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Targeting ALK in patients with advanced Non Small Cell Lung Cancer: Biology, diagnostic and therapeutic options
Critical Reviews in Oncology/Hematology 89 (2014) 358–365
Targeting ALK in patients with advanced Non Small Cell Lung Cancer: Biology, diagnostic and therapeutic options Chiara Lazzari a,∗ , Gianluca Spitaleri a , Chiara Catania a , Massimo Barberis b , Cristina Noberasco a , Mariacarmela Santarpia a , Angelo Delmonte a , Francesca Toffalorio a , Fabio Conforti a , Tommaso Martino De Pas a a
European Institute of Oncology, Division of Thoracic Oncology, Italy b European Institute of Oncology, Division of Pathology, Italy Accepted 17 September 2013
Contents 1. 2. 3. 4. 5. 6. 7.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EML4-ALK biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Crizotinib . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acquired resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Therapeutic strategies at crizotinib recurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract The discovery of EML4-ALK fusion gene in a subgroup of patients with lung adenocarcinoma led to the development of a new class of agents, the ALK inhibitors, and dramatically improved the clinical outcome of these patients. The striking results from clinical trials with crizotinib, the first ALK inhibitor evaluated, allowed the accelerated approval of crizotinib from the USA Food and Drug Administration (FDA). Despite the high initial results, patients acquire resistance to crizotinib, and different next generation ALK kinase inhibitors have been developed. In the current review, we will analyze the biology of EML4-ALK gene, the acquired resistance mechanisms to crizotinib, the therapeutic strategies, currently under evaluation, designed to overcome crizotinib resistance, and the open issues that need to be addressed in order to improve outcome in ALK+ Non Small Cell Lung Cancer (NSCLC) patients. © 2013 Elsevier Ireland Ltd. All rights reserved. Keywords: Non Small Cell Lung Cancer; EML4-ALK fusion gene; Crizotinib
1. Introduction
∗ Corresponding author at: European Institute of Oncology, via Ripamonti 435, 20131 Milan, Italy. Tel.: +39 0255210169. E-mail address: [email protected] (C. Lazzari).
1040-8428/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.critrevonc.2013.09.003
Non Small Cell Lung Cancer (NSCLC) is one of the leading causes of tumor-related deaths in the world. Chemotherapy is the treatment of choice in the majority of patients, and it is usually based on platinum doublet in front-line treatment and on mono-chemotherapy in the
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subsequent lines. Despite chemotherapy, the prognosis of NSCLC patients still remains poor, reaching a median survival of about 10 months in the metastatic setting. In 2007, the EML4-ALK fusion gene was discovered in a subset of patients with lung adenocarcinoma [1]. In a Phase I study, crizotinib, a dual ALK and MET inhibitor, showed an objective response rate (ORR) of 60% and a median progression-free Survival (PFS) at 6 months of 72% in patients carrying the ALK translocation [2,3]. A Phase III trial comparing crizotinib with second line standard chemotherapy (docetaxel or pemetrexed) has recently been closed and showed an improvement in terms of PFS and ORR for crizotinib compared with second line standard chemotherapy [4], while another Phase III study, comparing crizotinib with first line cisplatin pemetrexed chemotherapy is ongoing (NCT01639001). In August 2011, crizotinib was approved by the Food and Drug Administration (FDA) for the treatment of NSCLC patients harboring the EML4-ALK fusion gene, and in October 2012 the European Medicines Agency (EMA) gave conditional marketing authorization for the use of crizotinib in previously treated ALK+ NSCLC patients. Despite the initial high ORR to crizotinib, patients develop resistance. In the current review, we will focus on the biology of EML4-ALK gene, the acquired resistance mechanisms to crizotinib and the therapeutic strategies, currently under evaluation, designed to overcome crizotinib resistance.
2. EML4-ALK biology The EML4-ALK fusion gene was isolated for the first time in DNA from a 62 year-old man with lung adenocarcinoma [1]. The EML4-ALK gene is the result of a chromosome rearrangement between the N-terminal portion of the echinoderm microtubule associated protein-like 4 (EML4) gene and the tyrosine kinase (TK) domain of the anaplastic lymphoma kinase (ALK) gene. Both are located in the short arm of the chromosome 2 (2p), and have opposite orientations. An inversion of the two genes occurs within the chromosome 2p, leading to a chimeric oncoprotein with constitutive TK activity and oncogenic transforming activity in nude mice. The EML4 portion contains different regions: the coiled coil N terminal basic region, the hydrophobic HELP domain and the WD repeat. In vitro studies showed that the basic domain contribute most to the transforming activity of the fusion protein, since its deletion leads to a 84% decrease of the catalytic activity of the protein itself. This region, in fact, is involved in the EML4-ALK dimerization [1], while the HELP domain seems to mediate tubulin binding and the WD repeat is involved in protein–protein interaction. The EML4-ALK dimerization activates 3 interconnected downstream pathways, including the MAPK pathway (Ras/Raf/MEK/ERK), the JAK/STAT pathway and the PI3K/AKT pathway [5]. Twenty-seven ALK fusion variants have been described, 21 of those regarding the EML4, since truncations of EML4 may occur at different exons (2, 6, 13, 14, 15, 17, 18 and 20)
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[6–8]. In all variants, the TK domain of the ALK gene begins at exon 20 [9]. The most common fusion proteins are variant 1 (33%) and variants 3a/b (29%), that consist of exons 1–13 and 1–6 of EML4, joined to exon 20 of ALK [1]. Variants 3a and 3b derive from an alternative splicing of 33 bp that occurs within exon 6 [8,10,11]. Moreover, additional fusion partners of ALK have been described in NSCLC patients: TGF, KLC1 and KIKF5B [12]. The EML4-ALK translocation has been found in approximately 5% of NSCLC patients, more frequently in never or light smokers, and in younger patients (median age 52 years) with adenocarcinoma histology [13]. Moreover, among the different subtypes of adenocarcinoma, the EML4ALK translocation seems to be more frequently expressed in patients with the acinar, papillary, cribriform, mucinproducing or signet ring patterns [14–16].
