The identification of oncogenic genomic alterations is expected to facilitate the development of new molecularly targeted therapies for cancer. EML4 (echinoderm microtubule-associated protein-like 4)-ALK (anaplastic lymphoma kinase) was recently identified as a transforming fusion gene in non-small cell lung cancer (NSCLC). A small-molecule tyrosine kinase inhibitor of ALK, crizotinib, shows pronounced clinical activity in the treatment of patients with NSCLC positive for EML4-ALK, and it has rapidly entered into daily clinical practice. This review focuses on the biology and clinical features of, as well as diagnostic testing for, EML4-ALK-positive NSCLC. Current data on the efficacy and toxicity of crizotinib are also examined, and future directions for the treatment of NSCLC positive for ALK rearrangement are addressed. (Cancer Sci, doi: 10.1111/j.1349-7006.2012.02327.x, 2012)
Lung cancer is the leading cause of cancer deaths worldwide. Given that the efficacy of conventional chemotherapeutic agents with regard to improving clinical outcome in lung cancer patients is limited, target-based therapies are being pursued as potential treatment alternatives. Somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) have been associated with tumor responsiveness to EGFR tyrosine kinase inhibitors (TKIs) in a subset of individuals with non-small cell lung cancer (NSCLC).[1-3] Such findings suggest that the use of molecularly targeted therapy in genetically defined subsets of cancer patients may prove to be an effective strategy for the treatment of many cancers including NSCLC. Given that lung cancer is a common type of cancer, the identification of even small subsets of lung cancer patients harboring specific genetic abnormalities will translate into the provision of large cohorts for targeted therapy.
Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase whose gene was initially identified as part of the NPM-ALK fusion gene that contributes to the pathogenesis of a subset of anaplastic large cell lymphoma cases. ALK plays a key role in development, but it is not expressed in most, if not all, adult tissues. In 2007, a fusion of ALK with the echinoderm microtubule-associated protein-like 4 gene (EML4) was identified by a Japanese group led by Hiroyuki Mano as a result of the screening of a cDNA library derived from the tumor of a 62-year-old Japanese man with adenocarcinoma of the lung. The EML4-ALK fusion oncogene arises from an inversion on the short arm of chromosome 2 that joins the 5′ region of EML4 (encoding the NH2-terminal portion of EML4) to the 3′ region of ALK (encoding the COOH-terminal portion of ALK) (Fig. 1). The chromosomal inversion does not always occur in the same precise location, however, giving rise to multiple EML4-ALK variants, all of which contain the same intracellular tyrosine kinase domain of ALK but different truncations of EML4. Fusion of ALK with other genes, including TFG and KIF5B, has also been identified in NSCLC, although these fusions appear to be much less common than EML4-ALK (Fig. 1).[6, 7] ALK fusion proteins, including EML4-ALK, undergo ligand-independent dimerization mediated by the coiled-coil domain of the fusion partner, resulting in constitutive activation of the ALK tyrosine kinase. The EML4-ALK fusion protein exhibits marked transforming activity both in vitro and in vivo. In a transgenic mouse model, lung-specific expression of EML4-ALK thus results in the development of multiple lung adenocarcinomas.
Inhibition of the kinase activity of ALK by an ALK-TKI has been found to suppress the growth of and to induce apoptosis in EML4-ALK-positive lung cancer cells. Treatment of EML4-ALK transgenic mice with ALK inhibitors also resulted in tumor shrinkage. The first clinically available ALK-TKI, crizotinib, has shown pronounced clinical efficacy in the treatment of patients with advanced NSCLC positive for EML4-ALK. The clinical development of this drug occurred over a remarkably short period of time, from the initial identification of the EML4-ALK translocation as an oncogene in 2007, with validation of the fusion protein as a clinical target for NSCLC in 2010, to the approval of crizotinib by the FDA in 2011.
