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Antitumor Activity of Crizotinib in Lung Cancers Harboring a MET Exon 14 Alteration

MET exon 14 alterations are oncogenic drivers of non-small cell lung cancers (NSCLCs).1 These alterations are associated with increased MET activity and preclinical sensitivity to MET inhibition.2 Crizotinib is a multikinase inhibitor with potent activity against MET.3 The antitumor activity and safety of crizotinib were assessed in 69 patients with advanced NSCLCs harboring MET exon 14 alterations in an expansion cohort of an open-label phase 1 study of crizotinib (NCT00585195). The confirmed objective response rate was 32% (95% confidence interval [CI], 21–45) among 65 response-evaluable patients. Objective responses were observed independent of the molecular heterogeneity that characterizes these cancers and did not vary by MET exon 14 alteration splice site region and mutation type, concurrent increased MET copy number, or the detection of a MET exon 14 alteration in ctDNA. The median duration of response was 9.1 months (95% CI, 6.4–12.7). The median progression-free survival was 7.3 months (95% CI, 5.4–9.1). MET exon 14 alteration defines a molecular subgroup of NSCLCs for which MET inhibition with crizotinib is active. These results address an unmet need for targeted therapy in patients with MET exon 14-altered lung cancers and adds to an expanding list of genomically-driven therapies for oncogenic subsets of NSCLC.

Crizotinib is a multi-tyrosine kinase inhibitor that is approved for the treatment of ALK-or ROS1-rearranged advanced NSCLCs. Apart from its activity against ALK and ROS1, it has potent activity against MET and low nanomolar potency in cell lines that harbor MET exon 14 alterations 3,13 . In 2015, we reported the first cases of response to crizotinib in patients with MET exon 14-altered lung cancers in a small retrospective series 14 . This established early proof of principle of the activity of a MET inhibitor in these cancers and was followed by additional reports 4,7,13,[15][16][17][18][19][20] . In this paper, we evaluated the antitumor activity of crizotinib in MET exon 14-altered advanced NSCLCs in an expansion cohort of the PROFILE 1001 trial. In contrast to prior reports, this was the first prospective study to characterize the activity of a MET inhibitor in a dedicated cohort of patients with MET exon 14-altered NSCLCs.
From 11 September 2014 through 26 January 2018, 69 patients with MET-exon-14-altered NSCLCs were enrolled in the study and treated with crizotinib (Table 1 and Extended Data Fig. 1). The median age was 72 years. The majority (61%) were former smokers. Most patients (84%) had adenocarcinoma; 9% had sarcomatoid carcinoma. The majority of patients (62%) had received ≥1 previous lines of treatment for advanced disease. MET exon 14 alterations were identified by local testing, primarily using DNA-or RNA-based next-generation sequencing (NGS) in 96% of patients (Table 1 and Supplementary Table 1). The median duration of treatment was 7.4 months (95% CI, 5.5-9.1); 20 patients (29%) continued to receive crizotinib after the data cutoff date.