3. Diagnostics In order to detect the ALK rearrangement, different methods are currently under investigation, including fluorescence in situ hybridization (FISH), reverse transcriptase PCR (RTPCR) and immunohistochemistry (IHC). Vysis Dual color break apart FISH (Abbott Molecular, Des Plaines, IL) is the recommended diagnostic method approved by the FDA to identify ALK+ NSCLC, and is used to screen patients to enter the crizotinib and the ongoing trials with second ALK inhibitors. ALK FISH is considered positive if a split by more than 2 signal diameters is detected between the red and green signals labeling the 3 end of the ALK gene and the 5 end of EML4 in more than 15% of tumor cells, analyzing at least 4 fields. FISH is considered positive also in the presence of a single red pattern in more than 15% of tumor cells. Considering that EML4 and ALK are both located on chromosome 2p, and separated by only 12.5 megabases, false negative diagnoses may occur. Conversely, DNA stretching or nuclear sectioning may cause false positive results. Furthermore, FISH does not discriminate between the different ALK fusion types, while RT PCR can define the ALK partner and the fusion variant. Definitive results about how crizotinib sensitivity differs among the ALK variants have not yet been determined. Recently, in vitro studies have shown that EML4-ALK variant 2 seems the most sensitive to ALK inhibition, followed by variants 1 and 3b, that exhibit an intermediate sensitivity and variant 3a, that appears the least sensitive. Crizotinib sensitivity seems to be inversely correlated with the half-lives of the fusion types, variant 2 being the less stable and the most susceptible to ALK inhibition due to a more rapid protein degradation [17]. These data have not yet been confirmed in clinical practice: only 29 of the 82 patients enrolled in the Phase I crizotinib trial had an adequate amount of tissue to perform RT-PCR, but no association between EML4-ALK variants and (Response Evaluation Criteria in Solid Tumors) RECIST criteria was found [2]. One of the secondary end
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points of the current crizotinib studies is to determine the types of the fusion variants and the ALK expression, but analyses are still ongoing. RT-PCR seems to be particularly useful for the identification of variant 1 [18–20]. In fact, since variant 1 originates from a 2p inversion without any loss in the 5 fragment, it may be a challenge to detect the split signal by FISH. However, the limit of RT-PCR as a diagnostic method is that it cannot define unknown ALK rearrangements. The presence of ALK rearrangements is a rare event, while NSCLC tumor has a high incidence. Therefore routine testing for ALK using FISH only may not be feasible, because of cost, required expertise and the labor-intensive nature of performing and interpreting the test. Immunohistochemistry can be a useful technique to pre-screen cases reducing the burden of FISH testing to a manageable number. The use of IHC has been explored [21]. Different ALK antibodies (ALK1, 5A4 and DF53) have been evaluated, and results have been controversial. The ALK1 antibody is not directed against all the epitopes of the ALK variants and this limits its application in routine practice. The 5A4 and DF53 antibodies are still under study, and they are not yet commercially available. In a cohort of 46 patients with lung adenocarcinoma, FISH, RT-PCR and IHC were compared in terms of sensitivity and specificity [18]. The primers used to perform RT-PCR were designed to detect only variants 1 and 3a/3b, and the ALK1 antibody was used for IHC. Concordance among the 3 methods was 100% for variants 3a/3b, while no concordance was observed for variant 1. RT-PCR was shown to be the most sensitive test, while the sensitivity for IHC was poor, and only 2 ALK IHC+ cases were confirmed by both RT-PCR and FISH. Both tumor patients carried the variant 3a/3b. More recent studies strongly underlined the robustness of immunohistochemistry in identifying ALK-rearranged tumors [22–24]. In a multicentre study, Selinger et al. showed that when high-affinity antibodies are used with a highly sensitive detection method, ALK immunohistochemistry may provide an effective pre-screening technique to complement ALK FISH testing [25]. In fact, the use of immunohistochemistry could reduce the number of cases requiring ALK FISH testing, which is a more expensive and time consuming technique that requires considerable expertise. In their study, the 5A4 antibody showed high efficiency: for every 100 adenocarcinomas requiring determination of ALK status, FISH testing would only be required in three cases (3%), of which one would be expected to be positive. Moreover, immunohistochemistry gave reliable results regardless of the specific fusion gene and allowed the simultaneous morphological comparison. Some unresolved problems regard rare cases positive at immunohistochemistry, but FISH negative. One of these cases showed a dramatic response to crizotinib [26]. Sun et al. reported that the subtle signal separation because of the chromosomal inversion producing the EML4–ALK translocation variant 1, confirmed by reverse transcription-polymerase chain reaction, is more likely to be missed by FISH [26]. Intrisic limitations of immunohistochemistry must be kept in mind: the variable intensity of staining, the same
heterogeneity in staining and the actual lack of shared protocols. This technique probably will be widely used for screening, but every positive case or cases with inconclusive or controversial staining should be tested by FISH, that remains the gold standard to candidate the patient to crizotinib or other ALK inhibitors.
4. Crizotinib Crizotinib (Xalkori; Pfizer), an orally small weight molecule ALK and c-MET inhibitor, is the treatment of choice for ALK+ tumor patients. Its absorption is modestly reduced (14%) with meal consumption, and it is metabolized in the liver by cytochrome P450 CYP3A4/5 (Table 1). The first human phase I PF1001 trial was originally designed as a dose escalation study, with an expanded molecular cohort for MET-amplified tumors [2]. The dose-limiting toxicity (DLT) was defined at 300 mg twice daily (b.i.d.), and it was related to grade 3 fatigue. The maximum tolerated dose (MTD) and recommended phase II dose was defined at 250 mg b.i.d. Based on the identification of ALK rearrangement in NSCLC and preclinical response to ALK tyrosine kinase inhibition (12), 2 ALK+ tumor patients (one with an inflammatory myofibroblastic tumor and one with NSCLC) were enrolled, and both achieved a partial response. An additional cohort of ALK rearranged NSCLC patients entered at the recommended dose (250 mg b.i.d.). Sixty-one percent of them had an objective response, with a median PFS of 9.7 months and a median duration of response of 49 weeks [2,3]. The Phase II PF1005 study confirmed these striking results: at the latest data lock 901 ALK+ NSCLC patients previously treated with more than one chemotherapy lines received crizotinib therapy. The overall response rate observed was 59.8% (95% CI 53.6–65.9), with a median duration of response of 10.5 months (95% CI 8.2–12.4) and a median PFS of 8.1 months (95% CI 6.8–9.7)[27]. A Phase III trial (PROFILE 1007), comparing crizotinib with second line chemotherapy (docetaxel or pemetrexed) has recently been closed [4]. Three hundred and forty seven advanced ALK rearranged NSCLC patients were enrolled. Patients in the crizotinib arm had an advantage in terms of PFS (7.7 months compared with 3.0 months in the chemotherapy group) and RR (65% vs. 19.5%). Data on OS are not yet mature, since only 40% of the events have occurred. Moreover, it should be considered that the trial was not designed to show an OS advantage since the patients in the chemotherapy arm were crossed-over to crizotinib at the time of progression, and 64% of them received crizotinib as a third line in the PROFILE 1005 single arm Phase II study [27]. With regard to safety data, the most common treatmentrelated events were: nausea, vomiting, diarrhea, peripheral edema, visual disturbance and hepatotoxicity. In the majority of cases, grade 1–2 events were registered, with the exception of 2 deaths due to liver toxicity. Recently, it was shown that
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Table 1 Crizotinib trials. Study
Phase
Patients
ORR
PFS
PROFILE 1001 [2,3]
Phase I
149 NSCLC ALK+ pts
60.8% (95%CI 52.3–68.9)
9.7 m (95%CI 7.7–12.8)
PROFILE 1014
Phase III
PROFILE 1007 [4]
Phase III (crizotinib vs. P or D)
Recruitment ongoing (NCT01639001) 334 NSCLC ALK+ crizotinib vs. CDDP or CBDCA + pemetrexed 347 NSCLC ALK+ pts
PROFILE 1005 [27]
Phase II (crizotinib)
901 NSCLC a ALK + pts
65% vs. 19.5% p < 0.0001 60% (95% CI 53.6–65.9)
7.7 vs. 3.0 m p < 0.0001 8.1 m (95% CI 6.8–9.7)
ORR: objective response rate; PFS: progression free survival; m: months; CDDP: cisplatin; CBDCA: carboplatin; P: pemetrexed; D: docetaxel. a The mature data refer to 261 patients.
crizotinib may cause a decrease in the testosterone levels in male patients, suggesting that testosterone should be measured and replaced during crizotinib treatment [27–29]. Finally, rare observed adverse events were the development of renal cysts and asymptomatic bradycardia.
