Lung Cancer 84 (2014) 110–115

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Review

Overcoming the resistance to Crizotinib in patients with Non-Small Cell Lung Cancer harboring EML4/ALK translocation Cesar A. Perez a,∗ , Michel Velez b,1 , Luis E. Raez c,2 , Edgardo S. Santos d,3 a Division of Medical Oncology and Hematology, James Graham Brown Cancer Center, University of Louisville, 529 S Jackson Street, Suite 426, Louisville, KY 40202, USA b Division of Hematology/Oncology, Sylvester Comprehensive Cancer Center, University of Miami, Leonard M. Miller School of Medicine, 1475 NW 12th Avenue, D8-4, Miami, FL 33136, USA c Memorial Cancer Institute, Memorial Health Care System, Boca Raton, FL, USA d Thoracic Oncology Program, Lynn Cancer Institute, Boca Raton, FL, USA

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Article history: Received 20 December 2013 Received in revised form 30 January 2014 Accepted 1 February 2014 Keywords: AP26113 AUY922 Alectinib Crizotinib EML-4/ALK translocation LDK378 L1196 mutation

a b s t r a c t The large knowledge learned in molecular biology specifically in the oncology field during the last ten years has resulted in fruitful results for the treatment of non-small cell lung cancer. The first pathway to be effectively targeted in lung cancer was the epidermal growth factor receptor. The acceptance of epidermal growth factor receptor mutation as a strong predictive biomarker in non-small cell lung carcinoma has encouraged the search for more targets. In 2011, regulatory entities granted conditional approval to an anaplastic lymphoma kinase inhibitor (crizotinib) based on an impressive overall response rate in previously treated non-small cell lung cancer patients whose tumors harbored EML4/ALK translocations. The landmark approval of crizotinib based on early promising clinical data highlights the remarkable success of molecular medicine in lung cancer therapeutics. The cumulative data developed after that approval has confirmed the appropriateness of this decision as recently reported phase III has now demonstrated. Unfortunately, resistance to this agent invariably develops and we now face the challenge of understanding several resistance pathways and overcoming them with new and more potent compounds. New agents in clinical development such as alectinib, LDK378, AP26113, and AUY922 have not only demonstrated promising activity in crizotinib resistant patients, but also crossing new pharmacokinetic boundaries in ALK inhibition as potent CNS penetration. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Non-small cell lung cancer (NSCLC) has not been exempted of the successes seen in tumor biology and molecular medicine. The disease is now being targeted by several different small molecules and inhibitors of different pathways. Lung cancer continues to be the first cancer-related mortality in the United States. In 2013, a total of 228,190 new lung cancer cases are expected and 159,480

∗ Corresponding author at: Division of Medical Oncology and Hematology, James Graham Brown Cancer Center, University of Louisville, 529 S Jackson Street, Suite 426, Louisville, KY 40202, USA. Tel.: +1 502 562 4369; fax: +1 502 5626811. E-mail addresses: [email protected], [email protected] (C.A. Perez), [email protected] (M. Velez), [email protected] (L.E. Raez), [email protected] (E.S. Santos). 1 Tel.: +1 305 243 6554; fax: +1 305 243 3289. 2 Herbert Wertheim College of Medicine, Florida International University, 801 N. Flamingo Road, Suite 11, Pembroke Pines, FL 33028, USA. Tel.: +1 954 844 6868; fax: +1 954 443 4747. 3 Charles E. Schmidt College of Medicine, Florida Atlantic University, 701 NW 13 Street, 3rd Floor, Rm 3007, Boca Raton, FL 33486, USA. Tel.: +1 561 955 5156; fax: +1 561 955 4145. 0169-5002/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lungcan.2014.02.001

