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CLINICAL CANCER RESEARCH | PRECISION MEDICINE AND IMAGING

MET Alterations Are a Recurring and ActionableResistance Mechanism in ALK-Positive Lung Cancer A C

Ibiayi Dagogo-Jack1,2, Satoshi Yoda1,2, Jochen K. Lennerz3, Adam Langenbucher1, Jessica J. Lin1,2,Marguerite M. Rooney1, Kylie Prutisto-Chang1, Audris Oh1, Nathaniel A. Adams1, Beow Y. Yeap1,Emily Chin1, Andrew Do1, Hetal D. Marble3, Sara E. Stevens1, Subba R. Digumarthy4, Ashish Saxena5,Rebecca J. Nagy6, Cyril H. Benes1,2, Christopher G. Azzoli1,2, Michael S. Lawrence1, Justin F. Gainor1,2,Alice T. Shaw1,2, and Aaron N. Hata1,2

ABSTRACT◥

Purpose: Most ALK-positive lung cancers will develop ALK-independent resistance after treatment with next-generation ALKinhibitors. MET amplification has been described in patients pro-gressing onALK inhibitors, but frequency of this event has not beencomprehensively assessed.

Experimental Design: We performed FISH and/or next-generation sequencing on 207 posttreatment tissue (n ¼ 101) orplasma (n ¼ 106) specimens from patients with ALK-positive lungcancer to detect MET genetic alterations. We evaluated ALKinhibitor sensitivity in cell lines withMET alterations and assessedantitumor activity of ALK/MET blockade in ALK-positive cell linesand 2 patients with MET-driven resistance.

Results: MET amplification was detected in 15% of tumorbiopsies frompatients relapsing on next-generationALK inhibitors,including 12% and 22% of biopsies from patients progressing onsecond-generation inhibitors or lorlatinib, respectively. Patients

treated with a second-generation ALK inhibitor in the first-linesetting were more likely to develop MET amplification than thosewho had received next-generation ALK inhibitors after crizotinib(P¼ 0.019). Two tumor specimens harbored an identical ST7-METrearrangement, one of which had concurrent MET amplification.Expressing ST7-MET in the sensitive H3122 ALK-positive cell lineinduced resistance to ALK inhibitors that was reversed with dualALK/MET inhibition. MET inhibition resensitized a patient-derived cell line harboring both ST7-MET and MET amplificationto ALK inhibitors. Two patients with ALK-positive lung cancer andacquired MET alterations achieved rapid responses to ALK/METcombination therapy.

Conclusions: Treatment with next-generation ALK inhibitors,particularly in the first-line setting, may lead to MET-drivenresistance. Patients with acquired MET alterations may deriveclinical benefit from therapies that target both ALK and MET.

IntroductionALK-rearranged (i.e., ALK-positive) non–small cell lung cancers

(NSCLC) depend on constitutive ALK signaling for growth andsurvival (1). This oncogene dependency underlies the marked sensi-tivity of these tumors to tyrosine kinase inhibitors (TKI) targetingALK (2, 3). Historically, the ALK/ROS1/MET TKI crizotinib was thestandard first-line therapy for advanced ALK-positive NSCLC (4).However, crizotinib has recently been supplanted by more potent and

selective second-generation ALK TKIs (e.g., ceritinib, alectinib, andbrigatinib; refs. 2, 3, 5). In addition, the third-generation ALK TKIlorlatinib was recently approved for patients previously treated with asecond-generation TKI (6).

Despite initial sensitivity to ALK TKIs, ALK-positive tumorsinvariably develop resistance. In approximately one-half of cases,resistance to second-generation ALK TKIs is due to secondary muta-tions in the ALK kinase domain (7). These tumors remain ALK-dependent and responsive to treatment with lorlatinib (8). Theremaining cases have presumably developed ALK-independent resis-tance mechanisms, most often due to activation of alternative orbypass signaling pathways (7, 9). Recent studies of lorlatinib resistancesuggest an even more complicated array of resistance mechanisms,including diverse compound ALK mutations in one-third of patientsand ALK-independent resistance mechanisms in the remaining two-thirds of patients (9, 10).

To date, ALK-independent resistance mechanisms remain poorlycharacterized. In preclinical studies, multiple bypassmechanisms havebeen described, including activation of MET, EGFR, SRC, and IGF-1R (11–13). Of these, MET is a particularly attractive target given theavailability of potentMET TKIs. In addition, MET activation has beenpreviously studied as a bypass pathway in EGFR-mutant NSCLCwhere MET amplification has been reported in 5%–20% of resistantcases (14, 15). In phase I/II studies, cotargeting EGFR and MET canreinduce responses in resistant EGFR-mutant tumors with METamplification (16). In contrast to EGFR-mutant NSCLC, relativelylittle is known about the contribution of aberrant MET activation toresistance in ALK-positive NSCLC. Several case reports suggest that

