the presence of cquired crizotinib resistnce [152, 156]. Therefore, HSP90 inhibitors lone or in combintion with other therpy (ALK inhibitors, chemotherpy) hve the potentil to overcome cquired resistnce to crizotinib independent of specific secondry ALK muttions nd/or ALK fusion vrints [78]. Clinicl trils testing this hypothesis hve commenced. In the PROFILE 1001 crizotinib study, 8 of 133 evluble ptients with ALK-rerrnged NSCLC did not experience tumor shrinkge with crizotinib nd 5 ptients hd disese progression s best response, indicting the potentil existence of primry resistnce to crizotinib [118]. EML4-ALK is postulted to led to ntipoptosis by decresing the level of propoptotic BIM through ctivtion of ERK nd incresing the level of ntipoptotic survivin through the ctivtion of STAT3 [42]. Indeed, the pretretment BIM level hs been shown to predict response to wide rry of RTK inhibitors, with high pretretment BIM level ssocited with better response to RTK inhibitors nd improved clinicl outcome [157]. Another potentil primry mechnism is the existence of bypss pthwys. It hs been demonstrted in vitro tht ctivtion of the EGFR pthwy cn led to crizotinib resistnce [51, 146]. In the future, it my be importnt to mesure BIM levels nd to scertin the presence of EGFR muttions in ALK-rerrnged NSCLC prior to crizotinib tretment [51, 71]. CRIZOTINIB ACTIVITY IN OTHER ALK-DEPENDENT TUMORS AND MET-AMPLIFIED TUMORS Crizotinib ws initilly developed s MET inhibitor, nd the moleculr enriched cohort of PROFILE 1001 ws initilly designed to screen for MET-mplified tumors (e.g., gstric crcinom, non-brrett s gstroesophogel junction [GEJ] cncer) or MET-mutted tumors (e.g., squmous cell crcinom of the hed nd neck, spordic nd hereditry ppillry renl cell crcinom). Other MET-dependent tumors, such s lveolr soft prt srcom nd lveolr rhbdomyosrcom, were lso eligible upon histologic confirmtion. The criteri for MET mplifiction in PROFILE 1001 followed the Americn Society of Clinicl Oncology/CAP guidelines for definition of gene mplifiction (MET/CEP7 copy number rtio 2.2). As it turned out, true MET mplifiction in GEJ is n extremely rre event (2%), with ggressive tumor behvior tht severely limited ptient enrollment into the crizotinib clinicl tril. To dte, only two ptients with METmplified GEJ derived clinicl benefit on crizotinib, with time to progression being 105 nd 112 dys, respectively [158]. Similrly, de novo MET-mplified NSCLC is rre; no obvious clinicopthologic chrcteristics for this moleculr subset of NSCLC hve been identified. Recently, the U.S. Lung Cncer Muttion Consortium reported rte of 4.4% with MET/CEP7 cutoff t 2.2 nd ssocition with femle sex [58]. To dte, there is report of one ptient with de novo highly MET-mplified NSCLC chieving confirmed prtil response with crizotinib [159], with ongoing response for 19 months (S.-H.I. Ou, unpublished dt). Additionlly, one ptient with MET-mplified glioblstom chieved rpid rdiogrphic response on crizotinib [160]. Promising ntitumor ctivity hs lso been observed with crizotinib in ptients with tretment-resistnt ALK-positive ALCL [161]. The PROFILE 1013 study (NCT ) is enrolling ptients with ALK-positive tumors other thn NSCLC to better ssess the ctivity of crizotinib in wide rnge of mlignncies. In ddition, the Europen Orgniztion for Reserch nd Tretment of Cncer re in the process of inititing study to explore crizotinib in ptients with dvnced

20 1370 Crizotinib: The Second Decde of Trgeted Therpy tumors induced by csul lterntions of ALK nd MET (Tble 12). Currently, the U.S. Children s Oncology Group is conducting phse I study in peditric ptients with refrctory solid tumors nd ALCL. A recent report showed tht seven of eight enrolled ptients with ALCL experienced complete response. Among seven ptients with ALK-positive IMT, three chieved prtil response, one cheived prolonged stble disese ( 24 months), nd the other three were too erly in tretment to be ssessed. Two peditric ptients with ALK-positive NSCLC were enrolled. One chieved prtil response nd