deaths have been estimated [1]. In the recent years, clinicians are starting to routinely include molecular analyses of tumors from patients with NSCLC since cumulative data has shown more favorable toxicity profile, response rate and progression-free survival (PFS) in favor of targeted therapy over conventional, cytotoxic and “blind or untargeted” chemotherapy. Thus, it is crucial to test our patients for those targets such as epidermal growth factor receptor (EGFR) mutations, echinoderm microtubule associated protein like-4/anaplastic lymphoma kinase (EML-4/ALK) translocations, and recently ROS-1 translocations. EGFR exon 19 deletion and L858R mutations, EML-4/ALK translocation, EGFR exon 20 insertions, and ROS-1 translocations are found more commonly in similar phenotypic population: adenocarcinoma, younger patients, Asian ethnicity, and never smokers [2–4]. Thus, we cannot treat those patients appropriately if we do not perform these genetic tests. In this manuscript we are reviewing how crizotinib was established as a targeted agent in NSCLC and how to overcome the resistance to this therapeutic agent with several new agents in development. Today, the term “alk-omas” has been coined to those tumors regardless of the histologic type which harbor ALK translocation. The EML-4 and ALK (already known in anaplastic

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Fig. 1. The EML4-ALK translocation located in chromosome 2p encodes for a constitutively active kinase with transforming capacity. The aberrantly expressed fusion protein activates survival and proliferation pathways as the Ras/Mek/Erk and PI3K/Akt cascades. The fusion protein can be inhibited by the kinase inhibitor crizotinib (Cr), but an acquired mutation as the gatekeeper L1196 can be resistant to crizotinib. Other mechanisms of resistance to crizotinib are secondary EGFR mutations and overexpression of the EML4-ALK fusion protein.

non-Hodgkin’s lymphoma) genes were identified in NSCLC by Soda et al. [5]. This alteration occurs from chromosome 2p inversion and is found in 3–13% of NSCLC patients [6]. The inversion of chromosome 2 leads to the fusion gene, and subsequently, a fusion protein that induces a constitutive activation of the intracellular domain of ALK; therefore, a downstream cascade of events that lead to carcinogenesis (Fig. 1) [5]. Most of the patients harboring EML4-ALK rearranged tumors are younger than other patients with lung cancer, usually never smokers or light smokers (less than 10 pack/year) [5,7,8], most of the cases are adenocarcinomas and do not simultaneously carry other genetic abnormalities such as EGFR mutations which are also seen in never smokers or light smokers. [6]. Histologically, these ALKomas tend to have a mucinous cribiform pattern in 56% of the cases and as much as 43% had a solid signet-ring pattern sometimes seen in gastrointestinal tumors [9]. Lung adenocarcinomas which have these histologic characteristics and are negative for EGFR and K-Ras should be tested for EML-4/ALK translocation. Nonetheless, not a single histologic parameter was still completely sensitive or specific to predict ALK rearrangement. IHC has proven to be a reliable way for screening specimens in a daily routine practice because is less time consuming and cheaper than performing FISH analysis on the specimens [10]. However, FISH still remains the standard and only FDA approved tool for testing for EML-4/ALK rearrangement. 2. Crizotinib in NSCLC – the beginning Crizotinib (formerly known as PF-01241066), an inhibitor of ALK and c-MET receptor kinases, was the first ALK-targeted therapy tested in the clinical practice. A phase I, international, multicenter trial was conducted to investigate the safety, pharmacokinetics, pharmacodynamics, and antitumor activity of crizotinib in patients with advanced cancer [11]. That trial was already enrolling patients in the dose escalation phase when EML4-ALK in NSCLC was first reported in 2007. There were two patients with NSCLC harboring