1Massachusetts General Hospital Cancer Center and Department of Medicine,Massachusetts General Hospital, Boston, Massachusetts. 2Harvard MedicalSchool, Boston, Massachusetts. 3Department of Pathology, Center for Integrat-ed Diagnostics, Massachusetts General Hospital, Boston, Massachusetts.4Department of Radiology, Massachusetts General Hospital, Boston, Massachu-setts. 5Department of Medicine, Weill Cornell Medicine, New York, New York.6Guardant Health, Inc., Redwood City, California.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

I. Dagogo-Jack, S. Yoda, A.T. Shaw, and A.N. Hata contributed equally to thisarticle.

CorrespondingAuthors:Alice T. Shaw,Massachusetts General Hospital, 32 FruitStreet, Boston, MA 02114. Phone: 617-724-1134; Fax: 617-726-0453; E-mail:[email protected]; and Aaron N. Hata; E-mail: [email protected]

Clin Cancer Res 2020;26:2535–45

doi: 10.1158/1078-0432.CCR-19-3906

�2020 American Association for Cancer Research.

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MET amplification can mediate resistance to ALK TKIs and that theALK/ROS1/METTKI crizotinibmay be able to overcomeMET-drivenresistance (17, 18). However, larger studies are needed to validateMETas a clinically relevant driver of resistance in ALK-positive NSCLC.

Here we analyze tumor and/or blood specimens from 136 patientswith ALK-positive NSCLC relapsing on ALK TKIs. We identifygenetic alterations of MET in approximately 15% of resistant cases.In cell lines and in patients, MET activation drives resistance to ALKTKIs but confers sensitivity to dual ALK/MET blockade. These studiessupport the development of novel combinations targeting ALK andMET in ALK-positive NSCLC.

Materials and MethodsData collection

Resistant specimens were obtained from 136 patients with meta-static ALK-positive NSCLC who underwent molecular profilingbetween 2014 and 2019 (Fig. 1A). Medical records from these patientswere retrospectively reviewed to extract data on demographics, treat-ment histories, andmolecular profiling results. Datawere updated as ofAugust 31, 2019. This study was approved by the MassachusettsGeneral Hospital Institutional Review Board. Patient studies wereconducted according to the Declaration of Helsinki, the BelmontReport, and the U.S. Common Rule.

Molecular testingMET copy number was quantified in tissue using FISH or the

FoundationOne assay as described previously (19). For cases assessedby FISH, amplification was defined as a ratio ofMET to centromere 7(MET/CEP7) of≥2.2 (20). Cases withMET/CEP7≥ 4were classified ashigh-level amplification (20). For tumors genotyped with the Foun-dationOne assay, MET amplification was defined as ≥6 gene cop-ies (19). With the Guardant360 assay,MET amplification was definedas absolute plasma copy number ≥2.1 (21). As MET copy numberanalysis based on Massachusetts General Hospital (MGH) SNaPshotnext-generation sequencing (NGS) has not been formally validated,this assay was not used to detectMET amplification. MET rearrange-ments andmutations andALKmutations were detected in tissue using

either FoundationOne or the MGH SNaPshot/Solid FusionAssays (19, 22). Guardant360 was used to identify MET and ALKmutations in plasma, but is not designed to detect MET rearrange-ments (21). All patients included in this study provided writteninformed consent for molecular analysis.

Cell linesUsing methods described previously (13), we generated cell lines

from pleural fluid collected from MGH915 prior to lorlatinib(MGH915-3) and from a xenograft of pleural fluid at relapse onlorlatinib (MGH915-4). The cell lines were sequenced to confirm thepresence of ALK rearrangement, MET rearrangement, and METamplification. The ST7-MET fusion transcript was confirmed bysequencing a 700 bp region spanning the fusion breakpoint in cDNAgenerated from RNA extracted from MGH915-4. The correspondingST7-MET fusion sequence was constructed by fusing ST7 exon 1(NM_021908.3) to exons 2-21 ofMET (NM_000245.4) which includesthe full-length MET coding sequence. This resulting fusion sequenceincorporates the ST7 ATG start codon corresponding to position 41exon 1 followed by 151 bp of ST7 exon 1 and 14 bp ofMET exon 2 50 tothe wild-type MET ATG start codon. Wild-type MET sequence wasobtained from an open reading frame cDNA clone (GenScriptOHu28578, NM_001127500.2). The sequence was cloned into pENTTmcs (Addgene #25755), recombined with pSLIK-Neo (Addgene#25735) by using Gateway LR Clonase Enzyme (Invitrogen), andlentiviral particles were generated in HEK293FT cells by transfectionwith ViraPower packaging Mix (Invitrogen) following the manufac-turers’ instructions. H3122 cells were transduced by lentiviral infectionand selectedwithG418 (Gibco) for 5 days. The expression of ST7-METwas induced by doxycycline followed by addition of lorlatinib to selectthe ST7-MET–expressing resistant population. After selection, cellswere maintained with continuous culture in 1 mg/mL of doxycyclineand 300 nmol/L of lorlatinib. For drug sensitivity assays using DOX-controls, doxycycline was removed 3 days prior to the experiment toallow for ST7-MET expression levels to return to baseline.