Sixty-five patients were tumor-response evaluable. The objective response rate (ORR) was 32% (95% CI, 21-45). Among the 65 patients, 3 (5%) had a confirmed complete response, 18 (28%) had a confirmed partial response, 29 (45%) had stable disease and 4 (6%) had disease progression as their best overall response. Best overall response could not be determined for 11 patients (17%). These included 11 patients whose target lesions could not be measured at each follow-up time point or who did not undergo ≥1 post-baseline disease assessment secondary to toxicity, withdrawal of consent or death (Supplementary Table 2). Objective response by age, prior line of therapy, smoking history and histology is described in Supplementary Table 3. A decrease in target-lesion size Letters NATurE MEDICINE was observed in most patients (Fig. 1). The median time to tumor response was 7.6 weeks (range, 3.7 to 16.3), corresponding approximately to the first protocol-mandated tumor assessment. The median duration of response was 9.1 months (95% CI, 6.4-12.7). Of 21 patients with an objective response, 12 (57%) had a duration of response of ≥6 months.
MET exon 14 alterations were genomically diverse by local testing (Supplementary Table 4a). In terms of splice site, these alterations most commonly involved the splice donor site (57%), followed by the splice acceptor site (25%). The most frequent mutation type (regardless of splice site) was base substitution (51%) followed by indel (31%); a whole-exon deletion occurred in 1 (2%) patient. The genomic sequence of the MET exon 14 alteration (region/type) was unknown in 11 (17%) patients (included a MET rearrangement resulting in MET exon 14 exclusion; Supplementary Table 4a). Objective responses to crizotinib were observed independent of the MET exon 14 alteration splice site (ORR 12/37 (32%) for splice donor site and 5/16 (31%) for splice acceptor site) or mutation type (ORR 12/33 (36%) for base substitution and 5/20 (25%) for indel). The overall association of tumor response with MET exon 14 alteration (splice site region/mutation type, including patients with unknown MET exon 14 alteration status) was not significant (P = 0.65, Supplementary Table 4a and Fig. 1). Similar results were observed with central testing (P = 0.53, Supplementary Table 4b and Extended Data Fig. 2). Durable disease control was observed across different MET exon 14 alterations (Fig. 2).
Concurrent increased MET copy number was observed via local testing in 2 of 54 (4%) tumors in which testing was performed (Supplementary Table 5). While local testing reported these 2 cases as MET-amplified, the fluorescence in situ hybridization (FISH)positive case (MET/CEP7 ratio, 3.6) did not have high-level amplification (MET/CEP7 ratio of >4-5) 21 , and the fold change in the NGS-positive case was low (2.3-fold change). While the true impact of these concurrent copy-number changes remains unclear, both patients had durable disease control that exceeded 6 months (Fig. 2). Central testing of analyzable tumor tissue revealed an increase in MET copy number in 2 of 40 (5%) patients (Supplementary Table 5).