one chieved stble disese. Among the eight ptients with neuroblstom nd known ALK muttion, one experienced complete response nd two chieved stble disese. Of 19 ptients with ALK-unknown neuroblstom, one experienced complete response nd six hd prolonged stble disese [162]. ROS1 REARRANGMENT IN NSCLC Rerrngements in ROS1, nother humn RTK, in NSCLC were discovered by Rikov et l. in 2007 with the sme screening strtegy for ctivted tyrosine kinses tht identified ALKrerrnged NSCLC [19], with the initil incidence of ROSrerrnged NSCLC estimted to be 2% [163]. ROS1 is one of 58 humn receptor tyrosine kinse RTKs [164] nd is evolutionrily relted to ALK [165]; the mouse ALK nd ROS1 proteins shre 52% mino cid homology within the kinse domin [20]. No biologicl lignd hs been identified for ROS1 in humns, nd its norml physiologic function in humn is not well defined. Mle ROS1 knockout mice re infertile due to disruption of the differentition of the epididyml epithelium [166]. Rikov et l. identified two ROS1 fusion vrints in NSCLC, including SLC34A2-ROS1 in HCC78 cell line. More recently, five dditionl fusion prtners (TPM3, SDC4, EZR, LRIG3, FIG) to ROS1 hve been identified in NSCLC [56, 68, 166], representing 12 ROS1 fusion vrints in NSCLC. More importntly, the identifiction of SCL34A2-ROS1 in the HCC78 cell line gve the first indiction tht n ALK inhibitor my lso serve s ROS1 inhibitor. The potentil ROS1 inhibitory potentil by ALK inhibitors ws discovered by Mc- Dermott et l., who ttempted to identify potentil response mechnisms to ALK inhibition by screening the ctivity of TAE684, n ALK inhibitor in 603 different cell lines derived from vriety of humn tumors [40]. They identified 10 cell lines derived from NSCLC, ACLC, nd neuroblstom tht showed mximum inhibition by TAE684, including the HCC78 NSCLC cell line. No ALK bnormlity or detectble ALK protein expression ws found in HCC78 [40]. The discovery by Rikov et l. nd the mino cid homology in the kinse domin between ALK nd ROS1 led to the hypothesis tht ALK inhibitors could ct s ROS1 inhibitors. Consequently, ROS1-rerrnged tumors bsed on positive brek-prt FISH ssy were dded s n dditionl eligibility criterion for moleculrly enriched cohorts of PROFILE 1001 since My This modifiction provided the impetus to identify the clinicopthologic chrcteristics of ptients with ROS1-rerrnged NSCLC in order to implement screening strtegy to identify such ptients. Using home-grown brek-prt FISH ssy t Msschusetts Generl Hospitl, Bergethon et l. [167] screened 1,073 NSCLC tumor smples nd identified 18 (1.7%) with ROS1-rerrnged NSCLC nd 31 (2.9%) with ALK-rerrnged NSCLC, consistent with the previously reported low incidence of ROS1-rerrnged NSCLC [163]. Perhps not surprisingly, the clinicopthologic chrcteristics of ROS1-rerrnged NSCLC re very similr to ALK-rerrnged NSCLC: ptients re young ( similr medin ge of dignosis of pproximtely 50 yers of ge) never-smokers with denocrcinom [167]. These findings were independently demonstrted by Tkeuchi et l. [68]. It seems tht there is no difference in the prevlence of ROS1-rerrnged NSCLC between Asin nd non-asin ptients, with ROS1-rerrnged NSCLC found in pproximtely 1% of Est Asin ptients with denocrcinom [56, 68, 168]. Among the first 15 ptients (14 responses evluble) enrolled in the ROS1 NSCLC cohort of A , n ORR of 57% nd disese control rte of 79% t 8 weeks suggest tht crizotinib hs mrked ntitumor ctivities in ROS1-rerrnged NSCLC [169], similr to the ctivity of crizotinib initilly reported for ALK-rerrnged NSCLC [112]. Tken together, these dt suggest tht ROS1 rerrngement defines nother unique moleculrly defined subtype of lung cncer with heterogeneity, s well s vlidted driver muttion nd therpeutic trget. Whether the ctivity of crizotinib will be ble to be extended to other tumors with ROS1 rerrngement remins to be determined [170, 171]. This will serve s proof of principle demonstrtion tht ROS1 rerrngement lso function s driver muttion in those tumors. In ddition, optimized testing methods such s multiplexing