Drug sensitivity assays, cell proliferation assays, and Westernblot analysis

Cells (2,000) were plated in triplicate into 96-well plates. Viabilitywas determined by CellTiter-Glo (Promega) 3 days after drug treat-ment in drug sensitivity assays. Luminescence was measured with aSpectraMax M5 Multi-Mode Microplate Reader (Molecular Devices).GraphPad Prism (GraphPad Software) was used to analyze data. ForWestern blotting, a total of 0.25–2 � 106 cells was treated in 6-wellplates for 6 hours. Equal amounts of total cell lysate were processed forimmunoblotting.

Whole-genome sequencingWhole-genome sequencing (WGS) was performed as described in

the Supplementary Methods.

Statistical analysisFisher exact test was used to compareMET amplification frequency

between treatment groups. All P values were based on a two-sidedhypothesis and computed using Stata 12.1.

ResultsStudy population

To survey the landscape of molecular alterations associated withresistance to ALK TKIs, we analyzed 207 tissue (n¼ 101) and plasma

Translational Relevance

With the development of highly potent and selective next-generation ALK inhibitors, resistance is increasingly mediated byALK-independent mechanisms. Here through molecular profilingof more than 200 resistant tissue and plasma specimens, includingthe largest dataset of post-lorlatinib specimens to date, we iden-tified MET amplification in 15% of tumor biopsies from patientsrelapsing on next-generation ALK inhibitors and detected rare,novel ST7-MET rearrangements in two cases. Upregulation ofMET signaling through MET amplification and/or fusion of METwith ST7 decreased sensitivity of ALK-positive cells to ALKinhibitors. In cell lines, combined blockade of ALK and METovercame resistance driven by MET bypass signaling. Consistentwith these findings, 2 patients with ALK-positive lung cancer andacquiredMET alterations achieved rapid clinical and radiographicresponses to ALK/MET combination therapy. Our study demon-strates that MET bypass signaling is a recurring and actionablemechanism of resistance to ALK inhibitors, providing rationale forpursuing ALK/MET combination therapy in the clinic.

Dagogo-Jack et al.

Clin Cancer Res; 26(11) June 1, 2020 CLINICAL CANCER RESEARCH2536




(n ¼ 106) specimens from 136 patients with ALK-positive NSCLCrelapsing on ALK TKIs (Fig. 1A). Four assays were used for theanalysis (see Materials andMethods), including two tissue-based NGSassays (FoundationOne and MGH SNaPshot/Solid Fusion Assay;refs. 19, 22), a plasma NGS assay (Guardant360; ref. 21), and FISHfor MET amplification (Supplementary Table S1).

The clinicopathologic characteristics of the study patients wereconsistent with an ALK-positive NSCLC population (SupplementaryTable S2). Of the 207 specimens, 12 (6%) were from patients relapsingon crizotinib who had not been exposed to other ALK TKIs. Themajority of specimens (n ¼ 136/207, 66%) were obtained at progres-sion on a second-generation ALK TKI. The remaining 59 (29%)specimens were collected from patients relapsing on lorlatinib.Overall, ALK kinase domain mutations were detected in 34(47%) of 73 tissue biopsies genotyped by NGS, including 25/46(54%) specimens obtained after second-generation TKIs and 9/25(36%) post-lorlatinib biopsies (Supplementary Fig. S1). Theseresults suggest that a significant fraction of resistant cases lacksecondary ALK mutations and likely harbor ALK-independentmechanisms of resistance.

Genetic alterations of METMET amplification in tissue biopsies

To evaluate the role of MET as a resistance mechanism, we firstassessed MET copy number in 86 tumor biopsies using FISH or theFoundationOne assay (Fig. 1A; Supplementary Table S1). Eleven(13%) biopsies harboredMET amplification (Fig. 1B), including fourwith low-level MET amplification (MET/CEP7 2.4–3.9) and six with

high-levelMET amplification based on FISH (MET/CEP7 5.2 to >25)or NGS (16–19MET copies). One sample had focalMET amplificationbyNGS, and FISHwas too variable to estimate copy number. In 3 of 11cases, there was available tissue to assessMET copy number in a biopsyfrom an earlier timepoint, none of which had MET amplification,supporting the notion thatMET amplification was newly acquired. Nocoalterations in other genes potentially associated with resistance orbypass signaling were identified in the 11 biopsies. In addition, METamplification was mutually exclusive with ALK resistance mutations,with the exception of one case, MGH9226 (Fig. 1B; SupplementaryFig. S1). This patient developed ALK I1171N (without MET ampli-fication) in plasma after relapsing on first-line alectinib, and thenresponded to second-line brigatinib for 5.5 months. After relapsing onbrigatinib, tumor biopsy demonstrated persistence ofALK I1171N andacquisition of MET amplification, suggesting that MET was drivingresistance.