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In this study, we showed that crizotinib was active in patients with MET-exon-14-altered NSCLCs. This underscores the importance of testing for MET exon 14 alterations in the clinic. NGS using DNA-based hybrid capture is an ideal assay to use as it can detect a wide variety of mutations that result in MET exon 14 skipping or a comparable biology. It is important to note, however, that tumor RNA sequencing can detect MET exon 14 alterations in cancers where these were missed by DNA-based sequencing 26 . Beyond tumor-based testing, plasma-derived ctDNA assays can also be used to detect these alterations, as was demonstrated in this paper.
PROFILE 1001 was the first prospective trial to demonstrate that MET inhibition is active in MET exon 14-altered NSCLCs 27 . In this seminal dataset, crizotinib achieved an ORR of 32% (95% CI, 21-45), a median duration of response of 9.1 months, and a median PFS of 7.3 months. These outcomes clearly exceed those observed with second-line chemotherapy (ORR, 7-23%; median PFS, 2.4-4.5 months) 28,29 and are comparable with that of firstline platinum doublet-chemotherapy (ORR, 31-35%; median time to progression, 4.8-6.2 months) 30,31 . While these do not surpass the outcomes observed with select first-line chemoimmunotherapy combinations in unselected NSCLCs 32 , MET inhibition with crizotinib remains an important treatment option for METexon-14-altered NSCLCs, as is currently supported by National Comprehensive Cancer Network Guidelines 33 .
While robust, the ORR of crizotinib in MET-exon-14-altered NSCLCs was lower compared with the ORRs of ~60-80% achieved with targeted therapy for other NSCLC drivers 34,35 . As MET-exon-14-altered NSCLCs are heterogeneous, we posited that distinct subpopulations may be less likely to respond to crizotinib, contributing to a lower response rate. An exploratory analysis of this series showed, however, that this was not the case. First, mutation type and splice-site region did not affect outcomes, and MET copy number was not a major differentiating factor (recognizing that few tumors had concurrent MET amplification, and none had high-level MET amplification). Second, a strong association between concurrent genomic alterations and outcomes was not found, despite preclinical studies suggesting that select co-occurring alterations can confer resistance 13,36 . Finally, ctDNA status was not significantly associated with ORR, but the median PFS was significantly shorter in ctDNApositive versus ctDNA-negative patients. The latter should be interpreted with caution as the sample size was small and few events were observed. Other factors that potentially affect crizotinib outcomes should continue to be explored. It was recently shown, for example, that no responses were observed with crizotinib in MET-exon-14altered NSCLCs without MET protein expression 37 .
Ultimately, beyond these analyses, the type of MET inhibitor used to treat MET-exon-14-altered cancers may affect clinical outcomes. While crizotinib, a type Ia inhibitor, was the first MET tyrosine kinase inhibitor to be explored in MET-exon-14-altered NSCLCs, preliminary reports of prospective trials of type Ib tyrosine kinase inhibitors, such as capmatinib 38 , tepotinib 39 and savolitinib 40 , have subsequently emerged. These agents are more selective than crizotinib and are potentially more potent in preclinical models. The ORRs ranged from 40-55% in the post-platinum doublet or allcomers setting (for all 3 drugs) and ranged from 44-68% in treatment-naive patients (for tepotinib and capmatinib). Additional data Patients not evaluable for response, with early death and without measurable disease in the responseevaluable population of 65 patients were excluded from this plot. The bars indicate the best percentage change in the sum of target tumor measurements from baseline. As indicated by the asterisk, one patient was determined to have MET-exon-14-altered NSCLC by local testing and ROS1-rearranged (and MET wild type) NSCLC by central testing. Below the plot, local testing results for the MET exon 14 alteration splice-site region (a) and mutation type (b), and the presence of concurrent increased (↑) MET copy number (c), as well as the presence of a MET exon 14 alteration in ctDNA, by central testing (d) are depicted in relation to best response. Note that while the two cases with increased MET copy number were called MET-amplified by local testing, none of these tumors had high-level amplification. In row a, the splice acceptor region includes alterations in the splice acceptor region, polypyrimidine tract and branch point. Cases classified as unknown include MET exon 14 alterations for which DNA coding region information was not available, such as alterations detected using an rNA-based assay. In rows b and c, a white space indicates that no results were reported by the local assay, or that the reported results could not be analyzed for the biomarker of interest. In row d, a white space indicates that a plasma sample was not available for ctDNA analysis, such as in patients who were treated prior to an amendment that included plasma ctDNA collections.  While the cases with increased MET copy number were called MET-amplified by local testing, none of these tumors had high-level amplification. Arrows indicate patients for whom crizotinib therapy was ongoing at the time of data cutoff. As indicated by the asterisk, one patient was determined to have MET-exon-14-altered NSCLC by local testing and ROS1-rearranged NSCLC by central testing. In column a, the splice acceptor region includes alterations in the splice acceptor region, polypyrimidine tract and branching point. Cases classified as unknown include MET exon 14 alterations for which DNA-coding region information was not available, such as alterations detected using an rNA-based assay. In columns b and c, a white space indicates that no results were reported by the local assay, or that the reported results could not be analyzed for the biomarker of interest. In column d, a white space indicates that a plasma sample was not available for ctDNA analysis, such as in patients who were treated prior to an amendment that included plasma ctDNA collections.