5. Acquired resistance Despite the initial high response rate to crizotinib therapy, patients develop resistance. Different acquired resistance mechanisms, including secondary ALK mutations, ALK fusion gene amplification and activation of alternative signaling pathways, have been identified. Results come from the molecular analyses performed in a group of NSCLC patients, who underwent biopsies at the time of crizotinib recurrence [30,31]. In about 30% of cases secondary mutations, that may interfere with the drug binding or the ATP affinity, occur in the kinase domain of the ALK gene. So far, different mutations have been identified, L1196M and G1269A being the most common. L1196M is located in the gatekeeper residue, and it causes a steric hindrance for crizotinib binding [32], while G1269A occurs in the ATP binding pocket, and it confers crizotinib resistance in vitro [33]. Additional 2 point mutations, G1202R and S1206Y, located in the solvent front, alter crizotinib binding, while the 1151Tins increases the ATP affinity for the ALK kinase [33]. Two further mutations, C1156Y and L1152R, located in a non active site, do not directly interact either with crizotinib or the ATP. Recently, it was shown that C1156Y induces conformational changes in the binding cavity, thus decreasing the interactions between crizotinib and the ALK kinase, while L1152R reduces crizotinib-mediated inhibition of downstream AKT and ERK 1/2 [34,35]. The ALK mutants show different grade of crizotinib resistance, S1206Y being the least resistant mutation, while 1151Tins and L1196M are the most resistant mutations. Furthermore, in vitro studies showed that the types of mutations may affect second generation ALK-TKIs sensitivity [36].
Additional observed crizotinib acquired resistance mechanisms are represented by ALK amplification, either alone or in combination with a kinase domain mutation, and by the activation of alternative RTKs (KIT amplification, EGFR or k-RAS mutations) [30,31]. The biological significance of KIT amplification is currently under investigation. In vitro studies showed that the role of stroma derived stem cell factor (SCF) may be crucial for KIT activation. In fact, only in the presence of SCF, did the concomitant administration of crizotinib and imatinib lead to a markedly decreased proliferation of crizotinib sensitive cells overexpressing KIT, thus prompting the involvement of the tumor microenvironment [30]. Recently, in 3 out of 11 ALK+ tumor patients, k-RAS and EGFR mutations were found in the post-crizotinib treatment biopsy, suggesting that a second oncogenic driver may be present in the same cell or in separate clonal populations, and emerges under the selected pressure of crizotinib treatment. Furthermore, in 2 out of 11 re-biopsied patients, the ALK rearrangement was not confirmed at recurrence: L858R EGFR mutation was observed in one case, and no molecular resistance mechanism was shown in the other case [31]. Finally, in about 20% of patients, the molecular bases for crizotinib resistance still remains unknown, and additional studies are warranted.
6. Therapeutic strategies at crizotinib recurrence With the aim of overcoming crizotinib-acquired resistance, different therapeutic strategies, according to the type of progression (focal or systemic), are currently under investigation [37]. Approximately 33% of the patients enrolled in the PF1007 trial developed focal progression. If a clinical benefit was maintained, patients were allowed to continue crizotinib beyond RECIST defined progression, and to receive local treatments [4]. Median duration of crizotinib post progression was 15.9 weeks (2.9–73.4). Moreover, in approximately 40% of cases the occurrence of brain lesions was observed in the course of crizotinib treatment. The
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Table 2 Next generation ALK agents. Drug
Company
Study
Class of drug
Patients
ORR
AP26113 [39]
Ariad
Phase I
15 (8 with NSCLC)
PR in 4 out of 4 ALK+ NSCLC progressed at crizotinib
LDK378 [41]
Novartis
Phase I
Able to overcome in vitro ALK L1196M EGFR T790M ALK inhibitor
59 (50 ALK+ NSCLC)
RO5424802 [44] IPI504 [47]
Roche Infinity
Phase I Phase II
ALK inhibitor Hsp90 inhibitor
49 ALK+NSCLC 76 NSCLC
Ganetespib [48]
Synta Pharmaceuticals Corp Novartis
Phase II
Hsp90 inhibitor
96 NSCLC
81% in 37 ALK+ NSCLC progressed at crizotinib 85.7% PR in 2 out of 3 ALK+ crizotinib naive pts 50% in ALK+ crizotinib naive pts
Phase II
Hsp90 inhibitor
121 NSCLC
29% in ALK+ crizotinib naive pts
AUY922 [49]
ORR: objective response rate; PR: partial response.
reasons for crizotinib failure at brain site are not fully understood, and the available data are controversial. In the Phase II PROFILE 1005 study, 18 patients with asymptomatic, non irradiated brain metastases were enrolled, and only 2 of them developed progression. In the other cases, stable disease or partial response were observed [27]. However, in one patient with central nervous system progression under crizotinib therapy, crizotinib concentration was measured both in the plasma and in the cerebrospinal fluid (CSF). While plasma drug levels were 0.53 mol/L, crizotinib concentration in the CSF was 0.0014 mol/L, which is extremely lower than 0.24 mol/L crizotinib IC50, thus suggesting that the drug poorly penetrates the blood-brain barrier [38]. Different second generation ALK-TKIs have been recently evaluated in Phase I studies, and results seem promising (Table 2). Two Phase I studies have been recently closed: one with AP26113 (Ariad) and the other with LDK378 (Novartis). AP26113 is an oral TKI, that inhibits NSCLC cells expressing either the native ALK or the mutant L1196M ALK, as well as the mutated EGFR and the EGFR T790M [39]. In vitro studies showed that AP26113 is 10 fold more potent against the wild type ALK than crizotinib. Moreover, the IC50 of AP263 is lower than the IC50 of crizotinib both for the wild type (11 ± 4 nM compared with 39 ± 30 nM, respectively) and the L1196M mutant ALK (40 ± 19 nM compared with 1215 ± 708, respectively). Fifteen patients were enrolled in the trial, 8 of whom had a diagnosis of NSCLC (4 carried EGFR activated mutations and were refractory to EGFR-TKIs, while the other 4 were ALK+ and had failed crizotinib therapy) [39]. No serious adverse events and no dose-limiting toxicities (DLT) were observed. The dose of 120 mg/daily was chosen for the Phase II expansion study. Partial responses were shown in 4 out of 4 ALK + patients (1 at the dose of 60 mg/daily and 3 at the dose of 90 mg/daily). A Phase II study is going to start. Six cohorts are planned for the recommended phase 2 dose to generate those data, to decide how we are going to develop it. Those cohorts are (1) crizotinib-naive ALK-positive lung cancer patients, (2) ALK-positive lung cancer patients who failed to respond to crizotinib but not to other ALK inhibitors, (3) other
ALK-driven diseases such as inflammatory myelofibroblastic tumors (and there is a beautiful picture of a response in such a patient in the poster), (4) other molecularly driven targets of the drug (including ROS1), (5) a T790M-specific cohort, and (6) a dedicated ALK-positive brain metastases cohort. LDK378 is an oral selective ALK inhibitor, which induced tumor regression in EML4-ALK xenograft models [40]. Similarly to AP263, the IC50 of LDK378 was lower than the IC50 of crizotinib (0.00015 mol compared with 0.003 mol, respectively). Furthermore, LDK378 induced responses also in EML4-ALK xenografts expressing the C1156Y acquired mutation. Fifty nine patients were enrolled, 50 of whom were ALK+ NSCLC and 37 of them had previously received crizotinib. The maximum tolerated dose (MTD) was 750 mg/daily. Eighty one per cent of patients prior treated with crizotinib, and who received LDK378 at a dose ≥400 mg/daily had a response [41]. Two Phase II studies are currently ongoing, one in crizotinib naive patients, and the other in patients who progressed to crizotinib therapy. RO5424802 (CH5424802) (Roche) is the third second generation selective oral ALK inhibitor currently in development. It binds to the ATP site of ALK, and it inhibits the ALK pathway at nano-molar concentrations (IC50 1.9 nM) [42,43]. CH5424802 prevents the ALK autophosphorylation and preclinical studies showed that it is selective for the ALK kinase. It inhibits the ALK downstream pathway by blocking STAT3 and AKT both in NSCLC cell lines expressing the ALK rearrangement and in vivo model [42]. Furthermore, the compound showed antitumour activity against cell lines harboring the L1196M or C1156Y acquired mutations [42,43]. A Phase I and II dose escalation study in ALK+ NSCLC Japanese patients who had never been treated with an ALK inhibitor, has been recently closed [44]. Seventy patients (24 in the Phase I part and 46 in the Phase II part) were enrolled. No DLT was observed, and the recommended dose was defined at 300 mg twice daily. Despite the pharmacokinetics (PK) data in the Japanese population showed that at the dose of 300 mg twice daily, the plateau was not reached, the Japanese authority did not allow to explore the drug at higher dose levels because an increase in the concentration
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of the additive necessary to make the compound soluble was forbidden. On the other hand, the US Phase I study explored the safety of the drug at the dose of 900 mg. No DLT was registered. The PK analysis in the US population is still ongoing, but from the preliminary results it seems that the PK of RO5424802 is different in Western and Eastern patients. In the Phase II portion of the Japanese study, 93.5% of the patients developed an objective response [44]. Moreover, the available results showed that it is effective at brain site: 15 out of the 70 enrolled patients had brain metastases, and 12 were still receiving treatment without progression at the time of the analysis. A Phase II trial in crizotinib pretreated patients is ongoing. Finally, an alternative approach to overcome crizotinib resistance is represented by another class of drugs, the Hsp90 inhibitors, that seems to show promising activity in ALK+ NSCLC patients. The EML4-ALK protein is a client protein of the Hsp90 chaperone, that is responsible for the folding, the stability and the function of the EML4-ALK protein. Hence, the Hsp90 inhibition induces the misfolding of the EML4-ALK protein, thus favoring its degradation by the proteasome system [45,46]. In vitro studies showed that Hsp90 inhibitors have activity against different types of acquired ALK mutations and ALK amplification. Different compounds are currently under evaluation. In a Phase II study with IPI 504, among the 76 NSCLC patients enrolled, 2 out of 3 crizotinib-naïve ALK+ tumor patients achieved a partial response with a median PFS of 7 months [47]. In another Phase II trial, Ganetespib showed a disease control rate at 16 weeks in 88% of ALK+ patients, with an objective partial response in 50% of cases [48]. Finally, a recent Phase II study investigated the activity of AUY922 in 121 NSCLC patients progressing after more than one chemotherapy line [49]. Patients were characterized according to the molecular profile (ALK+, EGFR mutated, k-RAS mutated and wild type). Twenty nine per cent of ALK+ tumor patients and 20% of EGFR mutated tumor patients had a partial response.