EML4-ALK translocations treated with crizotinib who showed dramatic improvement in their symptoms during the dose escalation phase [11]. That observation led to a large prospective screening of NSCLC patients and recruitment of those with ALK-positive NSCLC into an expanded molecular cohort at the maximum tolerated dose (MTD) of 250 mg twice daily [11]. Thus, in a clinical trial conducted by Kwak et al., 1500 NSCLC patients were tested for EML4/ALK translocations and 82 patients were found to have ALK-positive tumors and entered into the clinical trial [11]. Herein, the overall response rate (ORR) was 57% and 27 patients (33%) who had stable disease (SD) [11]. Expanded results of this trial included a total of 143 ALK-positive NSCLC patients evaluable for response, with a ORR of 60.8% (87 of 143 patients) including three CR and 84 PR. Responses occurred fast with a median time to first documented objective response of 7.9 weeks (range 2.1–39.6); median duration of response was 49.1 weeks. Median PFS was 9.7 months (95% CI, 7.7–12.8). At the time of presentation, OS data was not mature. Noteworthy, 39 patients continued to receive crizotinib for more than two weeks after progression due to a perceived clinical benefit from the physician. Most of the adverse events (AE) were grade 1 or 2. The most common AEs were visual effects, nausea, diarrhea, constipation, vomiting, and peripheral edema [12]. The impact of crizotinib on OS remains to be determined; however, based on the >9-month PFS in a heavily pretreated population of NSCLC patients, the impact on OS is likely to be substantial. Based on these results, in October 2011 crizotinib received conditional approval by the United States Food and Drug Administration (US FDA) under their accelerated approval program, for treatment of patients with NSCLC whose tumors carry an ALK translocation and it was finally approved in November 2013. 3. Clinical trials using crizotinib – post-marketing approval In the Phase III trial PROFILE 1007 crizotinib was compared against pemetrexed or docetaxel in the second line setting [13].

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Patients with stage IIIB/IV ALK-positive NSCLC previously treated with one prior platinum-based doublet (n = 347) were randomized to receive crizotinib (n = 173) at 250 mg orally twice a day or chemotherapy (n = 174; 41% received docetaxel and 57% pemetrexed at standard doses). Patients with progressive disease on chemotherapy were offered crizotinib on another trial known as PROFILE 1005. The PROFILE 1007 study met its primary endpoint PFS when patients treated with crizotinib had a median PFS of 7.7 months versus 3.0 months for those treated with chemotherapy (HR: 049; 95% CI 0.37–0.64; P < 0.0001). ORR was also significantly higher for crizotinib arm vs. chemotherapy group (65% vs. 20%; P < 0.0001). The toxicity seen in this study for crizotinib was similar to other previously reported studies (diarrhea, visual disturbances, nausea, vomiting, and constipation) [13]. Therefore, the PROFILE 1007 trial results supported the superiority of crizotinib over the competitor treatments in patients whose tumors harbor EML4/ALK translocation. Based on this data, the US FDA granted regular approval for crizotinib on November 20, 2013. In the 5th Asia Pacific Lung Cancer Conference in Japan, Ou et al. presented the cumulative data in ALK-positive NSCLC patients treated with crizotinib beyond progression of disease [14]. Reported data is coming from two single arm studies PROFILE 1001 and 1005 were patients progressed on crizotinib but they were allowed to continue on this agent if there was evidence of ongoing clinical benefit in the investigator’s opinion. To be included in the analysis, patients must have at least more than two weeks on crizotinib post-progression of disease (post-PD). A total of 229 out of the 410 patients treated with crizotinib among the two studies had PD; of these, 60% (138 patients) qualified as post-PD. This group of patients represent a heterogeneous population, usually they have good ECOG performance status, great initial tumor response (ORR 70%) on crizotinib, and they tended to have new lesions which defined their PD especially brain (46%) and liver (26%). This particular group “crizotinib progressors” were able to remain on crizotinib for a substantial period of time post-PD; median duration of crizotinib therapy post-PD was 20 weeks, but 30% of these patients received therapy more than 6 months [14]. The ongoing open label phase III clinical trial PROFILE 1014 trial is comparing crizotinib against platinum-based doublet (cisplatin or carboplatin plus pemetrexed) in patients ALK-positive previously untreated for stage IIIB/IV NSCLC. The primary endpoint is PFS.