All 11 cases with MET amplification were patients relapsing onsecond- or third-generation ALK TKIs (Fig. 1B). MET amplificationwas identified in six (12%) of 52 biopsies taken after a second-generation ALK TKI and in five (22%) of 23 post-lorlatinib biopsies.MET amplificationwas not detected in patients relapsing on crizotinib.In addition, 6 (55%) of the 11 positive cases had never receivedcrizotinib (Fig. 1B and C). To determine whether the developmentof MET amplification may be impacted by prior crizotinib exposure,we evaluated the frequency of MET amplification according to priorTKI therapy. Tumors from patients previously treated with crizotinibfollowed by next-generation TKI(s) were significantly less likely toharborMET amplification than those from patients treated only with

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Summary of MET alterations in resistant ALK-positive NSCLC. A, Schematic depicts number of specimens analyzed using four different assays. Pie charts arecolor-coded to reflect whether specimens were tissue (blue) or plasma (purple). The ALK inhibitor received immediately prior to biopsy is shown. TheSNaPshot cohort includes specimens genotyped using both SNaPshot and Solid Fusion Assays. Asterisk (�) includes 28 cases analyzed by both FISH andSNaPshot/Solid Fusion Assay. One patient (MGH915) had molecular profiling of two disease sites at relapse on lorlatinib but is only counted once in theFISH cohort given identical findings at both sites. 2nd gen., second-generation. B, Table lists MET alterations identified in resistant specimens. � , MGH075had focal MET amplification that was too variable to estimate copy number or MET/CEP7. �� , Plasma testing evaluated MET mutations but did not assessfor MET rearrangement. CN, copy number; ex14 skip, exon 14 skipping; MET/CEP7, ratio of MET to centromere 7 probe. C, Bar graphs illustrate frequency ofMET amplification according to prior ALK inhibitors received. The P value corresponds to the comparison of MET amplification frequency in crizotinib-na€�veversus crizotinib-pretreated patients who received next-generation ALK inhibitors. 2nd-gen. ALKi, second-generation ALK inhibitor.

MET Alterations in ALK-Positive Lung Cancer

AACRJournals.org Clin Cancer Res; 26(11) June 1, 2020 2537




next-generation TKI(s) (9% vs. 33%, P ¼ 0.019; Fig. 1C). Thus, priorexposure to crizotinib, an ALK inhibitor with documented METactivity, may have suppressed emergence of MET-amplified clonesin these patients.

MET copy number gain by plasma genotypingWe then analyzed MET copy number in 106 plasma specimens

from patients relapsing on next-generation ALK TKIs using theGuardant360 assay (Fig. 1A).MET copy number gain was detectedin eight (7.5%) plasma specimens (Supplementary Fig. S2), oneof which was due to polysomy of chromosome 7. The seven caseswith focal amplification of MET included two (3%) of 77 plasmaspecimens from patients relapsing on a second-generation ALKTKI and five (17%) of 29 post-lorlatinib specimens (Fig. 1B).Among 23 patients with contemporaneous plasma and tissuespecimens, MET amplification was detected in both plasma andtissue in four cases (Supplementary Table S3). Eighteen pairsdemonstrated absence of MET amplification. One patient,MGH960, had low-level MET amplification (2.2 copies) in plasmawhich was not detected in a contemporaneous biopsy. Thus, whentissue was used as the reference, plasma genotyping demonstrated100% sensitivity, 95% specificity, and 80% positive predictive valuefor detecting MET amplification. The remaining 2 patients withhigh-level MET amplification (4.9–6.1 copies) in plasma did nothave a paired tumor biopsy.

Four of the seven specimens with focal MET amplificationharbored both an ALK mutation and MET amplification in plasma(Supplementary Fig. S2), including MGH9226, as described above.Apart fromMGH92260s ALK I1171Nmutation, the remaining threeplasma specimens contained the gatekeeper ALK L1196Mmutation.ALK L1196M is known to cause resistance to crizotinib but issensitive to next-generation ALK TKIs (7). Indeed, all 3 patients hadreceived crizotinib prior to the next-generation ALK TKI(s) andpresumably developed this secondary mutation after crizotinib

exposure. The known activity of next-generation ALK TKIs againstALK L1196M suggests that MET amplification rather than the ALKmutation was likely mediating drug resistance. In addition to METamplification (6.1 copies), MGH92240s plasma demonstrated KRASamplification (2.6 copies) and a PIK3CA E545K mutation (allelicfrequency 0.04%) which may have also contributed to resistance.The remaining plasma specimens did not contain other putativedrivers of resistance.

Other genetic alterations of METWe next analyzed resistant specimens for other genetic alterations

that could lead to aberrant MET activation. To identify MET muta-tions, we reviewed molecular profiling results from 179 tissue andplasma specimens genotyped by MGH SNaPshot/Solid Fusion NGS(n ¼ 43), FoundationOne (n ¼ 30), or Guardant360 (n ¼ 106;Supplementary Figs. S1 and S2). One plasma specimen from a patientrelapsing after sequential alectinib and brigatinib harboredMET exon14 skipping without MET amplification (Fig. 1B). Two additionalplasma specimens contained MET mutations (D1101H and G500C;Supplementary Fig. S2) of unknown significance.METmutations werenot detected in any of the tissue specimens.