re needed. SUMMARY In 2011, we mrked the beginning of the second decde of trgeted therpy in oncology. The development nd pprovl of crizotinib indictes tht brethtking dvnces re in store for the next 10 yers. Alredy, novel chromosoml trnsloctions involving receptor tyrosine kinses re now being discovered in other mjor epithelil mlignncies [68, 81, 83 84, ]. Although it took lmost 20 yers from the identifiction of the trnslocted ABL kinse in the Phildelphi chromosome in chronic myeloid leukemi [172] to the initil pprovl of imtinib s n ABL inhibitor in 2001 [173], it took only 4 yers from the discovery of ALK rerrngement in NSCLC to the pprovl of crizotinib for the tretment of dvnced ALKpositive NSCLC. The development of crizotinib put into focus severl previously rre nd reltively unknown moleculr subsets of mlignncy, such s ALK-rerrnged NSCLC nd IMT, in similr wy to the role of imtinib in defining gstrointestinl stroml tumor s unique moleculr subset of tumor. Similr to imtinib, crizotinib is lso multitrgeted kinse inhibitor, nd its emerging role s MET nd ROS1 inhibitor will likely spotlight further unique moleculr tumor subsets underpinned by these driver muttions. The reliztion of the

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First-SIGNAL: First-line single-gent iress versus gemcitbine nd cispltin tril in never-smokers with denocrcinom of the lung. J Clin Oncol 2012;30: Po W, Girrd N. New driver muttions in nonsmll-cell lung cncer. Lncet Oncol 2011;12: incresing incidence of chromosoml berrtions in solid tumors ws ctully predicted by Mitelmn et l. [174]. Going forwrd, the emerging complexities of resistnce mechnisms found in crizotinib-treted ALK-rerrnged NSCLC will crete dunting tsk [71, ]. To complicte the tsk further, progression on crizotinib occurred in diverse clinicl scenrios, such s development of new metstsis, progression of pre-existing lesions, or both. Therefore, the importnce of rebiopsying fter tretment progression cnnot be overemphsized [71, 144, 175]. Rebiopsying on tretment progression will likely be mjor prdigm switch in solid tumor oncology s we go forwrd in the second decde of personlized therpy. Although rebiopsying in solid tumor mlignncies will not be esy compred to repeted blood drws in chronic myeloid leukemi, it is only then tht rtionl combintion with trgeted therpies cn be implemented to dely or overcome resistnce in order to prolong survivl. The pprovl of crizotinib ws dependent on the simultneous pprovl of vlidted dignostic ssy. In contrst, when imtinib ws conditionlly pproved for CD117-positive gstrointestinl stroml tumor in 2001, one of the post pprovl commitments ws to develop CD117 IHC ssy in order to identify ptients for imtinib tretment [176]. The codevelopment of drug with compnion dignostic ssy hs spurred rpid development in the re of dignostic ssys for chromosoml trnsloction in solid tumors; it engenders n ongoing helthy nd vigorous debte s to the most sensitive, specific, nd cost-effective ssy for the screening of chromosoml berrtions. With the dvent of the second decde of moleculr trgeted therpy, the continul development of crizotinib points to more moleculr subsets of solid tumors being discovered nd defined, the incresing importnce of repet tissue cquisition nd nlysis during the clinicl mngement of solid tumors, rpid dvnces in the dignostic re of oncology, expnded trnsltionl reserch in oncology in the future, nd the need for close collbortion between clinicl oncologists, pthologists, nd trnsltionl scientists. ACKNOWLEDGMENTS We thnk Keith Wilner (Pfizer Globl Reserch, L Joll, CA, USA) for shepherding the initil nd ongoing clinicl development of crizotinib. This work ws supported by funding from Pfizer. Editoril ssistnce ws provided by Mrtin Quinn t Acumed (Tytherington, U.K.). AUTHOR CONTRIBUTIONS Conception/Design: Si-Hong Igntius Ou, Cynthi Hung Brtlett Collection nd/or ssembly of dt: Si-Hong Igntius Ou, Cynthi Hung Brlett, Mrie Mino-Kenudson, Jen Cui, A. 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