7. Conclusions The discovery of EML4-ALK fusion gene and the introduction of crizotinib dramatically improved survival in NSCLC ALK+ tumor patients. Future investigations are warranted to offer better therapeutic chances and improve prognosis in this population. With the approval of crizotinib, new ethical and scientific considerations are arising. According to the British National Institute for Health and Clinical Excellence (NICE), despite being a clinically effective treatment, crizotinib was not considered as a costeffective use of National Health Schemes (NHS) resources. A course of treatment with crizotinib would cost the NHS between £37,512 and £46,890 until disease progression. NICE determined that the cost per quality adjusted life year (QALY) for crizotinib would be £63,800 and £181,100
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when compared with docetaxel chemotherapy, and between £51,700 and £80,500 when compared with best supportive care, thus refusing the reimbursement of crizotinib. This generates the consideration if it is ethical not to give a patient the access to a drug that prolongs PFS, improves the Quality of Life, even though the Phase III randomized trial PF1007 did not show an increase in terms of OS. However, it should be noted that the PF1007 was not designed to show an OS difference. Moreover, approximately 64% of the patients receiving chemotherapy were crossed to crizotinib at progression, thus influencing the OS results. Furthermore, retrospective analyses in NSCLC ALK+ patients who did not receive crizotinib over the course of their treatments had a very poor prognosis, thus suggesting that crizotinib is able to influence the duration of life of these patients. With the up coming of crizotinib and the second generation ALK inhibitors, global therapeutical strategies are needed to plan the best approach and the optimal combination of targeted agents and chemotherapy in NSCLC ALK+ patients. An emerging issue is if, how and when treat ALK+ patients with cisplatin based chemotherapy. While the FDA approved crizotinib for the treatment of NSCLC ALK+ patients in any line, waiting for the results from the ongoing Phase III PF1014 trial that is comparing crizotinib to cisplatin pemetrexed chemotherapy in first line setting, the EMA granted the approval of crizotinib only in previously treated ALK+ patients. This is of striking relevance, especially with the development of second generation ALK inhibitors: which is the best therapeutic sequence among the different ALK agents need to be addressed, whether to employ crizotinib initially and then switch to second generation ALK inhibitors or vice versa. It is likely that the molecular mechanisms of resistance will differ between crizotinib and the newer ALK inhibitors, and probably the selected pressure of these agents may generate more aggressive tumors not susceptible to efficient crizotinib inhibition. Preclinical data showed that the ALK clonal cell populations harboring the acquired resistant mutations have faster proliferative rates than the wild type cell clones [30,31,50]. This is different from what is usually seen in EGFR-mutated NSCLC patients who develop EGFR T790M at EGFR TKIs recurrence, since the clones carrying EGFR T790M proliferate slower than the mutated EGFR without T790M [51], thus providing the molecular basis for continuing EGFR TKIs despite progression or for EGFR TKIs rechallenge after a period of chemotherapy treatment. The management of ALK+ tumor patients at crizotinib recurrence outside a clinical trial with a second generation ALK inhibitor is very challenging and it is a matter of debate whether a more durable disease control may be reached through continuing crizotinib treatment beyond progression or switching to a chemotherapy regimen. Retrospective analyses have demonstrated that pemetrexed is an active treatment in ALK+ tumors [52,53], even though the current data are based on naive crizotinib population, and we do not still know whether patients remain sensitive to pemetrexed after crizotinib treatment.
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Another open question is whether the percentage of ALK+ cells present in tumor tissue affects crizotinib sensitivity. According to the FDA recommendations, ALK translocation should be detected at least in 15% of tumor cells in order to consider the patient eligible to crizotinib treatment. Preclinical data showed that the type of the ALK variant is the most important biologic determinant to affect crizotinib sensitivity. Therefore, the biology of ALK translocation needs to be further explored in order to better understand how patient’s prognosis and crizotinib sensitivity differ according to the ALK variants. This is important especially with the arrival of more potent second generation ALK inhibitors. In conclusion, the ALK agents offer an active and effective treatment for ALK+ NSCLC patients. Several acquired mechanisms are involved in the resistance to crizotinib. Therefore, re-biopsy assessment at crizotinib failure should be recommended to determine the biologic mechanisms of resistance and identify the genotype directed therapy to overcome it. Finally, novel strategies to reduce brain relapse should be explored.
Conflict of interest statement The authors do not have conflict of interest to report.
Reviewers Solange Peters, M.D. Ph.D., Medecin Associé, CHUV, Oncology Department, Bugnon 46, Lausanne, Switzerland. Tony S.K. Mok, M.D., Professor, The Chinese University of Hong Kong, Department of Clinical Oncology, Prince of Wales Hospital, Hong Kong, China. Cesare Gridelli, M.D., S.G. Moscati Hospital-Avellino, Division of Medical Oncology, Via Circumvallazione, I83100 Avellino, Italy.
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Biographies Chiara Lazzari is a Medical Doctor, fellow in Oncology at the European Institute of Oncology. She works in the Division of Thoracic Oncology. She is involved in Phase I studies and translational research on lung cancer. From 2009 to 2010 she was fellow for the project: “Mass spectrometry to predict outcome in NSCLC patients” granted by Associazione Italiana per la Ricerca sul Cancro. She is ESMO member. She has been sub investigator in the Crizotinib trials. Tommaso Martino De Pas is Medical Oncologist with clinical and translational research experience. He is team leader of the Division of Thoracic Oncology at the European Institute of Oncology. His work is mainly focused on individualized therapies, translational research, and Phase I/II/III clinical trials (in collaboration with many important Cancer Centres). He has been principal investigator in the Crizotinib trials.
