The gold standard objective method for detection of a gene fusion is fluorescent in situ hybridisation (FISH) and this is the currently approved detection method for ALK + NSCLC by the United States Food and Drug Administration (FDA). FISH is a reliable but resource intensive technique for detection of ALK gene fusion that requires specific training and skill. The protein product of ALK gene fusion can also be detected using immunohistochemistry (IHC) since ALK protein is not normally expressed in lung tissue and IHC is less resource intense. A high level of IHC ALK expression has been shown to correlate strongly with the presence of ALK-fusion gene using FISH. Lower levels of ALK protein expression detected by IHC are more likely to represent false positive cases, that prove to be negative using FISH; and high concordance has been demonstrated between IHC negative and FISH negative cases. To date a number of antibodies have demonstrated reliability and utility but the best performing antibody is debated. A third method uses RT-PCR and this is anticipated to become more widespread in clinical diagnostic laboratories as technology improves. Currently the emerging consensus is to prescreen cases using IHC and confirm ALk status using FISH where resources limit the routine use of FISH for screening of all cases. The reader is referred to evidence-based guidance and recommendations for ALK testing [9].

Crizotinib is generally well tolerated and the safety profile has been consistent across Phase I, II and III studies [10, 16, 19]. Most adverse events attributed to crizotinib are low grade. Nausea, diarrhoea, vomiting, constipation and visual effects have been reported most commonly. Rarely have the reported adverse events led to dose interruption, reduction or discontinuation. In randomised comparison of crizotinib to chemotherapy higher rates of (mild) nausea were reported for crizotinib [19]. Importantly, routine prophylactic antiemetics were mandatory in the study protocol for chemotherapy but not for crizotinib. Other relatively common and usually low grade toxicities include peripheral oedema, dizziness, fatigue and decreased appetite. Endocrine effects, specifically testosterone depletion may be common and more rarely, hepatic and pulmonary effects have been observed. At the time of writing data continues to emerge on the clinical impact and significance of these effects. With respect to hepatic toxicity, transaminase elevations on crizotinib treatment have generally occurred within the first 2 months of treatment (median onset 40 days). Among 1,054 patients on the PROFILE 1001 and 1005 studies [15, 16] ALT elevations of more than 3, 5 and 10 times the upper limit of normal occurred in 15, 7.4 and 3 % of patients, respectively. Grade 3 and 4 events were more frequent for ALT elevation but total bilirubin elevation was less common. Currently it is recommended to monitor liver function at least once per month on treatment, more frequently if results are abnormal, and to interrupt dosing to resume treatment at a lower dose on normalisation of liver function.

A rapid decrease in total testosterone levels after initiating crizotinib has been observed [21, 22]. This is postulated to occur via a hypothalamic or pituitary effect and may account for some of the fatigue experienced by some patients. Testosterone levels were noted to normalise on discontinuation of crizotinib. There is a general lack of data on testosterone levels in patients with advanced lung cancer and/or the effects of other treatments such as chemotherapy on testosterone. Data from prospective, randomised trials (including the PROFILE 1014 trial [20] is awaited to further clarify the causality and sequelae of testosterone deficiency with respect to crizotinib.

Initial response rates to crizotinib are approximately 60 % in the ALK + NSCLC population which suggests primary resistance in a proportion of cases. At a cellular and molecular level in vitro, activation of the EGFR pathway has been observed to confer crizotinib resistance [23, 24]. Similarly, pretreatment levels of the proapoptotic protein BIM are described to influence response to crizotinib [25]. In general the reasons for primary resistance are not well understood. Relatively more progress has been made in understanding acquired resistance. Invariably, patients who initially respond to crizotinib treatment develop disease progression. Studies in vitro and in biopsies taken from patients with progressive disease after initial response have identified various molecular causes that have evolved during crizotinib treatment. Currently the most significant with respect to drug development is the occurrence of ALK gene mutations that alter crizotinib binding. ALKL1196M in the gatekeeper region, ALKS1206Y near the ribose binding pocket and ALKG1269A in the DFG motif, ALKC1156Y, ALKL1152R, ALKF1174L, ALK1151Tins and ALKG1202R mutations account for crizotinib resistance in approximately one third of patient samples tested ([26, 27] and [6] for review). Other identified mechanisms of acquired resistance include ALK gene copy number gain, loss of the ALK fusion gene, EGFR pathway activation including EGFR mutation, KRAS mutation and KIT amplification [28]. In addition to molecular mechanisms mediating resistance, crizotinib has poor penetration through the blood brain barrier and into the central nervous system (CNS). Consequently the CNS is a sanctuary site where ALK + NSCLC can progress [29].

LDK378 is a selective ALK inhibitor that is active against ALK gene fusion with the crizotinib resistant gatekeeper mutation C1156Y. In phase I evaluation [30] an overall response rate of 70 % was observed and a response rate of 73 % occurred among patients who had progressed on or were resistant to crizotinib. Responses were also observed in patients with untreated CNS metastases. In this study the progression free survival among all NSCLC patients (n = 123) was 8.6 months. The most common adverse events were nausea (72 %), diarrhoea (69 %), vomiting (50 %) and fatigue (31 %). LDK378 is generally well tolerated. The most common grade 3 and 4 adverse events in phase I were ALT elevation, diarrhoea and AST elevation that occurred in 12, 7 and 6 % of patients respectively. Several studies of LDK 378 are ongoing to evaluate its efficacy in crizotinib treated, crizotinib naïve, and CNS disease in patients with ALK + NSCLC. A phase II multicentre, single-arm study of LDK 378 aims to enrol 137 patients for the primary endpoint of response rate. Patients must have previously received chemotherapy and have progressed on crizotinib [31]). A similarly designed but separate study is enrolling patients who are crizotinib naïve [32]. Two randomised phase III studies are open to evaluate LDK378 versus first line chemotherapy [33] and second line chemotherapy [34] respectively.

AP26113 (Ariad) is a potent ALK inhibitor with activity against known resistance mutations to crizotinib. It is also active against ROS1 tyrosine kinase and mutant epidermal growth factor receptor (EGFR) including EGFR harbouring the T790M gatekeeper resistance mutation. Results of a first-in-human study have been reported [35]. The study design included cohorts of ALK inhibitor naïve ALK + NSCLC, crizotinib resistant ALK + NSCLC and ALK + NSCLC with active brain metastases. The response rate was 61 % (19/31) in patients who had previously received crizotinib and responses in brain metastases in crizotinib resistant ALK + NSCLC were observed. The toxicity profile of AP26113 appears similar to that of crizotinib with mild nausea, fatigue and diarrhoea most commonly. Rash has not been observed despite activity against mutant EGFR. Of concern, severe pulmonary symptoms of dyspnoea, hypoxia and pneumonitis occurred in approximately 10 % of patients in the early stages of clinical development. Onset was typically within the first few days of receiving AP26113 and was reversible with interruption of AP26113 and supportive treatments including steroids. As a consequence the dose of AP26113 (90–180 mg QD) was re-evaluated in a cohort of patients with narrowed inclusion criteria; specifically no supplemental oxygen therapy, no interstitial lung disease and ECOG PS 0, 1. With these narrowed inclusion criteria there were no pulmonary adverse events observed. A dose of 180 mg QD is the recommended phase II dose for further evaluation and full details of several planned trials, including randomised, are now awaited.