in chronic myelogenous leukemia and EGFR-mutated lung cancer. Similarly, a less pronounced but probably clinically important resistance to crizotinib can be secondary to less common mutations as the C1156Y, involving the substitution of a cysteine for a tyrosine in the position 1156 of the of the EML4-ALK receptor and the G1269A [15–19]. Mutations in the kinase domain of ALK currently account for approximately 25% of observed drug resistance [16–19]. Another mechanism of resistance to crizotinib that has been described is an increase or amplification of the number of rearranged EML4-ALK genes per cell relative to non-resistance cells [19]. It is hypothesized not all the ALK fusion proteins in a tumor can be therefore inhibited by clinically achievable doses of crizotinib, allowing then sufficient downstream signaling for tumor cell survival. The cells with amplified EML4-ALK seem to be resistant to intermediate doses of crizotinib. Although this has been demonstrated in vitro, the contribution to in vivo resistance is not as clear as the acquired mutations. As important as the intrinsic kinase domain resistance, activation of compensatory signaling pathways may also confer resistance to targeted ALK agents. Heat shock protein 90 (Hsp90) is a molecular chaperone that regulates the correct folding, stability, and function of numerous client proteins [20]. The EML4/ALK fusion protein is one of these client proteins and inhibition of Hsp90 causes regression of EML4-ALK-driven xenografts and murine lung adenocarcinomas [21]. This blockade might also overcome drug resistance mechanisms, giving therefore the rational to use Hsp90 inhibitors in crizotinib-resistance Alk-positive [20]. Acquired mutations in the EGFR and K-Ras after crizonitib therapy in EML4/ALK mutated tumors have also been described, but how much these contribute to the acquired resistance is unclear [19]. However, reports of the outgrowth of KRAS and EGFR mutant, ALK-negative tumors from patients with ALK-mutant NSCLC previously treated with crizotinib might demonstrate the emergence of a separate oncogenic driver as a resistance mechanism [22]. Hence, we have a great need for novel agents that can overcome these ALK and kinase inhibitors mechanisms of resistance. A new generation of ALK inhibitors have been developed and in early clinical studies, they have shown promising results.

5. Novel agents with ALK inhibition properties 5.1. AUY922

4. Mechanism of resistance to crizotinib Unfortunately, PFS in patients on crizotinib are short-lived despite of great clinical and radiographic responses, although cases with more than 24 months of clinical benefit have been reported [12]. Patients exposed to crizotinib may have an intrinsic resistance, demonstrated sometimes by a fast progression of the disease while on therapy, or acquired resistance to inhibitors after an initial response [11]. Several mechanisms of acquired resistance to ALK inhibitors have been described, but the three well-recognized are the development of mutations of the ALK kinase domain, amplification of the EML4-ALK gene, and activation of alternative signaling pathways. Probably the most important resistance mechanism to these agents is the development of mutations clustering around the ATP binding site of the EML4/ALK translocation. A substitution of leucine for a methionine at position 1196 (L1196) of the kinase domain of ALK gives rise to a change in the ATP-binding pocket of the receptor, placing an amino acid with a bulky side chain at this “gatekeeper” location and interfering with the binding of inhibitor. [15] This mutation is analog to mutations at threonine at position 315 in ABL and at position 790 in the EGFR receptor; the latter are the most frequently described acquired resistance mutations

A novel isoxazole-based heat shock protein 90 (Hsp90) inhibitor has been shown to cause the degradation of multiple oncogenic cellular proteins. Preclinical data suggest broad antitumor activity including xenografts for breast, ovarian, prostate cancer, melanoma, glioblastoma, and multiple myeloma. This novel compound not only causes tumor regression but decreases invasion and angiogenesis. Hsp90 is a molecular chaperone which supports multiple client proteins essential for apoptosis, cell-cycle regulation and proliferation, thus targeting multiple signaling pathways [23,24]. Single agent AUY922 was first studied in humans with advanced solid tumors, in a phase I dose finding study conducted by Samuel et al. [25]. AUY922 was administered as an intravenous infusion once weekly. Ninety-six patients were enrolled in this trial, and received AUY922 at doses between 2 and 70 mg/m2 , with 23 patients in the 70 mg/m2 dose group. The most frequently reported AEs possibly related to AUY922 were diarrhea in 55%, nausea in 35%, fatigue in 32%, night blindness in 20%, and vomiting in 19% of patients. Visual symptoms mainly grade 1–2 and mostly reversible included blurred vision, flashing, and delayed dark/light accommodation were reported starting at 40 mg/m2 , and increased in frequency or severity with dose. Dose limiting toxicities (DLTs), all grade 3, included atrial flutter, anorexia, fatigue, diarrhea, asthenia,