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and longer follow-up will determine whether these agents are markedly different in activity compared with crizotinib. Furthermore, a careful analysis of relative toxicity will be important given that increased anti-MET activity could result in higher rates of on-target adverse events (for example, peripheral edema) in patients. In summary, crizotinib was active in patients with advanced MET-exon-14-altered NSCLC. The results of PROFILE 1001 established a clinical paradigm for the treatment of MET-exon-14altered lung cancers with MET-directed targeted therapy, paving the way for additional research to move forward in this space with the goal of regulatory approval of one or more MET inhibitors for this genomic indication.

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Patients.
Eligible patients had histologically confirmed NSCLC that harbored a MET exon 14 alteration (local molecular testing; Supplementary Table 1). Increased MET copy number was determined by assay-defined thresholds for both local and central molecular testing (FoundationOne CDx) 7,41 . Other eligibility criteria included an age of ≥18 years (≥20 years for Japanese patients), an Eastern Cooperative Oncology Group performance status 42 of 0 to 1 (2 was allowed with sponsor approval), adequate organ function and measurable disease (non-measurable disease was allowed with sponsor approval) per Response Evaluation Criteria in Solid Tumors (RECIST), version 1.0 (ref. 43 ). For a full list of inclusion/exclusion criteria, see Supplementary Information.
The protocol (see Supplementary Information) was approved by the institutional review board or independent ethics committee at each site and complied with the International Ethical Guidelines for Biomedical Research Involving Human Subjects, Good Clinical Practice guidelines, the Declaration of Helsinki and local laws. All patients provided written informed consent.
Study design and treatment. PROFILE 1001 is an ongoing open-label phase 1 study with a dose-escalation phase followed by a Recommended Phase 2 Dose (RP2D) expansion phase 44 . Details on dose escalation and results from the expansion phase in ALK-rearranged and ROS1-rearranged NSCLCs have been published 23,24,45 . The study protocol was amended (amendment no. 21, 7 April 2015) to allow the retrospective and prospective enrollment of patients with MET-exon-14-altered advanced NSCLCs in a separate expansion cohort, including sample collection for circulating nucleic acid analysis and documenting survival information every 3 months after discontinuation of study treatment up to at least 1 year after the first crizotinib dose for the last patient enrolled into the MET exon 14 cohort. Subsequent protocol amendments (nos. 22 and 23) are described in the statistical analysis section. Objective response was the primary end point. Duration of response (the time from the first documentation of objective tumor response (complete response or partial response) that was subsequently confirmed, to the documentation of objective tumor progression or death on study due to any cause, whichever occurred first), time to tumor response (time from first dose to first documentation of objective tumor response (complete response or partial response) that was subsequently confirmed), PFS (time from first dose to first documentation of tumor progression or death on study due to any cause, whichever occurred first), OS (time from first dose to death due to any cause) and safety were additional end points.
Crizotinib was administered orally at the standard starting dose of 250 mg twice daily in continuous 28-d cycles. Disease progression or clinical deterioration, unacceptable toxicity, consent withdrawal or death were reasons for treatment discontinuation. Treatment could be continued beyond progression at the investigator's discretion and with sponsor approval.

Study assessments.
Patients underwent baseline tumor imaging with computed tomography or magnetic resonance imaging (chest, abdomen and pelvis). Brain/bone scans were obtained if disease at these sites was suspected. Tumor assessments were performed every 8 weeks and could be performed every 16 and 24 weeks starting with cycles 15 and 24, respectively. Response was confirmed ≥4 weeks after initial response documentation.
Adverse events were assessed from the first day of study treatment until ≥28 d after the last dose or until all serious/drug-related toxicities resolved or were determined to be stable/chronic, whichever was later. The type, incidence, and severity of adverse events were determined by the Common Terminology Criteria for Adverse Events, version 3.0 (http://ctep.cancer.gov/protocolDevelopment/ electronic_applications/docs/ctcaev3.pdf).