ERK and STAT3 are the Principal Signaling Pathways Activated by EML4-ALK
EML4-ALK undergoes constitutive oligomerization mediated by the coiled-coil domain of the EML4 portion of the fusion protein, and it manifests marked oncogenic activity both in vitro and in vivo. The intracellular signaling pathways activated by EML4-ALK to induce tumor growth have not been fully characterized, however. To investigate the signaling pathways that operate downstream of EML4-ALK, we established mouse fibroblastic (NIH 3T3) cell lines that stably express EML4-ALK variants 1 or 3, which are the most common EML4-ALK variants, together accounting for approximately 60% of EML4-ALK-positive lung cancer cases. Consistent with previous observations, these EML4-ALK variants exhibited marked transforming activity both in vitro and in vivo. Immunoblot analysis revealed that the phosphorylation (activation) of both the mitogen-activated protein kinase (MAPK) ERK (extracellular signal-regulated kinase) and signal transducer and activator of transcription 3 (STAT3) was markedly increased in the cells expressing either variant of EML4-ALK, compared with that apparent in 3T3-Mock cells transfected with the empty vector, whereas the phosphorylation level of the protein kinase AKT was not affected by the expression of EML4-ALK. These data suggest that both ERK and STAT3 signaling pathways, but not the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway, are the principal downstream pathways activated by EML4-ALK in lung cancer cells (Fig. 2).
Effects of ALK Inhibition on Apoptosis-Related Proteins in EML4-ALK-Positive Lung Cancer Cells
Preclinical studies have shown that treatment of NSCLC cell lines expressing EML4-ALK with ALK inhibitors suppresses cell proliferation and induces apoptosis. Indeed, treatment of cells expressing EML4-ALK with TAE684, a selective and highly potent ALK inhibitor, induced marked apoptosis accompanied by inhibition of ERK and STAT3 phosphorylation (but not that of AKT phosphorylation), supporting the notion that ERK and STAT3 signaling pathways function downstream of EML4-ALK. We further examined the effects of ALK inhibition on the expression of apoptosis-related proteins in such cells and found that TAE684 induced BIM expression in a manner dependent on inhibition of the ERK pathway. BIM is a key proapoptotic member of the Bcl-2 family of proteins and initiates apoptosis signaling by binding to and antagonizing the function of prosurvival members of the Bcl-2 family. We found that knockdown of BIM by RNAi resulted in significant inhibition of TAE684-induced apoptosis in EML4-ALK-positive cells, suggesting that BIM induction plays a pivotal role in ALK inhibitor-induced apoptosis in such cells. These findings are consistent with the earlier observations that EGFR-TKIs induce BIM expression via inhibition of the MEK-ERK pathway and that BIM induction plays a key role in EGFR-TKI-induced apoptosis in EGFR mutation-positive NSCLC cells.
We also showed that ALK inhibition results in downregulation of survivin through inhibition of the STAT3 signaling pathway in EML4-ALK-positive lung cancer cells. Survivin is a member of the inhibitor of apoptosis protein (IAP) family and protects cells from apoptosis by either directly or indirectly inhibiting the activation of effector caspases. Forced expression of a constitutively active form of STAT3 resulted in upregulation of survivin expression and attenuated TAE684-induced apoptosis in EML4-ALK-positive lung cancer cells, suggesting that inhibition of STAT3-survivin signaling contributes to ALK inhibitor-induced apoptosis. Together, these data indicate that inhibition of both ERK-BIM and STAT3-survivin signaling pathways is responsible for ALK inhibitor-induced apoptosis in EML4-ALK-positive lung cancer cells (Fig. 3).
Clinicopathologic Features of EML4-ALK-Positive NSCLC
Although the frequency of ALK rearrangements is only approximately 5% in unselected NSCLC patients, knowledge of the associated clinicopathologic features of this subset of patients might be expected to facilitate their identification. One of the most striking features of individuals with EML4-ALK-positive NSCLC is their young age at disease onset, with a median age in the mid-50s. Similar to EGFR mutations, the prevalence of EML4-ALK is higher in NSCLC patients who have never smoked or are light smokers as well as in those with adenocarcinoma associated with a variety of histological features including acinar, papillary, cribriform, mucin-producing, and signet-ring patterns. ALK rearrangements also tend to be mutually exclusive with EGFR and KRAS mutations. These findings suggest that NSCLC patients with clinical characteristics associated with EGFR mutations, but who test negative for such mutations, are more likely to harbor EML4-ALK. However, although EML4-ALK is associated with several key clinicopathologic features, it is important to recognize that ALK rearrangements also occur in lung cancers of smokers and elderly patients. To date, it is thus not possible to reliably predict the presence or absence of ALK rearrangement on the basis of clinical characteristics alone.