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and darkening of vision. The maximal MTD was 70 mg/m2 . Median duration of exposure was 7.0 weeks with some patients receiving up to 80 weeks. SD was reported in 16 patients and 9 patients reported partial metabolic response in FDG-PET scans. Dose-related induction of Hsp70, indicating inhibition of Hsp90 was seen [25]. Hsp90 is relevant in NSCLC pathogenesis and it has been demonstrated that mutant EGFR is an Hsp90 client, suggesting Hsp90 inhibition as a novel treatment strategy. ErbB2, mutant B-Raf, or mutant or overexpressed c-Met can also lead to lung cancer proliferation and are also degraded on Hsp90 inhibition. AUY922 activity was evaluated in vitro on 21 different NSCLC cell lines, where it showed strong anti-proliferative effect in EGFR mutated lines, which were either sensitive or resistant to kinase inhibitors. Noteworthy for this review is the fact that AUY922 has shown activity in those lines with wild type EGFR as well as EML4/ALK and K-Ras mutated NSCLC [26]. A phase II study of AUY922 was conducted in patients with previously treated, advanced NSCLC, stratified by molecular status. Patients with advanced NSCLC who progressed following greater than or equal to one prior line of chemotherapy received AUY922 70 mg/m2 as a once-weekly, one hour infusion. Patients were assigned to four groups: ALK-positive, EGFR mutant, K-Ras mutant or wild-type EGFR/K-Ras/ALK (triple negative). The primary endpoint included ORR or SD at 18 weeks. Secondary endpoints included OS, PFS, as well as safety and tolerability. A total of 121 patients had been treated including 22 ALK-positive, some previously treated with crizotinib, 35 EGFR mutants, 28 K-Ras mutants, and 33 patients with “triple negative” phenotypic profile. A small group of 3 patients was deemed to have an undetermined mutational status. Most patients had been heavily pretreated and more than half had at least three prior regimens [27]. Interestingly, clinical activity of AUY922 was seen mainly in patients with ALKpositive tumors and EGFR mutant NSCLCs, with PR seen in 29% and 20%, respectively. Four out of six ALK-positive responders were crizotinib-naive. Estimated median PFS rates were 42% and 34% at 18 weeks in ALK-positive and EGFR mutant patients, respectively. Patients who had evidence of EGFR mutations and had only been previously treated with erlotinib attained a median PFS rate at 18 weeks of 45% vs. 21% in those patients who had not received a TKI as their immediate pre-AUY922 therapy. The most frequent AEs were eye disorders (77%), diarrhea (74%), and nausea (46%). Most AEs were grade 1 or 2; grade 3 and 4 AEs were rare. Thus, the authors concluded that AUY922 is safe and effective in a heavily pretreated patient population with ALK- and EGFR-mutant NSCLC tumors; there was an increase in PFS in those patients who had been recently treated with TKI for EGFR mutated NSCLC [25]. Based on the above mentioned data, there are multiple studies underway involving patients with NSCLC using AUY922 either alone or in combination with other targeted agents, especially for those with either ALK- or EGFR mutated tumors.