In two (3%) of 73 tissue specimens, we detected a rearrangementfusing exons 2–21 of MET with exon 1 of ST7 (suppressor oftumorigenicity 7), both of which reside in close proximity on chro-mosome 7q (Fig. 2A). Both cases were detected using an RNA-basedNGS assay (MGHSolid FusionAssay; ref. 22). In the first case, the ST7-MET fusionwas seen inMGH92840s pericardial fluid at progression onfirst-line alectinib without concurrentMET amplification. The patienthad primary progression on second-line lorlatinib at which timemolecular profiling of pleural fluid did not demonstrate the rearrange-ment but revealed low-level MET amplification (MET/CEP7 2.4,Fig. 1B). In the second case, concomitant ST7-MET and high-levelMET amplification were detected in MGH9150s pleural fluid afterfailure of lorlatinib (Fig. 1B), neither of which were present prior

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Acquired resistance due to ST7-MET and MET amplification. A, Sanger sequencing of the RT-PCR product in MGH9150s lorlatinib-resistant pleural fluid sampleshows fusion of ST7 exon 1 to MET exon 2. B and C, Cell viability after 3 days of monotherapy or combination therapy, as indicated. Viability was determinedusing CellTiter-Glo. Data are mean � SEM of three biological replicates. D, Immunoblotting to assess phosphorylation of ALK, MET, and downstream targetsin cells treated with lorlatinib and the MET TKIs shown. The arrowhead at 120 kDa indicates the band for phospho-ALK. Cells were treated with each drugat 300 nmol/L for 6 hours.

Dagogo-Jack et al.





to lorlatinib. Interestingly, contemporaneous biopsy of an axillarynode showed only high-level MET amplification and no detectableST7-MET.

Functional validation of ST7-MET as a targetable mechanism ofresistance

To evaluate the functional role of MET in driving resistance toALK TKIs, we established cell lines from MGH9150s pre-lorlatiniband post-lorlatinib malignant pleural effusion (MGH915-3 andMGH915-4, respectively). Consistent with the pleural fluid ana-lysis, the MGH915-4 cell line harbored both MET amplification(MET/chromosome 7 ratio 4.2) and the ST7-MET rearrangement,but MGH915-3 did not (Supplementary Fig. S3A and S3B). HighMET expression was confirmed in MGH915-4 cell line at themRNA and protein level (Supplementary Fig. S3B and S3C). Incell viability studies, the MGH915-4 cell line was resistant tosecond- and third-generation ALK TKIs, none of which have METactivity, but was sensitive to crizotinib (Fig. 2B). Treatment withMET-specific TKIs capmatinib or savolitinib, none of which haveALK activity, partially suppressed proliferation (SupplementaryFig. S3D). However, the combination of the ALK-selective TKIlorlatinib with capmatinib, savolitinib, or crizotinib potentlysuppressed cell proliferation (Fig. 2C; Supplementary Fig. S3D).Consistent with the cell growth assays, lorlatinib monotherapypotently inhibited ALK phosphorylation in immunoblotting stud-ies, but failed to inhibit downstream ERK or PI3K/AKT signaling(Fig. 2D). Only dual inhibition of ALK and MET by crizotinib orby the combination of lorlatinib plus a MET TKI effectively sup-pressed both ALK and downstream signaling pathways (Fig. 2D;Supplementary Fig. S3E).

As MGH915-4 harbors the ST7-MET rearrangement, we nextevaluated whether ST7-MET rearrangement could drive resistance toALK inhibition. The sensitive ALK-positive cell line H3122 wastransduced with lentivirus expressing a TET-inducible ST7-METconstruct corresponding to the ST7-MET fusion identified inMGH915-4 (Fig. 2A; Supplementary Fig. S3C). Whereas the H3122cells without expression of ST7-METwere highly sensitive to lorlatiniband other ALK TKIs, doxycycline-induced expression of ST7-MET inH3122 cells caused resistance to all ALK TKIs except crizotinib(Fig. 3A). Treatment with MET TKIs restored sensitivity to lorlatinib(Fig. 3B). Similarly, treatment with a selective MET TKI alone did notsuppress proliferation of H3122 cells expressing ST7-MET, but theaddition of lorlatinib to the MET TKIs resensitized the cells totreatment (Fig. 3B). The combination of lorlatinib plus crizotinibhad greater antiproliferative effect than crizotinib alone, likely due tomore potent inhibition of ALK (Fig. 3B).

We next examined the effect of ST7-MET expression on ALKand MET signaling pathways. In the absence of doxycyclineinduction of ST7-MET, we observed very little MET phosphor-ylation, and suppression of ALK phosphorylation with ALK TKIswas sufficient to inhibit downstream MAPK and PI3K signaling(Fig. 3C). Upon doxycycline-induced expression of ST7-MET, weobserved increased phospho-MET, which could be suppressedwith MET TKIs. Consistent with the effects on cell viability, thecombination of MET plus ALK inhibitor, but not either agentalone, was sufficient to inhibit downstream signaling (Fig. 3C). Ofnote, we were unable to distinguish between ST7-MET andendogenous MET by Western blot analysis due to their similarsizes and lack of suitable antibodies capable of detecting ST7exon 1. However, upon doxycycline induction of ST7-MET, wedetected an increase in two bands corresponding to immature