Molecular Diagnosis of EML4-ALK-Positive NSCLC
Break-apart FISH analysis has been applied to diagnostic testing for ALK rearrangement in clinical trials of the ALK kinase inhibitor crizotinib. In such analysis, the 5′ and 3′ ends of ALK are differentially labeled with red and green fluorescent probes. In normal cells, the two probes are detected as overlapping red and green (yellowish) signals. In tumor cells with ALK rearrangement, however, the red and green signals are separated (Fig. 4). This assay detects ALK rearrangements regardless of the ALK fusion partner or the specific EML4-ALK variant. It was also recently approved by the FDA. Break-apart FISH analysis is thus provisionally considered to be the standard diagnostic test for ALK rearrangement. It is not without potential problems, however. Given that EML4 and ALK loci are located in relatively close proximity on chromosome 2p, detection of the EML4-ALK fusion gene on the basis of the gap between the red and green probes is sometimes difficult. Indeed, false negative results have been experienced, possibly as a result of the poor quality of tested tumor specimens. Furthermore, FISH is a relatively low-throughput assay and costly, and it may therefore not be ideal as a screening test for detecting a relatively small subset of NSCLC patients.
Immunohistochemical analysis is a widely available diagnostic tool in daily clinical practice. Given that ALK is not expressed in normal lung tissue, the detection of any level of ALK expression is expected to result from ALK rearrangement. Initial attempts to detect ALK fusion proteins in NSCLC specimens were disappointing, however, because of the low level of fusion protein expression. The subsequent incorporation both of techniques to enhance the immunohistochemical signal and of more sensitive ALK antibodies has improved the ability to detect ALK fusion proteins in NSCLC tissue. Nevertheless, published studies have yielded widely differing results for immunohistochemical analysis of ALK fusion proteins, with such results being influenced by the manner in which the tumor specimens are prepared and the detection system adopted. Immuohistochemistry remains a potentially cost-effective and rapid screening tool for the identification of patients with ALK rearrangement-positive NSCLC, but it still requires optimization of both the staining method and the ALK antibodies.
Reverse transcription-PCR analysis is potentially the most sensitive diagnostic method for identification of ALK rearrangement in NSCLC. It is also the only technique capable of distinguishing between the different EML4-ALK variants. However, this procedure requires an adequate amount of RNA of sufficient quality, which is difficult to obtain from formalin-fixed, paraffin-embedded specimens of tumor tissue. RNA extracted from such specimens is highly degraded and less amenable to RT-PCR compared with that isolated from nonfixed, freshly frozen tissue. RT-PCR analysis is thus more readily applied to cytology samples such as pleural effusion.
Crizotinib: The First Clinically Available ALK-TKI
Although originally developed as an inhibitor of c-MET, crizotinib (PF-02341066) is an orally active small-molecule TKI of ALK. Crizotinib competes with ATP for binding to the tyrosine kinase pocket of ALK and thereby inhibits tyrosine phosphorylation of the activated enzyme at nanomolar concentrations. A dose-escalation phase I trial of crizotinib in patients with advanced solid tumors established 250 mg twice daily as the maximum tolerated dose. Fatigue was the dose-limiting toxicity, occurring at grade 3 in two of the six patients in the cohort treated with 300 mg twice daily. Two patients with EML4-ALK-positive NSCLC who were treated with crizotinib showed a dramatic amelioration of their symptoms, prompting a large-scale prospective screening for NSCLC with ALK rearrangement and enrollment of patients into an expanded molecularly defined cohort.