5.2. Ganetespib Ganetespib (STA-9090) is a second-generation triazolone heterocyclic Hsp90 inhibitor that binds to the ATP-binding domain at the N-terminus of Hsp90 and acts as a potent Hsp90 inhibitor by inducing degradation of multiple oncogenic Hsp90 client proteins including HER2/neu, mutated EGFR, c-Kit, PDGFR␣, c-Met and ALK, among others [28]. In an open-label phase II, ganetespib was administered to 3 cohorts of patients with molecularly defined NSCLCs, including tumors harboring mutant EGFR and mutant KRAS [29]. Although there was no significant clinical activity in the EGFR and KRAS mutant cohorts, there were 4 PRs seen in the third cohort and all of these were retrospectively found to have an ALK rearrangement. Four other patients with ALK rearrangement were also treated on study, 3 of them had SD and the other

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progressive disease. All 4 responders were, however, crizotinib naïve [29]. Ganetespib has been found to inhibit crizotinib-resistant cells in vitro that were also insensitive to other ALK inhibitors as CH5424802, ASP3026, and TAE684 [20]. Interestingly, the agent also overcame secondary ALK mutations as the L1196M gatekeeper in vitro [20]. Clinically, ganetespib increased the response rates and survival when added to docetaxel in the phase IIb/3 GALAXY trial, but its clinical activity in patients with ALK rearrangement after crizotinib failure has not been demonstrated in a cohort of patients. The phase 2 CHIARA trial, a single arm study of ganetespib in crizotinib-naïve patients with ALK-Positive NSCLC is currently enrolling patients [30]. 5.3. AP26113 AP26113 is a novel, synthetic, orally-active kinase inhibitor which potently inhibits mutant activated forms of ALK and EGFR cell lines. This is based on preclinical studies that showed that AP26113 in vitro has 100-fold selectivity for ALK-positive versus ALK-negative lines; in vivo, AP26113 produced regressions induced by daily oral dosing of 10–25 mg/kg in anaplastic large cell lymphoma and NSCLC models. Pharmacokinetic studies at these dose levels defined efficacious trough concentration of 10–27 ng/mL, consistent with levels required for potent inhibition of growth in vitro. It also showed strong antitumor activity associated with sustained inhibition of p-ALK. Pharmacokinetic studies showed moderate in vitro plasma protein binding, no inhibition of major CYP isoforms and to be orally bioavailable across multiple species with tolerability at and above predicted efficacious plasma levels [31]. Moreover, AP26113 is active against several ALK mutations including the gatekeeper mutation L1196M [32]. Recently, updated results of a phase 1/2 open-label, multicenter study of AP26113 in patients with advanced malignancies was reported. Of the 55 patients enrolled, 47 had NSCLC and 24 of these were ALK positive tumors. Of the 16 patients with ALK positive NSCLC previously treated with crizotinib, 12 had a response (75%). The longest response was 40 weeks and the patient was still on the drug when the updated results of the trial were reported. Of note, 4 of 5 patients with ALK positive tumors and untreated or progressing CNS lesions at baseline had evidence of radiographic improvement with AP26113, demonstrating the CNS penetration of the agent. Patients with EGFR mutant NSCLC were also included, most of them previously treated with EGFR TKI, and although only one out of the 18 evaluable had a response, 7 had stable disease including 4 with a T790M mutation. The drug was well tolerated with the most common grade 3/4 treatment-related AEs being pneumonia in 5% of the patients. DLTs were grade 3 ALT elevation in one patient and grade 4 dyspnea on another. Common grade 1/2 AEs seen included nausea (36%), fatigue (40%), and diarrhea (33%). The MTD still has not been determined, but the recommended phase 2 dose was 180 mg daily. The planned phase II expansion will include 4 cohorts including ALK-positive NSCLC patients who are naïve or resistant to prior ALK-targeted therapy, also EGFR mutated NSCLC patients who are resistant to EGFR targeted therapy, and finally other cancers with abnormalities in ALK gene [33]. 5.4. LDK378 LDK378 is a potent and selective inhibitor of ALK. In enzymatic and cell-based assays, it exhibits increased potency with lower IC50 values when compared to crizotinib, with increase selectivity and no significant inhibition of Met [34]. A Phase I study was conducted with the primary objective of determining MTD and safety profile of LDK378 in patients with ALK-positive advanced malignancies, administered orally once-daily on a continuous 21-day schedule. The study included patients that were both treatment-naïve and