pro-MET (175 kDa) and the mature MET beta-chain (145 kDa),with an increase in phosphorylation of mature MET only afterdoxycycline induction (Fig. 3C). To compare the functional effectof ST7-MET relative to amplification of wild-type MET, we alsogenerated H3122 cells overexpressing wild-type MET. Similar toST7-MET, doxycycline-induced expression of MET caused resis-tance to all ALK inhibitors except crizotinib, and the combinationof ALK and MET inhibitors suppressed downstream signalingand cell proliferation (Supplementary Fig. S4). In our overexpres-sion system, the effect of ST7-MET and wild-type MET werecomparable. While the nature of our experimental system pre-cludes the ability to discriminate between increased MET activityresulting from a hyperfunctional ST7-MET protein or increasedMET expression level, these studies mimic the MGH915-4 clinicalscenario and suggest that resistance resulting from increasedexpression of ST7-MET is potentially targetable.

Clinical activity of combined ALK/MET inhibition in MET-drivenresistanceCrizotinib monotherapy

Based on our preclinical findings, we treated two resistant ALK-positive patients with combination ALK/MET TKIs. The first patient,MGH939, received first-line alectinib and relapsed after 9 months(Fig. 4A). Biopsy of a progressing lung lesion demonstrated no ALKmutations. However, high-level MET amplification with MET/CEP7 > 25 was identified (Fig. 1B). Similarly, plasma testing showedno detectable ALK mutations, but did show evidence of MET ampli-fication (2.5 copies). The patient was treated with carboplatin/peme-trexed for 5 cycles and then transitioned to crizotinib after diseaseprogression. Scans performed 5 weeks after initiating crizotinib dem-onstrated significant response to therapy (Fig. 4B). However, thepatient relapsed 10 weeks later. Biopsy of a chest wall mass confirmedpersistent MET amplification. There was insufficient tissue for NGS,but plasma testing revealed multiple ALK resistance mutations thatwere not detected pre-crizotinib/post-alectinib, in addition to theknownMET amplification. Thesefindings suggest that while crizotinibis active against both ALK and MET, its activity may ultimately belimited by secondary ALK mutations.

Combination lorlatinib plus crizotinibThe second patient, MGH915, was treated with first-line cer-

itinib with response lasting 31 months. Subsequent therapiesincluded alectinib-based combinations, chemotherapy plus immu-notherapy, chemotherapy with alectinib, and lorlatinib (Fig. 5A).As noted previously, at the time of relapse on lorlatinib, analysis ofpleural fluid demonstrated ST7-MET and high-level MET ampli-fication (MET/CEP7 ratio 5.2, Fig. 5B). A concurrent biopsy of agrowing axillary node also revealed MET amplification (MET/CEP7 ratio 5.7) but did not demonstrate the MET rearrangement.On the basis of these findings, the patient received the combinationof lorlatinib and crizotinib. Due to potential overlapping toxicities,the lorlatinib dose was reduced to 50 mg when crizotinib 250 mgtwice a day was added. After 2 weeks on the combination, lorlatinibwas escalated to 75 mg daily. The patient tolerated combinedtherapy well with side effects of grade 1 nausea and grade 1 lipidelevation. He experienced rapid symptomatic improvement, andfirst restaging scans confirmed improvement in disease (Fig. 5C).However, after 3 months, the patient developed progression of lungand axillary metastases (Fig. 5C). Biopsy of an axillary nodedemonstrated persistent high-level MET amplification withMET/CEP7 ratio >25 (Fig. 5B).






Convergent evolution of MET alterations during treatment withALK inhibitors

To investigate the evolutionary origin of MET alterations, weperformed WGS on MGH9150s serial tumor specimens andpatient-derived models (Fig. 6A; Supplementary Fig. S5A andS5B). We first focused on the MET locus and observed that thepost-lorlatinib specimens (MGH915-4) harbored a focal amplifi-cation (copy number 14-16) extending from the intergenic regionpreceding the MET gene to intron 1 of ST7 (Fig. 6B). This regionwas contained within a larger approximately 3 Mb amplification(Supplementary Fig. S5C), consistent with an initial tandem dupli-cation of the MET-ST7 locus, followed by amplification of theresulting ST7-MET and wild-type MET loci (�7 copieseach; Fig. 6C). The duplication placed exon 1 of ST7 22kb

upstream of the MET transcriptional start site, likely generatinga chimeric read-through fusion whereby mRNA processing causesthe first exon of ST7 to be spliced to MET exon 2. Neither thetandem duplication nor MET amplification was present in the pre-lorlatinib (post-alectinib) pleural effusion (MGH915-3). In thepost-lorlatinib/crizotinib combination sample (MGH915-9), weobserved an independent MET amplification event that did notinvolve the amplified 3 Mb region housing the ST7-MET tandemduplication (Fig. 6B; Supplementary Fig. S5C). When all non-METmutation clusters and MET alterations were assessed, there wassignificant overlap between mutations in the post-alectinib pleuraleffusion (MGH915-3) and the post-lorlatinib/crizotinib axillarynode (MGH915-9), but MET amplification only occurred inMGH915-9 (Fig. 6D). The post-lorlatinib pleural effusion

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Expression of ST7-MET confers resistance to ALK TKIs and can be overcome by dual ALK and MET inhibition. A and B, Cell viability of H3122 cells with or withoutdoxycycline-induced ST7-MET expression (DOXþ and DOX�, respectively) was measured after 3 days of monotherapy or combination therapy, as indicated.Viability was determined using CellTiter-Glo. Data are mean � SEM of three biological replicates. C, Immunoblotting to assess phosphorylation of ALK, MET, anddownstream targets in cells treated with lorlatinib and the MET TKIs shown. The arrowheads at 175 and 145 kDa indicate the band for pro-MET and MET beta-chain,respectively. Cells were treated with each drug at 300 nmol/L for 6 hours. DOX, doxycycline.