Pronounced Clinical Activity of Crizotinib for EML4-ALK-Positive Advanced NSCLC
Crizotinib was the first ALK inhibitor to be tested in the clinical setting. Information on the clinical activity of this drug in patients with EML4-ALK-positive advanced NSCLC was updated at the Annual Meeting of the American Society of Clinical Oncology in 2011. Results were reported for 119 patients with advanced NSCLC whose tumors were confirmed to be positive for ALK rearrangement by break-apart FISH analysis. The median age of the subjects was 51 years (range, 21–79 years), which is younger than is typical for advanced NSCLC patients overall. Most had an adenocarcinoma histology, and 72% had never smoked. The patients received crizotinib continuously at a dose of 250 mg twice daily. Among 116 evaluable patients, the objective response rate (ORR) was 61% (95% confidence interval [CI], 52–71%), with 55% of the objective tumor responses being achieved during the first 8 weeks of treatment. The ORR was independent of age, sex, Eastern Cooperative Oncology Group performance status, and number of prior treatment regimens. The clinical response was even more favorable among Asian patients (n =34), with an ORR of 82% (95% CI, 66–93%). Among the 15 Japanese patients included in the efficacy-evaluation population, 14 showed a partial response, yielding an ORR of 93% (95% CI, 68–100%). Clinically significant responses to crizotinib are thus observed across ethnicities. Among the 119 patients evaluable for progression-free survival (PFS), the median PFS was 9.2 months, a remarkably long period for previously treated advanced NSCLC patients. On the basis of its dramatic clinical activity, crizotinib was approved by the FDA for treatment of ALK rearrangement-positive NSCLC in August 2011.
Survival Benefit of Crizotinib Treatment for EML4-ALK-Positive Advanced NSCLC
To date, the impact of crizotinib on overall survival (OS) remains to be determined, given the lack of available data from randomized controlled trials. Two global randomized phase III trials are currently underway to compare crizotinib with the standard of care (systemic chemotherapy) in patients with advanced ALK rearrangement-positive NSCLC (Fig. 5). One of these trials is a phase III registration trial testing crizotinib versus second-line therapy (pemetrexed or docetaxel) in previously treated patients with advanced NSCLC positive for ALK rearrangement (PROFILE 1007, NCT00932893). The other is a phase III trial testing crizotinib versus first-line therapy (pemetrexed-cisplatin or pemetrexed-carboplatin) in treatment-naive patients with advanced NSCLC positive for ALK rearrangement (PROFILE 1014, NCT01154140). On the basis of the marked antitumor activity of crizotinib, patients assigned to the chemotherapy arm of each trial can cross over to the crizotinib arm after the onset of progressive disease. The primary end point of both phase III trials is PFS. It will therefore be difficult to obtain OS data for crizotinib-naive patients with EML4-ALK-positive NSCLC being treated with conventional chemotherapy in a prospective cohort study. A retrospective comparison of crizotinib-treated patients enrolled in a phase I clinical trial (NCT00585195) and crizotinib-naive controls screened during the same time period revealed that the survival outcome of crizotinib-naive patients with advanced NSCLC positive for ALK rearrangement was similar to that of clinically comparable patients who were negative for ALK rearrangement, suggesting that ALK rearrangement is not a prognostic factor in advanced NSCLC. Furthermore, among patients with ALK-rearranged advanced NSCLC, OS was significantly longer for those treated with crizotinib than for clinically comparable, crizotinib-naive controls, suggesting that crizotinib can prolong OS in patients with advanced NSCLC positive for ALK rearrangement.
Even before abovementioned phase III studies (PROFILE 1007 and PROFILE 1014) were completed; however, crizotinib was approved for patients with ALK-rearranged NSCLC by the FDA in August 2011. On the basis of its pronounced clinical activity, the National Comprehensive Cancer Network guidelines also already recommend crizotinib as a first-line systemic therapy for advanced NSCLC positive for ALK rearrangement.