Central biomarker analyses.
MET exon 14 alterations were retrospectively evaluated by central testing on archival tumor tissue using a validated DNA-based next-generation sequencing (NGS; FoundationOne CDx, Foundation Medicine, Cambridge, MA) assay. MET exon 14 alteration status was retrospectively evaluated by ctDNA analyses using a validated assay (PlasmaSELECT-R64, Personal Genome Diagnostics) in baseline or end-of-treatment plasma samples. The same assays identified concurrent genomic alterations in tumor tissue and ctDNA.
Study oversight. The study was designed jointly by the investigators and representatives of the sponsor, Pfizer Incorporated. All authors were involved in data analysis and manuscript preparation and attest to the completeness and accuracy of the data, analyses and study protocol adherence.
Statistical analysis. It was originally anticipated that 33 patients would need to be enrolled in the MET exon 14-altered NSCLC group. This would achieve a power of ≥90% to test the null hypothesis that the ORR of crizotinib would be ≤10%, versus the alternative hypothesis that the ORR would be >10% (onesided alpha level of 0.05, single-stage design). For the alternative hypothesis, the target ORR was 30%. On this basis, the null hypothesis would be rejected if ≥7 objective responses were observed among the first 33 evaluable patients (protocol amendment no. 22, 27 April 2016). As of 1 August 2016, there were 11 objective responses (among 28 evaluable patients); thus, the null hypothesis was rejected.
To report antitumor activity and safety with greater precision, an increase in the sample size beyond the original 33 required for rejection of the null hypothesis was planned (protocol amendment no. 23, 21 February 2017). Thus, the sample size was increased to 68 patients as of 15 May 2017. Because the null hypothesis was already rejected with the first 33 patients enrolled, adjustment of type I error rate was not considered necessary for subsequent analyses. This dataset met the planned cutoff of an expanded sample size of at least 68 patients for formal reporting. As of 7 December 2017, the sample size was augmented to 81 patients; however, because enrollment is still ongoing (at the data cutoff date of 31 January 2018), a final analysis will be conducted after the last patient accrued completes the last survival follow-up visit. Patients who received ≥1 dose of crizotinib were included in the safety population and analyses of PFS and OS. The responseevaluable population was defined as all patients in the safety population who had an adequate baseline and ≥1 post-baseline disease assessment (≥6 weeks from the first dose), or who withdrew from the study, progressed or died. CIs for the ORR were estimated using an exact binomial method based on the F-distribution. A Kaplan-Meier analysis of time-to-event data to estimate medians and the Brookmeyer-Crowley method to calculate two-sided 95% CIs for the median were used. Only descriptive statistics were produced for time to tumor response. The associations of tumor response with splice site region/mutation type or with ctDNA results were evaluated using two-sided Fisher's exact tests. Hazard ratios and associated 95% CIs were estimated by Cox regression models and 2-sided log-rank tests were performed to compare the duration of treatment and PFS between patients who were ctDNA-positive versus ctDNA-negative. P values were not adjusted for multiple comparisons. Study database data were analyzed with the use of SAS statistical software, version 9.2 or later (SAS Institute). The data cutoff date was 31 January 2018.
Reporting Summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
Upon request, and subject to certain criteria, conditions and exceptions (see https://www.pfizer.com/science/clinical-trials/trial-data-and-results for more information), Pfizer will provide access to individual de-identified patient data from Pfizer-sponsored global interventional clinical studies conducted for medicines, vaccines and medical devices (1) for indications that have been approved in the United States and/or European Union or (2) in programs that have been terminated (that is, development for all indications has been discontinued). Pfizer will also consider requests for the protocol, data dictionary and statistical analysis plan. Data may be requested from Pfizer trials 24 months after study completion. The de-identified patient data will be made available to researchers whose proposals meet the research criteria and other conditions, and for which an exception does not apply, via a secure portal. To gain access, data requestors must enter into a data access agreement with Pfizer.

October 2018
Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences
Behavioural & social sciences Ecological, evolutionary & environmental sciences For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
It was originally anticipated that 33 patients would need to be enrolled in the MET exon 14-altered NSCLC group. This would achieve a power of ≥90% to test the null hypothesis that the ORR of crizotinib would be ≤10%, versus the alternative hypothesis that the ORR would be >10% (one-sided alpha level of 0.05, single-stage design). For the alternative hypothesis, the target ORR was 30%. As of August 01, 2016, there were 11 objective responses (among 28 evaluable patients), which exceeded the 7 required to reject the null hypothesis. To permit a more accurate assessment of antitumor activity and safety, the sample size was expanded to 68, and later to 81 patients. The number of additional patients was not based on statistical considerations.

Replication
First report in human subjects-findings have not been replicated Randomization All patients with MET exon 14 advanced NSCLC enrolled in PROFILE 1001 were treated with crizotinib 250 mg BID, randomization was not applied.
Blinding PROFILE 1001 is an ongoing single-arm phase I trial with an open-label design. Blinding is not standard practice in single arm phase 1 trials of oncologic therapies Reporting for specific materials, systems and methods