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(MGH915-4), however, represented a distinct branch of the phy-logenetic tree which included more than 3,400 private mutations inaddition toMET amplification and ST7-MET. Thus, reconstructionof phylogenetic relationships supports convergent evolution ofindependent genomic events leading to MET-driven acquiredresistance to ALK TKIs.

DiscussionWe analyzedmore than 200 tissue and plasma specimens to identify

genetic alterations leading to MET bypass signaling in resistant ALK-positive NSCLC.We detectedMET amplification and/or aMET fusionin 15% of tumors after failure of a next-generation ALK TKI. Impor-tantly, resistant cell line models and patients harboring MET ampli-fication or rearrangement could be resensitized to treatment with dualALK/MET inhibition.

MET gene amplification was initially discovered as a bypass mech-anism in EGFR-mutant NSCLC in 2007 and has subsequently beenidentified in up to 20% of resistant specimens (14, 23). In this study, wedemonstrate that the prevalence ofMET alterations in patients relaps-ing on ALK TKIs may be comparable to that seen in EGFR-mutantNSCLC. Interestingly, the incidence of MET amplification in EGFR-mutant NSCLC is higher after exposure to the third-generation EGFRTKI osimertinib compared with less potent EGFR TKIs (14, 15).Similarly, in our series, we observed that the frequency of METamplification was highest in tumors resistant to the third generation,broad-spectrum ALK TKI lorlatinib (Fig. 1C). These findings suggestthat an association may exist between TKI potency and likelihood ofdeveloping ALK-independent resistance mechanisms such as METamplification. In addition to TKI potency, TKI selectivity may alsoimpact the evolution of resistance. In support of this hypothesis, weobservedMET amplification in one-third of cases that received front-

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Clinical response to crizotinib in a patient with ALK-positive lung cancer and acquired MET amplification: A, Timeline illustrates treatments received andmolecular profiling results from serial tissue and plasma specimens analyzed during MGH9390s disease course. AMP, amplification; qns, quantity not sufficient;v3, EML4-ALK variant 3, or fusion of exon 6 of EML4 to exon 20 of ALK. B, Positron emission CT scans depict metabolic response to treatment in the chestwall and liver during treatment with crizotinib.






line second-generation ALK TKIs (which have no activity againstMET) compared with less than 10% of patients initially treated withcrizotinib.

MET signaling can be activated through a variety of mechanisms,including copy number gain, mutation, fusions, and ligand upregula-

tion (24). In this study, we found that copy number gain was mostfrequently used to activate MET bypass signaling. The range of METcopies in tissue was broad, spanningMET/CEP7 ratios of 2.4 to greaterthan 25 (Fig. 1B). In cases where MET amplification was detected inpaired tissue and plasma specimens, the number of MET copies in

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Clinical activity of dual ALK/MET TKIs in MET-driven resistance. A, Timeline of treatments and biopsies for MGH915. LN, lymph node; n.d., not performed; R, right;v1, EML4-ALK variant 1, or fusion of exon 13 of EML4 to exon 20 of ALK. B, FISH images from serial biopsies demonstrate progressive increase inMET copy number. LN,lymph node; MET/CEP7, ratio of MET to centromere 7 probe. C, CT scans depicting radiologic response to combined lorlatinib and crizotinib, with decrease in pleuralfluid and lung mass (red arrow, top) and decrease in size of right axillary node (bottom, red arrow). The patient later relapsed at these same sites due to resistance.

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plasmawas consistently lower than in tissue. TumorDNA represents asmall fraction of total cell-free DNA and some disease sites aremore likely to shed tumor DNA than others. Thus, calculation ofMET copy number in plasma is challenging. The relationship betweenMET copy number and dependency on MET bypass signaling inresistant ALK-positive NSCLC remains to be determined. WhileNSCLCs with de novo high-level MET amplification appear to bemore sensitive to crizotinib than NSCLCs with low-levelMET ampli-fication, MET/CEP7 ratio of 4–5 is an imperfect threshold for pre-dicting sensitivity to crizotinib and some tumors with low-levelamplification respond to crizotinib (20). In the context of acquiredresistance, the relationship between MET copies and sensitivity toMET TKIs may be more complex. Specifically, in resistant ALK-positiveNSCLCs incompletely inhibited byALKTKIs, it is conceivablethat even low-level MET amplification may be adequate to restoreproliferative signals. Furthermore, intratumoral molecular heteroge-neity resulting from exposure to multiple lines of therapy may impact

response to MET TKIs even among ALK-positive tumors with high-level MET amplification.