Safety of Crizotinib in Patients with EML4-ALK-Positive Advanced NSCLC
Crizotinib has been shown to be generally well tolerated. The most common treatment-related adverse events are gastrointestinal toxicities of grade 1 or 2 including nausea, vomiting, and diarrhea. Dysgeusia occurs in a subset of patients, sometimes resulting in appetite loss. Visual disorders including visual impairment, photopsia, blurred vision, vitreous floaters, photophobia, and diplopia have been reported in more than half of patients. Visual disturbances described as “trails of light” that occur when accommodating from dark to light have also been noted. All visual disorders have been of grade 1, with no evidence of ocular pathology having been obtained in any patient. The percentage of Japanese patients showing visual disorders was found to be 67% (10 of 15 patients in the A8081001 study). These disorders usually begin within 2 weeks of the onset of drug administration. Physicians should thus caution patients about the possible danger of driving with such vision disorders.
Pneumonitis or interstitial lung disease
Life-threatening or fatal interstitial lung disease (ILD) has been attributed to crizotinib treatment with a frequency of four in 255 patients (1.6%) across clinical studies. Patients should be carefully monitored for pulmonary symptoms and radiographic findings indicative of ILD, and crizotinib should be permanently discontinued in individuals diagnosed with treatment-related ILD. Given that drug-induced ILD has a high associated mortality, a systematic survey allowing direct determination of the prevalence of, and identification of risk factors for, crizotinib-induced ILD is warranted.
An increase in alanine aminotransferase (ALT) level of grade 3 or 4 has been observed in approximately 7% of patients treated with crizotinib. Such increases are usually asymptomatic and reversible on interruption of drug administration, but crizotinib-induced hepatotoxicity with a fatal outcome has been reported in <1% of patients across clinical trials. Liver function tests, including those for ALT and total bilirubin, should be performed once a month or as clinically indicated, with more frequent repeat testing for patients who develop an ALT elevation of grade 2–4.
QT interval prolongation
Prolongation of the QT interval has been observed with a frequency of 1.6% in patients treated with crizotinib across clinical studies. In the event that such a prolongation of grade 3 is observed, crizotinib should be temporarily suspended until recovery to grade 1 or less, when it should be resumed at a lower dose. If a QT interval prolongation of grade 4 is detected, crizotinib should be permanently discontinued.
Acquired resistance to crizotinib
Despite the great benefits of crizotinib treatment for ALK-rearranged NSCLC, all such treated patients ultimately develop drug resistance, as has been observed with other effective small-molecule kinase inhibitors such as the EGFR-TKIs gefitinib and erlotinib. Secondary mutations are a common cause of acquired drug resistance to kinase inhibitors, and such mutations were identified in a Japanese patient with ALK-rearranged NSCLC who relapsed after 5 months of crizotinib treatment. Detailed sequencing of DNA encoding the tyrosine kinase domain of ALK in the relapsed tumor revealed the presence of two mutations (C1156Y and L1196M). The L1196M mutation in ALK corresponds to the T790M “gatekeeper” mutation in EGFR that confers resistance to gefitinib and erlotinib in EGFR mutation-positive NSCLC, suggesting that L1196 of ALK is also a gatekeeper residue. Establishment of a cell line from malignant pleural effusion of a patient who had developed acquired resistance to crizotinib revealed that the cells harbored an L1152R secondary mutation in ALK. Although both L1152 and C1156 are located at a distance from the crizotinib binding site of ALK, cells engineered to express EML4-ALK with either of these secondary mutations were found to be resistant to crizotinib. Structural and biochemical studies of each of these mutations will be necessary to further our understanding of how they give rise to crizotinib resistance.
We and others have shown that activation of alternative signaling pathways by ligand stimulation contributes to resistance to molecularly targeted therapy in several cancer models.[20-22] Ligand-dependent activation of the EGFR signaling pathway can thus bypass the continued block of ALK signaling and contribute to ALK inhibitor resistance. Concurrent inhibition of both EGFR and ALK is therefore a potential strategy for overcoming such acquired resistance. The relative contributions of secondary mutations and activation of alternative signaling pathways to crizotinib resistance remain unknown. Although challenging, repeated biopsy and molecular analysis of tumors that recur after crizotinib treatment should be required in clinical trials for treatment strategies to overcome acquired resistance.