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also those who progressed on standard therapy. Oral absorption of LDK378 was rapid and demonstrated to have a half-life of 36 h, therefore amenable for daily dosing administration. The MTD was found to be at the 750 mg daily dose level. At time of data cutoff, discontinuations were due to AEs in one patient and disease progression in 19 patients. The most frequent AEs were nausea in 59% of the patients, vomiting in 54%, and diarrhea in 48%. The most frequent grade 3/4 AE was diarrhea in 9% of the patients and one patient developed interstitial lung disease at the dose of 750 mg. All AEs were reversible upon discontinuation of therapy. Of the 56 patients included, 50 had ALK-positive NSCLC, defined as tumor with FISH positive in ≥15% of tumor cells. A total of 47 patients with NSCLC were evaluable for response; 51% of them achieved a PR. Interestingly, of the 26 patients with NSCLC who had progressed following crizotinib and were treated at ≥400 mg/day, there were 21 (81%) responses [35]. Thus, LDK378 has now received breakthrough therapy designation by the US FDA for the treatment of patients with ALK positive metastatic NSCLC who had progressed during treatment with, or were intolerant to, crizotinib. 5.5. Alectinib (CH5424802) Alectinib is a potent, selective oral ALK inhibitor with preferential antitumor activity against NSCLC cells expressing EML4/ALK fusion in vitro and in vivo. More importantly, alectinib inhibited ALK L1196M, which corresponds to the gatekeeper mutation conferring common resistance to kinase inhibitors, and blocked EML4/ALKL1196M-driven cell growth [36]. A Phase I/II trial has recently been presented using alectinib twice daily orally in patients with ALK-positive NSCLC until progressive disease or toxicity was observed. In the phase I portion, dose was escalated using an accelerated titration scheme. During that phase, 15 patients were treated with alectinib. At doses of 300 mg twice daily, the highest dose level defined in the protocol, MTD was not reached since a DLT was not determined. The most frequent toxicity was grade 1 myalgia and grade 3 reported toxicities were hypophosphatemia, neutropenia and increased in serum creatinine kinase. All patients (at all dose levels) achieved tumor regression and at dose levels 240 mg twice daily or more under fasting conditions, all seven patients with measurable lesions achieved a PR [37]. The phase II portion of the trial was later presented at the 2012 European Society of Medical Oncology (ESMO) annual congress. A total of 34 patients with no prior ALK inhibitor therapy were treated with alectinib at 300 mg twice daily. The reported response rate (RR) in the first 15 evaluable patients was 73.3% with one CR and ten PR. No treatment-related AEs led to dose reductions. Main treatment-related AEs among 34 patients were liver function test abnormalities, neutropenia, rash, nausea and myalgia, most of them grade 1 except for neutropenia [38]. Another phase I trial enrolled patients with ALK positive NSCLC who failed crizotinib using doses up to 900 mg twice daily of alectinib [39]. Again no DLTs were observed even at the highest dose tested. Most common grade 3/4 adverse effects were GGT increase in 3 patients and neutrophil increase in 2 patients. Of the 31 evaluable patients, PR was 48% and SD 34%. A total of 16 patients had CNS disease at baseline; within 3–6 weeks of treatment, patients treated with alectinib had a response in the brain lesions, with leptomeningeal responses also reported [40]. The phase II study was determined to be 600 mg BID. 6. Conclusion Certainly, the accelerated approval of crizotinib has benefited many patients whose tumors harbor this novel translocation EML4/ALK. In this period of time, when researchers have developed

Table 1 Response rates of novel ALK kinase inhibitors in patients with ALK positive NSCLC after progression on crizotinib. Drug