Targeting ALK in patients with advanced Non Small Cell Lung Cancer: Biology, diagnostic and therapeutic options Chiara Lazzari a,∗ , Gianluca Spitaleri a , Chiara Catania a , Massimo Barberis b , Cristina Noberasco a , Mariacarmela Santarpia a , Angelo Delmonte a , Francesca Toffalorio a , Fabio Conforti a , Tommaso Martino De Pas a a
European Institute of Oncology, Division of Thoracic Oncology, Italy b European Institute of Oncology, Division of Pathology, Italy Accepted 17 September 2013
Contents 1. 2. 3. 4. 5. 6. 7.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EML4-ALK biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Crizotinib . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acquired resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Therapeutic strategies at crizotinib recurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abstract The discovery of EML4-ALK fusion gene in a subgroup of patients with lung adenocarcinoma led to the development of a new class of agents, the ALK inhibitors, and dramatically improved the clinical outcome of these patients. The striking results from clinical trials with crizotinib, the first ALK inhibitor evaluated, allowed the accelerated approval of crizotinib from the USA Food and Drug Administration (FDA). Despite the high initial results, patients acquire resistance to crizotinib, and different next generation ALK kinase inhibitors have been developed. In the current review, we will analyze the biology of EML4-ALK gene, the acquired resistance mechanisms to crizotinib, the therapeutic strategies, currently under evaluation, designed to overcome crizotinib resistance, and the open issues that need to be addressed in order to improve outcome in ALK+ Non Small Cell Lung Cancer (NSCLC) patients. © 2013 Elsevier Ireland Ltd. All rights reserved. Keywords: Non Small Cell Lung Cancer; EML4-ALK fusion gene; Crizotinib
1. Introduction
∗ Corresponding author at: European Institute of Oncology, via Ripamonti 435, 20131 Milan, Italy. Tel.: +39 0255210169. E-mail address: [email protected] (C. Lazzari).
1040-8428/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.critrevonc.2013.09.003
Non Small Cell Lung Cancer (NSCLC) is one of the leading causes of tumor-related deaths in the world. Chemotherapy is the treatment of choice in the majority of patients, and it is usually based on platinum doublet in front-line treatment and on mono-chemotherapy in the
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subsequent lines. Despite chemotherapy, the prognosis of NSCLC patients still remains poor, reaching a median survival of about 10 months in the metastatic setting. In 2007, the EML4-ALK fusion gene was discovered in a subset of patients with lung adenocarcinoma [1]. In a Phase I study, crizotinib, a dual ALK and MET inhibitor, showed an objective response rate (ORR) of 60% and a median progression-free Survival (PFS) at 6 months of 72% in patients carrying the ALK translocation [2,3]. A Phase III trial comparing crizotinib with second line standard chemotherapy (docetaxel or pemetrexed) has recently been closed and showed an improvement in terms of PFS and ORR for crizotinib compared with second line standard chemotherapy [4], while another Phase III study, comparing crizotinib with first line cisplatin pemetrexed chemotherapy is ongoing (NCT01639001). In August 2011, crizotinib was approved by the Food and Drug Administration (FDA) for the treatment of NSCLC patients harboring the EML4-ALK fusion gene, and in October 2012 the European Medicines Agency (EMA) gave conditional marketing authorization for the use of crizotinib in previously treated ALK+ NSCLC patients. Despite the initial high ORR to crizotinib, patients develop resistance. In the current review, we will focus on the biology of EML4-ALK gene, the acquired resistance mechanisms to crizotinib and the therapeutic strategies, currently under evaluation, designed to overcome crizotinib resistance.
2. EML4-ALK biology The EML4-ALK fusion gene was isolated for the first time in DNA from a 62 year-old man with lung adenocarcinoma [1]. The EML4-ALK gene is the result of a chromosome rearrangement between the N-terminal portion of the echinoderm microtubule associated protein-like 4 (EML4) gene and the tyrosine kinase (TK) domain of the anaplastic lymphoma kinase (ALK) gene. Both are located in the short arm of the chromosome 2 (2p), and have opposite orientations. An inversion of the two genes occurs within the chromosome 2p, leading to a chimeric oncoprotein with constitutive TK activity and oncogenic transforming activity in nude mice. The EML4 portion contains different regions: the coiled coil N terminal basic region, the hydrophobic HELP domain and the WD repeat. In vitro studies showed that the basic domain contribute most to the transforming activity of the fusion protein, since its deletion leads to a 84% decrease of the catalytic activity of the protein itself. This region, in fact, is involved in the EML4-ALK dimerization [1], while the HELP domain seems to mediate tubulin binding and the WD repeat is involved in protein–protein interaction. The EML4-ALK dimerization activates 3 interconnected downstream pathways, including the MAPK pathway (Ras/Raf/MEK/ERK), the JAK/STAT pathway and the PI3K/AKT pathway [5]. Twenty-seven ALK fusion variants have been described, 21 of those regarding the EML4, since truncations of EML4 may occur at different exons (2, 6, 13, 14, 15, 17, 18 and 20)
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[6–8]. In all variants, the TK domain of the ALK gene begins at exon 20 [9]. The most common fusion proteins are variant 1 (33%) and variants 3a/b (29%), that consist of exons 1–13 and 1–6 of EML4, joined to exon 20 of ALK [1]. Variants 3a and 3b derive from an alternative splicing of 33 bp that occurs within exon 6 [8,10,11]. Moreover, additional fusion partners of ALK have been described in NSCLC patients: TGF, KLC1 and KIKF5B [12]. The EML4-ALK translocation has been found in approximately 5% of NSCLC patients, more frequently in never or light smokers, and in younger patients (median age 52 years) with adenocarcinoma histology [13]. Moreover, among the different subtypes of adenocarcinoma, the EML4ALK translocation seems to be more frequently expressed in patients with the acinar, papillary, cribriform, mucinproducing or signet ring patterns [14–16].