New ALK kinase inhibitors under clinical development
Several new ALK-TKIs are currently under development, some of which are entering early clinical testing. CH5424802 (Chugai Pharmaceuticals; development code, AF802) is a potent, selective, and orally available ALK inhibitor that shows much higher selectivity for ALK than does crizotinib. Specific kinase inhibition by this compound appears to be related to its number of hinge hydrogen bonds. Crystallographic analysis thus revealed that CH5424802 possesses one hinge hydrogen bond, as do the TKIs erlotinib, imatinib, and lapatinib, whereas other ALK inhibitors including crizotinib form two or three such bonds. CH5424802 also shows substantial efficacy in vivo with EML4-ALK-positive tumors that also harbor the putative gatekeeper mutation L1196M, providing potential therapeutic opportunities for patients who develop acquired resistance to crizotinib due to secondary ALK mutations. The drug AF802 is also under investigation in a phase I/II clinical trial for crizotinib-naive patients with ALK-rearranged advanced NSCLC in Japan. Finally, ASP3026, the lead product of Astellas Pharma, is a small-molecule kinase inhibitor that targets ALK and other tyrosine kinases including ROS. This compound induced tumor regression in an EML4-ALK-dependent mouse xenograft model. Dose-escalation phase I studies of ASP3026 are ongoing for unselected patients with solid tumors both in Japan and in the United States.
Potential combination treatment strategies
Combination therapy with different inhibitors of intracellular signaling pathways is an option for overcoming crizotinib resistance. We examined the effects of the ALK-TKIs TAE684 and crizotinib on the growth of the NSCLC cell lines H3122 and H2228, both of which harbor EML4-ALK, and we found that each drug markedly inhibited the proliferation of H3122 cells at low concentrations but affected the growth of H2228 cells only at high concentrations. Immunoblot analysis revealed that TAE684 inhibited STAT3 phosphorylation and induced downregulation of survivin, but that it failed to inhibit ERK phosphorylation and to upregulate BIM, in H2228 cells. Inhibition of both STAT3 and ERK pathways by the combination of either ALK-TKI TAE684 and the MEK inhibitor AZD6244 resulted in a pronounced proapoptotic effect in H2228 cells, consistent with our earlier observation that simultaneous upstream interruption of the STAT3-survivin and MEK-ERK-BIM pathways mediates ALK-TKI-induced apoptosis. Although most patients with NSCLC positive for EML4-ALK derive benefit from treatment with ALK-TKIs, the clinical efficacy of these drugs varies greatly among such individuals. The molecular mechanism underlying the sustained activation of the ERK signaling pathway in the presence of an ALK inhibitor in H2228 cells remains unclear, but our data provide a rationale for combination therapy with ALK and MEK inhibitors in EML4-ALK-positive NSCLC patients for whom ALK inhibitors alone are ineffective.
Personalized treatment algorithm for advanced NSCLC
Delivery of effective personalized therapies to patients with advanced NSCLC requires evaluation for both EGFR mutations and ALK rearrangements in routine molecular diagnostic testing (Fig. 6). Ideally, tumors should be tested simultaneously for these genetic abnormalities. Otherwise, a stepwise approach could be adopted, with EGFR mutations being tested for first; given that such mutations and ALK rearrangements are generally mutually exclusive, ALK testing would then be instituted only for patients with wild-type EGFR. If a tumor is positive for an EGFR mutation (exon 19 deletion or L858R), single-agent treatment with an EGFR-TKI should be recommended as soon as possible. If the tumor is positive for ALK rearrangement, single-agent treatment with crizotinib would be the preferred option. However, a subset of tumors remains that does not fall into either of these two molecularly specified categories. For such patients, tumor histology (squamous cell carcinoma or nonsquamous carcinoma) forms the basis for selection of the appropriate chemotherapy regimen in terms of toxicity and efficacy. Ongoing efforts to discover other “driver mutations” in NSCLC should eventually provide additional personalized treatment options for this challenging disease.