RF2D

DLTs

n

RRb

AP26113 LDK378 Alectinib

180 mg daily 750 mg daily 300 mg BIDa

Not yet seen ↑ALT ↓serum PO4 Not determined

16 26 31

75% 81% 48%

a b

RF2D Recommended Phase II trial dose. Of evaluable patients; CZT: Crizotinib.

novel agents which are active against previously treated crizotinib patients or patients progressing on this agent, this ALK inhibitor has also shown activity on another translocation that shares 90 percent of its biological pathway and signaling, ROS-1. To date, patients with adenocarcinoma histology former or never smokers are being routinely tested for EGFR, EML4/ALK and ROS1 genetic abnormalities. Crizotinib has shown to be superior than conventional cytotoxic agents in phase III randomized clinical trials in second line therapy for ALK positive NSCLC and we are eagerly awaiting for the results of PROFILE 1014 in the frontline setting which compare crizotinib vs. cisplatin or carboplatin plus pemetrexed in patients whose tumors harbor EML4/ALK translocation. However, most of the patients showed disease progression within a year of therapy. Fortunately, the novel generation of ALK inhibitors such as alectinib and AP21113 as well as the Hsp90 inhibitors AUY922 and ganestepib are promising alternatives with high RRs for those tumors which acquired resistance to crizotinib (Table 1). Moreover, these agents might provide us with an option to treat the ‘tumor escape’ phenomenon seen in patients with good systemic response but CNS progression, and perhaps, a better toxicity profile. Based on the encouraging data available, we strongly recommend patients with ALK-positive NSCLC to be enrolled in clinical trials with these novel agents after crizotinib failure. Conflict of interest statement Dr. Raez has received research funding from Pfizer, Novartis, Genentech/Roche, Syntha and Aztra-Zeneca. Dr. Santos is part of the speaker bureau of Pfizer, Lilly, Genentech, Millenium, Celgene, Amgen and Boehringer-Ingelheim. Dr. Perez and Dr. Velez have no potential conflicts of interest. References [1] Cancer Facts and Figures 2013. Available at: http://www.cancer.org/research/ cancerfactsfigures/cancerfactsfigures/cancer-facts-figures-2013 [2] Takahashi T, Sonobe M, Kobayashi M, Yoshizawa A, Menju T, Nakayama E, et al. Clinicopathologic features of non-small-cell lung cancer with EML4-ALK fusion gene. Ann Surg Oncol 2009;17:889–97. [3] Shaw AT, Solomon B. Targeting anaplastic lymphoma kinase in lung cancer. Clin Cancer Res 2011;17(8):2081–6. [4] Oxnard GR, Lo PC, Nishino M, Dahlberg SE, Lindenman NI, Butaney M, et al. Natural history and molecular characteristics of lung cancers harboring EGFR exon 20 insertions. J Thorac Oncol 2013;8(2):179–84. [5] Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 2007;448(7153):561–6. [6] Horn L, Pao W. EML4-ALK: honing in on a new target in non-small-cell lung cancer. J Clin Oncol 2009;27:4232–5. [7] Shaw AT, Yeap BY, Mino-Kenudson M, Digumarthy SR, Costa DB, Heist RS, et al. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK. J Clin Oncol 2009;27(26):4247–53. [8] Koivunen JP, Mermel C, Zejnullahu K, Murphy C, Lifshits E, Holmes AJ, et al. EML4-ALK fusion gene and efficacy of an ALK kinase inhibitor in lung cancer. Clin Cancer Res 2008;14:4275–83. [9] Yoshida A, Tsuta K, Nakamura H, Kohno T, Takahashi F, Asamura H, et al. Comprehensive histologic analysis of ALK-rearranged lung carcinomas. Am J Surg Pathol 2011;35:1226–34. [10] McLeer-Florin A, Moro-Sibilot D, Melis A, Salameire D, Lefebvre C, Ceccaldi F, et al. Dual IHC and FISH testing for ALK gene rearrangement in lung adenocarcinomas in a routine practice: a French study. J Thorac Oncol 2012;7(2):348–54.

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