3. Diagnostics In order to detect the ALK rearrangement, different methods are currently under investigation, including fluorescence in situ hybridization (FISH), reverse transcriptase PCR (RTPCR) and immunohistochemistry (IHC). Vysis Dual color break apart FISH (Abbott Molecular, Des Plaines, IL) is the recommended diagnostic method approved by the FDA to identify ALK+ NSCLC, and is used to screen patients to enter the crizotinib and the ongoing trials with second ALK inhibitors. ALK FISH is considered positive if a split by more than 2 signal diameters is detected between the red and green signals labeling the 3 end of the ALK gene and the 5 end of EML4 in more than 15% of tumor cells, analyzing at least 4 fields. FISH is considered positive also in the presence of a single red pattern in more than 15% of tumor cells. Considering that EML4 and ALK are both located on chromosome 2p, and separated by only 12.5 megabases, false negative diagnoses may occur. Conversely, DNA stretching or nuclear sectioning may cause false positive results. Furthermore, FISH does not discriminate between the different ALK fusion types, while RT PCR can define the ALK partner and the fusion variant. Definitive results about how crizotinib sensitivity differs among the ALK variants have not yet been determined. Recently, in vitro studies have shown that EML4-ALK variant 2 seems the most sensitive to ALK inhibition, followed by variants 1 and 3b, that exhibit an intermediate sensitivity and variant 3a, that appears the least sensitive. Crizotinib sensitivity seems to be inversely correlated with the half-lives of the fusion types, variant 2 being the less stable and the most susceptible to ALK inhibition due to a more rapid protein degradation [17]. These data have not yet been confirmed in clinical practice: only 29 of the 82 patients enrolled in the Phase I crizotinib trial had an adequate amount of tissue to perform RT-PCR, but no association between EML4-ALK variants and (Response Evaluation Criteria in Solid Tumors) RECIST criteria was found [2]. One of the secondary end
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points of the current crizotinib studies is to determine the types of the fusion variants and the ALK expression, but analyses are still ongoing. RT-PCR seems to be particularly useful for the identification of variant 1 [18–20]. In fact, since variant 1 originates from a 2p inversion without any loss in the 5 fragment, it may be a challenge to detect the split signal by FISH. However, the limit of RT-PCR as a diagnostic method is that it cannot define unknown ALK rearrangements. The presence of ALK rearrangements is a rare event, while NSCLC tumor has a high incidence. Therefore routine testing for ALK using FISH only may not be feasible, because of cost, required expertise and the labor-intensive nature of performing and interpreting the test. Immunohistochemistry can be a useful technique to pre-screen cases reducing the burden of FISH testing to a manageable number. The use of IHC has been explored [21]. Different ALK antibodies (ALK1, 5A4 and DF53) have been evaluated, and results have been controversial. The ALK1 antibody is not directed against all the epitopes of the ALK variants and this limits its application in routine practice. The 5A4 and DF53 antibodies are still under study, and they are not yet commercially available. In a cohort of 46 patients with lung adenocarcinoma, FISH, RT-PCR and IHC were compared in terms of sensitivity and specificity [18]. The primers used to perform RT-PCR were designed to detect only variants 1 and 3a/3b, and the ALK1 antibody was used for IHC. Concordance among the 3 methods was 100% for variants 3a/3b, while no concordance was observed for variant 1. RT-PCR was shown to be the most sensitive test, while the sensitivity for IHC was poor, and only 2 ALK IHC+ cases were confirmed by both RT-PCR and FISH. Both tumor patients carried the variant 3a/3b. More recent studies strongly underlined the robustness of immunohistochemistry in identifying ALK-rearranged tumors [22–24]. In a multicentre study, Selinger et al. showed that when high-affinity antibodies are used with a highly sensitive detection method, ALK immunohistochemistry may provide an effective pre-screening technique to complement ALK FISH testing [25]. In fact, the use of immunohistochemistry could reduce the number of cases requiring ALK FISH testing, which is a more expensive and time consuming technique that requires considerable expertise. In their study, the 5A4 antibody showed high efficiency: for every 100 adenocarcinomas requiring determination of ALK status, FISH testing would only be required in three cases (3%), of which one would be expected to be positive. Moreover, immunohistochemistry gave reliable results regardless of the specific fusion gene and allowed the simultaneous morphological comparison. Some unresolved problems regard rare cases positive at immunohistochemistry, but FISH negative. One of these cases showed a dramatic response to crizotinib [26]. Sun et al. reported that the subtle signal separation because of the chromosomal inversion producing the EML4–ALK translocation variant 1, confirmed by reverse transcription-polymerase chain reaction, is more likely to be missed by FISH [26]. Intrisic limitations of immunohistochemistry must be kept in mind: the variable intensity of staining, the same
heterogeneity in staining and the actual lack of shared protocols. This technique probably will be widely used for screening, but every positive case or cases with inconclusive or controversial staining should be tested by FISH, that remains the gold standard to candidate the patient to crizotinib or other ALK inhibitors.
4. Crizotinib Crizotinib (Xalkori; Pfizer), an orally small weight molecule ALK and c-MET inhibitor, is the treatment of choice for ALK+ tumor patients. Its absorption is modestly reduced (14%) with meal consumption, and it is metabolized in the liver by cytochrome P450 CYP3A4/5 (Table 1). The first human phase I PF1001 trial was originally designed as a dose escalation study, with an expanded molecular cohort for MET-amplified tumors [2]. The dose-limiting toxicity (DLT) was defined at 300 mg twice daily (b.i.d.), and it was related to grade 3 fatigue. The maximum tolerated dose (MTD) and recommended phase II dose was defined at 250 mg b.i.d. Based on the identification of ALK rearrangement in NSCLC and preclinical response to ALK tyrosine kinase inhibition (12), 2 ALK+ tumor patients (one with an inflammatory myofibroblastic tumor and one with NSCLC) were enrolled, and both achieved a partial response. An additional cohort of ALK rearranged NSCLC patients entered at the recommended dose (250 mg b.i.d.). Sixty-one percent of them had an objective response, with a median PFS of 9.7 months and a median duration of response of 49 weeks [2,3]. The Phase II PF1005 study confirmed these striking results: at the latest data lock 901 ALK+ NSCLC patients previously treated with more than one chemotherapy lines received crizotinib therapy. The overall response rate observed was 59.8% (95% CI 53.6–65.9), with a median duration of response of 10.5 months (95% CI 8.2–12.4) and a median PFS of 8.1 months (95% CI 6.8–9.7)[27]. A Phase III trial (PROFILE 1007), comparing crizotinib with second line chemotherapy (docetaxel or pemetrexed) has recently been closed [4]. Three hundred and forty seven advanced ALK rearranged NSCLC patients were enrolled. Patients in the crizotinib arm had an advantage in terms of PFS (7.7 months compared with 3.0 months in the chemotherapy group) and RR (65% vs. 19.5%). Data on OS are not yet mature, since only 40% of the events have occurred. Moreover, it should be considered that the trial was not designed to show an OS advantage since the patients in the chemotherapy arm were crossed-over to crizotinib at the time of progression, and 64% of them received crizotinib as a third line in the PROFILE 1005 single arm Phase II study [27]. With regard to safety data, the most common treatmentrelated events were: nausea, vomiting, diarrhea, peripheral edema, visual disturbance and hepatotoxicity. In the majority of cases, grade 1–2 events were registered, with the exception of 2 deaths due to liver toxicity. Recently, it was shown that
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Table 1 Crizotinib trials. Study
Phase
Patients
ORR
PFS
PROFILE 1001 [2,3]
Phase I
149 NSCLC ALK+ pts
60.8% (95%CI 52.3–68.9)
9.7 m (95%CI 7.7–12.8)
PROFILE 1014
Phase III
PROFILE 1007 [4]
Phase III (crizotinib vs. P or D)
Recruitment ongoing (NCT01639001) 334 NSCLC ALK+ crizotinib vs. CDDP or CBDCA + pemetrexed 347 NSCLC ALK+ pts
PROFILE 1005 [27]
Phase II (crizotinib)
901 NSCLC a ALK + pts
65% vs. 19.5% p < 0.0001 60% (95% CI 53.6–65.9)
7.7 vs. 3.0 m p < 0.0001 8.1 m (95% CI 6.8–9.7)
ORR: objective response rate; PFS: progression free survival; m: months; CDDP: cisplatin; CBDCA: carboplatin; P: pemetrexed; D: docetaxel. a The mature data refer to 261 patients.
crizotinib may cause a decrease in the testosterone levels in male patients, suggesting that testosterone should be measured and replaced during crizotinib treatment [27–29]. Finally, rare observed adverse events were the development of renal cysts and asymptomatic bradycardia.
5. Acquired resistance Despite the initial high response rate to crizotinib therapy, patients develop resistance. Different acquired resistance mechanisms, including secondary ALK mutations, ALK fusion gene amplification and activation of alternative signaling pathways, have been identified. Results come from the molecular analyses performed in a group of NSCLC patients, who underwent biopsies at the time of crizotinib recurrence [30,31]. In about 30% of cases secondary mutations, that may interfere with the drug binding or the ATP affinity, occur in the kinase domain of the ALK gene. So far, different mutations have been identified, L1196M and G1269A being the most common. L1196M is located in the gatekeeper residue, and it causes a steric hindrance for crizotinib binding [32], while G1269A occurs in the ATP binding pocket, and it confers crizotinib resistance in vitro [33]. Additional 2 point mutations, G1202R and S1206Y, located in the solvent front, alter crizotinib binding, while the 1151Tins increases the ATP affinity for the ALK kinase [33]. Two further mutations, C1156Y and L1152R, located in a non active site, do not directly interact either with crizotinib or the ATP. Recently, it was shown that C1156Y induces conformational changes in the binding cavity, thus decreasing the interactions between crizotinib and the ALK kinase, while L1152R reduces crizotinib-mediated inhibition of downstream AKT and ERK 1/2 [34,35]. The ALK mutants show different grade of crizotinib resistance, S1206Y being the least resistant mutation, while 1151Tins and L1196M are the most resistant mutations. Furthermore, in vitro studies showed that the types of mutations may affect second generation ALK-TKIs sensitivity [36].
Additional observed crizotinib acquired resistance mechanisms are represented by ALK amplification, either alone or in combination with a kinase domain mutation, and by the activation of alternative RTKs (KIT amplification, EGFR or k-RAS mutations) [30,31]. The biological significance of KIT amplification is currently under investigation. In vitro studies showed that the role of stroma derived stem cell factor (SCF) may be crucial for KIT activation. In fact, only in the presence of SCF, did the concomitant administration of crizotinib and imatinib lead to a markedly decreased proliferation of crizotinib sensitive cells overexpressing KIT, thus prompting the involvement of the tumor microenvironment [30]. Recently, in 3 out of 11 ALK+ tumor patients, k-RAS and EGFR mutations were found in the post-crizotinib treatment biopsy, suggesting that a second oncogenic driver may be present in the same cell or in separate clonal populations, and emerges under the selected pressure of crizotinib treatment. Furthermore, in 2 out of 11 re-biopsied patients, the ALK rearrangement was not confirmed at recurrence: L858R EGFR mutation was observed in one case, and no molecular resistance mechanism was shown in the other case [31]. Finally, in about 20% of patients, the molecular bases for crizotinib resistance still remains unknown, and additional studies are warranted.
6. Therapeutic strategies at crizotinib recurrence With the aim of overcoming crizotinib-acquired resistance, different therapeutic strategies, according to the type of progression (focal or systemic), are currently under investigation [37]. Approximately 33% of the patients enrolled in the PF1007 trial developed focal progression. If a clinical benefit was maintained, patients were allowed to continue crizotinib beyond RECIST defined progression, and to receive local treatments [4]. Median duration of crizotinib post progression was 15.9 weeks (2.9–73.4). Moreover, in approximately 40% of cases the occurrence of brain lesions was observed in the course of crizotinib treatment. The
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Table 2 Next generation ALK agents. Drug
Company
Study
Class of drug
Patients
ORR
AP26113 [39]
Ariad
Phase I
15 (8 with NSCLC)
PR in 4 out of 4 ALK+ NSCLC progressed at crizotinib
LDK378 [41]
Novartis
Phase I
Able to overcome in vitro ALK L1196M EGFR T790M ALK inhibitor
59 (50 ALK+ NSCLC)
RO5424802 [44] IPI504 [47]
Roche Infinity
Phase I Phase II
ALK inhibitor Hsp90 inhibitor
49 ALK+NSCLC 76 NSCLC
Ganetespib [48]
Synta Pharmaceuticals Corp Novartis
Phase II
Hsp90 inhibitor
96 NSCLC
81% in 37 ALK+ NSCLC progressed at crizotinib 85.7% PR in 2 out of 3 ALK+ crizotinib naive pts 50% in ALK+ crizotinib naive pts
Phase II
Hsp90 inhibitor
121 NSCLC
29% in ALK+ crizotinib naive pts
AUY922 [49]
ORR: objective response rate; PR: partial response.
reasons for crizotinib failure at brain site are not fully understood, and the available data are controversial. In the Phase II PROFILE 1005 study, 18 patients with asymptomatic, non irradiated brain metastases were enrolled, and only 2 of them developed progression. In the other cases, stable disease or partial response were observed [27]. However, in one patient with central nervous system progression under crizotinib therapy, crizotinib concentration was measured both in the plasma and in the cerebrospinal fluid (CSF). While plasma drug levels were 0.53 mol/L, crizotinib concentration in the CSF was 0.0014 mol/L, which is extremely lower than 0.24 mol/L crizotinib IC50, thus suggesting that the drug poorly penetrates the blood-brain barrier [38]. Different second generation ALK-TKIs have been recently evaluated in Phase I studies, and results seem promising (Table 2). Two Phase I studies have been recently closed: one with AP26113 (Ariad) and the other with LDK378 (Novartis). AP26113 is an oral TKI, that inhibits NSCLC cells expressing either the native ALK or the mutant L1196M ALK, as well as the mutated EGFR and the EGFR T790M [39]. In vitro studies showed that AP26113 is 10 fold more potent against the wild type ALK than crizotinib. Moreover, the IC50 of AP263 is lower than the IC50 of crizotinib both for the wild type (11 ± 4 nM compared with 39 ± 30 nM, respectively) and the L1196M mutant ALK (40 ± 19 nM compared with 1215 ± 708, respectively). Fifteen patients were enrolled in the trial, 8 of whom had a diagnosis of NSCLC (4 carried EGFR activated mutations and were refractory to EGFR-TKIs, while the other 4 were ALK+ and had failed crizotinib therapy) [39]. No serious adverse events and no dose-limiting toxicities (DLT) were observed. The dose of 120 mg/daily was chosen for the Phase II expansion study. Partial responses were shown in 4 out of 4 ALK + patients (1 at the dose of 60 mg/daily and 3 at the dose of 90 mg/daily). A Phase II study is going to start. Six cohorts are planned for the recommended phase 2 dose to generate those data, to decide how we are going to develop it. Those cohorts are (1) crizotinib-naive ALK-positive lung cancer patients, (2) ALK-positive lung cancer patients who failed to respond to crizotinib but not to other ALK inhibitors, (3) other
ALK-driven diseases such as inflammatory myelofibroblastic tumors (and there is a beautiful picture of a response in such a patient in the poster), (4) other molecularly driven targets of the drug (including ROS1), (5) a T790M-specific cohort, and (6) a dedicated ALK-positive brain metastases cohort. LDK378 is an oral selective ALK inhibitor, which induced tumor regression in EML4-ALK xenograft models [40]. Similarly to AP263, the IC50 of LDK378 was lower than the IC50 of crizotinib (0.00015 mol compared with 0.003 mol, respectively). Furthermore, LDK378 induced responses also in EML4-ALK xenografts expressing the C1156Y acquired mutation. Fifty nine patients were enrolled, 50 of whom were ALK+ NSCLC and 37 of them had previously received crizotinib. The maximum tolerated dose (MTD) was 750 mg/daily. Eighty one per cent of patients prior treated with crizotinib, and who received LDK378 at a dose ≥400 mg/daily had a response [41]. Two Phase II studies are currently ongoing, one in crizotinib naive patients, and the other in patients who progressed to crizotinib therapy. RO5424802 (CH5424802) (Roche) is the third second generation selective oral ALK inhibitor currently in development. It binds to the ATP site of ALK, and it inhibits the ALK pathway at nano-molar concentrations (IC50 1.9 nM) [42,43]. CH5424802 prevents the ALK autophosphorylation and preclinical studies showed that it is selective for the ALK kinase. It inhibits the ALK downstream pathway by blocking STAT3 and AKT both in NSCLC cell lines expressing the ALK rearrangement and in vivo model [42]. Furthermore, the compound showed antitumour activity against cell lines harboring the L1196M or C1156Y acquired mutations [42,43]. A Phase I and II dose escalation study in ALK+ NSCLC Japanese patients who had never been treated with an ALK inhibitor, has been recently closed [44]. Seventy patients (24 in the Phase I part and 46 in the Phase II part) were enrolled. No DLT was observed, and the recommended dose was defined at 300 mg twice daily. Despite the pharmacokinetics (PK) data in the Japanese population showed that at the dose of 300 mg twice daily, the plateau was not reached, the Japanese authority did not allow to explore the drug at higher dose levels because an increase in the concentration
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of the additive necessary to make the compound soluble was forbidden. On the other hand, the US Phase I study explored the safety of the drug at the dose of 900 mg. No DLT was registered. The PK analysis in the US population is still ongoing, but from the preliminary results it seems that the PK of RO5424802 is different in Western and Eastern patients. In the Phase II portion of the Japanese study, 93.5% of the patients developed an objective response [44]. Moreover, the available results showed that it is effective at brain site: 15 out of the 70 enrolled patients had brain metastases, and 12 were still receiving treatment without progression at the time of the analysis. A Phase II trial in crizotinib pretreated patients is ongoing. Finally, an alternative approach to overcome crizotinib resistance is represented by another class of drugs, the Hsp90 inhibitors, that seems to show promising activity in ALK+ NSCLC patients. The EML4-ALK protein is a client protein of the Hsp90 chaperone, that is responsible for the folding, the stability and the function of the EML4-ALK protein. Hence, the Hsp90 inhibition induces the misfolding of the EML4-ALK protein, thus favoring its degradation by the proteasome system [45,46]. In vitro studies showed that Hsp90 inhibitors have activity against different types of acquired ALK mutations and ALK amplification. Different compounds are currently under evaluation. In a Phase II study with IPI 504, among the 76 NSCLC patients enrolled, 2 out of 3 crizotinib-naïve ALK+ tumor patients achieved a partial response with a median PFS of 7 months [47]. In another Phase II trial, Ganetespib showed a disease control rate at 16 weeks in 88% of ALK+ patients, with an objective partial response in 50% of cases [48]. Finally, a recent Phase II study investigated the activity of AUY922 in 121 NSCLC patients progressing after more than one chemotherapy line [49]. Patients were characterized according to the molecular profile (ALK+, EGFR mutated, k-RAS mutated and wild type). Twenty nine per cent of ALK+ tumor patients and 20% of EGFR mutated tumor patients had a partial response.
7. Conclusions The discovery of EML4-ALK fusion gene and the introduction of crizotinib dramatically improved survival in NSCLC ALK+ tumor patients. Future investigations are warranted to offer better therapeutic chances and improve prognosis in this population. With the approval of crizotinib, new ethical and scientific considerations are arising. According to the British National Institute for Health and Clinical Excellence (NICE), despite being a clinically effective treatment, crizotinib was not considered as a costeffective use of National Health Schemes (NHS) resources. A course of treatment with crizotinib would cost the NHS between £37,512 and £46,890 until disease progression. NICE determined that the cost per quality adjusted life year (QALY) for crizotinib would be £63,800 and £181,100
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when compared with docetaxel chemotherapy, and between £51,700 and £80,500 when compared with best supportive care, thus refusing the reimbursement of crizotinib. This generates the consideration if it is ethical not to give a patient the access to a drug that prolongs PFS, improves the Quality of Life, even though the Phase III randomized trial PF1007 did not show an increase in terms of OS. However, it should be noted that the PF1007 was not designed to show an OS difference. Moreover, approximately 64% of the patients receiving chemotherapy were crossed to crizotinib at progression, thus influencing the OS results. Furthermore, retrospective analyses in NSCLC ALK+ patients who did not receive crizotinib over the course of their treatments had a very poor prognosis, thus suggesting that crizotinib is able to influence the duration of life of these patients. With the up coming of crizotinib and the second generation ALK inhibitors, global therapeutical strategies are needed to plan the best approach and the optimal combination of targeted agents and chemotherapy in NSCLC ALK+ patients. An emerging issue is if, how and when treat ALK+ patients with cisplatin based chemotherapy. While the FDA approved crizotinib for the treatment of NSCLC ALK+ patients in any line, waiting for the results from the ongoing Phase III PF1014 trial that is comparing crizotinib to cisplatin pemetrexed chemotherapy in first line setting, the EMA granted the approval of crizotinib only in previously treated ALK+ patients. This is of striking relevance, especially with the development of second generation ALK inhibitors: which is the best therapeutic sequence among the different ALK agents need to be addressed, whether to employ crizotinib initially and then switch to second generation ALK inhibitors or vice versa. It is likely that the molecular mechanisms of resistance will differ between crizotinib and the newer ALK inhibitors, and probably the selected pressure of these agents may generate more aggressive tumors not susceptible to efficient crizotinib inhibition. Preclinical data showed that the ALK clonal cell populations harboring the acquired resistant mutations have faster proliferative rates than the wild type cell clones [30,31,50]. This is different from what is usually seen in EGFR-mutated NSCLC patients who develop EGFR T790M at EGFR TKIs recurrence, since the clones carrying EGFR T790M proliferate slower than the mutated EGFR without T790M [51], thus providing the molecular basis for continuing EGFR TKIs despite progression or for EGFR TKIs rechallenge after a period of chemotherapy treatment. The management of ALK+ tumor patients at crizotinib recurrence outside a clinical trial with a second generation ALK inhibitor is very challenging and it is a matter of debate whether a more durable disease control may be reached through continuing crizotinib treatment beyond progression or switching to a chemotherapy regimen. Retrospective analyses have demonstrated that pemetrexed is an active treatment in ALK+ tumors [52,53], even though the current data are based on naive crizotinib population, and we do not still know whether patients remain sensitive to pemetrexed after crizotinib treatment.
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Another open question is whether the percentage of ALK+ cells present in tumor tissue affects crizotinib sensitivity. According to the FDA recommendations, ALK translocation should be detected at least in 15% of tumor cells in order to consider the patient eligible to crizotinib treatment. Preclinical data showed that the type of the ALK variant is the most important biologic determinant to affect crizotinib sensitivity. Therefore, the biology of ALK translocation needs to be further explored in order to better understand how patient’s prognosis and crizotinib sensitivity differ according to the ALK variants. This is important especially with the arrival of more potent second generation ALK inhibitors. In conclusion, the ALK agents offer an active and effective treatment for ALK+ NSCLC patients. Several acquired mechanisms are involved in the resistance to crizotinib. Therefore, re-biopsy assessment at crizotinib failure should be recommended to determine the biologic mechanisms of resistance and identify the genotype directed therapy to overcome it. Finally, novel strategies to reduce brain relapse should be explored.
Conflict of interest statement The authors do not have conflict of interest to report.
Reviewers Solange Peters, M.D. Ph.D., Medecin Associé, CHUV, Oncology Department, Bugnon 46, Lausanne, Switzerland. Tony S.K. Mok, M.D., Professor, The Chinese University of Hong Kong, Department of Clinical Oncology, Prince of Wales Hospital, Hong Kong, China. Cesare Gridelli, M.D., S.G. Moscati Hospital-Avellino, Division of Medical Oncology, Via Circumvallazione, I83100 Avellino, Italy.
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Biographies Chiara Lazzari is a Medical Doctor, fellow in Oncology at the European Institute of Oncology. She works in the Division of Thoracic Oncology. She is involved in Phase I studies and translational research on lung cancer. From 2009 to 2010 she was fellow for the project: “Mass spectrometry to predict outcome in NSCLC patients” granted by Associazione Italiana per la Ricerca sul Cancro. She is ESMO member. She has been sub investigator in the Crizotinib trials. Tommaso Martino De Pas is Medical Oncologist with clinical and translational research experience. He is team leader of the Division of Thoracic Oncology at the European Institute of Oncology. His work is mainly focused on individualized therapies, translational research, and Phase I/II/III clinical trials (in collaboration with many important Cancer Centres). He has been principal investigator in the Crizotinib trials.