In addition to MET amplification, we detected identical ST7-METrearrangements in 2 patients relapsing on next-generation ALK TKIs.Rare ST7-MET rearrangements with different breakpoints have beendescribed in gliomas and ovarian cancer, but until now the pathoge-nicity of these fusions was unknown (25, 26). MGH9150s patient-derived cell line harbored both ST7-MET and MET amplification,preventing us from assessing the independent contribution of eachMET alteration to ALK TKI resistance. We demonstrate that intro-ducing ST7-MET into a sensitive ALK cell line is sufficient to conferresistance to ALK-targeted therapy. However, as engineering the cellsto express the ST7-MET rearrangement also increases the total expres-sion ST7-MET, we are unable to conclusively determine whether thefusion alone (in the absence of overexpression) is sufficient to driveresistance. Interestingly, ST7-MET was only identified in one ofMGH9150s two contemporaneous biopsies, while high-level MET

Figure 6.

Convergent evolution upon MET activation drives resistance to lorlatinib. A, Metastatic disease sites analyzed by WGS. B, Copy number profiles of METlocus demonstrate amplification encompassing MET and first exon of ST7 in post-lorlatinib samples. Circos plots depicting chromosomal structure areshown on right. The EML4-ALK rearrangement and MET locus are indicated. C, Proposed sequence of genomic alterations leading to generation of ST7-METfusion and amplification. D, Phylogenetic relationship of serial tumor samples based on shared and private mutations and MET alterations: MGH915-1(pretreatment tumor biopsy), MGH915-3 (cell line derived from post-alectinib pleural effusion), MGH914-4 (post-lorlatinib pleural effusion and patient-derived xenograft), MGH915-9 (post-lorlatinib/crizotinib axillary lymph node biopsy). Number of somatic mutations and selected mutations private to eachbranch point are indicated.






amplification was present in both of the resistant disease sites. InMGH92840s case, ST7-MET was identified in pericardial fluid whilelow-levelMET amplification without the rearrangement was detectedin pleural fluid 2 months later. These findings suggest that a singletumor can acquire distinct resistance alterations that converge onMET activation and also highlight the potential for intratumoralheterogeneity of MET genetic alterations.

In 2 patients with MET-driven resistance, inhibiting both ALK andMET led to clinical responses. However, despite initial responses, bothpatients relapsed after approximately 3 months. At relapse on crizo-tinib monotherapy, MGH9390s plasma analysis demonstrated multi-ple crizotinib-resistant (but next-generation ALK TKI-sensitive) ALKmutations, providing rationale for exploring combinations pairingmore potent next-generation ALK TKIs with a MET TKI. Indeed,MGH915 did receive combination therapy with lorlatinib and crizo-tinib, but still had a short-lived response. Repeat biopsy after relapse onthe combination revealed persistentMET amplification without addi-tional genetic alterations associated with resistance to ALK and/orMET TKIs (Figs. 3B and 4A). Interestingly, the MET/CEP7 ratioincreased from 5.7 to greater than 25 at relapse on the lorlatinib/crizotinib combination. As there can be cell-to-cell variability inMETcopy number which may be difficult to capture by FISH, it is possiblethat there was expansion of a more highly MET amplified subpopu-lation under the selective pressure of crizotinib. In this scenario, it isconceivable that the MET copy number increase was not the primarydriver of relapse inMGH9150s case. Resistant cellsmay have developedother alterations (including nongenetic aberrations) that decreasedsensitivity to dual ALK/MET combination treatment. To inform theclinical development of ALK/MET TKI combinations, additionalstudies will be required to characterize molecular determinants ofresistance to ALK/MET combination therapies, to determine whethersensitivity to combination therapy is impacted by line of therapy, andto assess antitumor activity of other more potent MET TKIs likecapmatinib and savolitinib (Supplementary Fig. S3D). Indeed, there isclinical rationale for exploring ALK/MET combinations besides lor-latinib/crizotinib. For example, while cross-trial comparisons suggestthatmost adverse events, including nausea/vomiting and edema, occurat comparable rates with the three MET TKIs evaluated in cell lines(crizotinib, capmatinib, savolitinib; refs. 4, 27, 28), the blood–brainbarrier penetration of capmatinib is superior to crizotinib. Capmatinibmay therefore be a more optimal partner for lorlatinib in patients withbrain metastases.

This study has several important limitations. First, due to limitedtissue availability, we could not perform analyses for all MET altera-tions in all specimens. In addition, we used several assays to detectMET alterations, each with its own limitations. Second, patients in ourstudy did not consistently undergo repeat biopsy each time theyrelapsed on an ALK TKI, precluding definitive timing of the acqui-sition of theMET alteration in the majority of cases. Third, we limitedour analysis to genetic alterations ofMET in resistant tumors and didnot examine nongenetic mechanisms of MET activation, includingupregulation of MET's ligand hepatocyte growth factor. Fourth, in theWGS analysis, clonal relationships were inferred from patient-derivedcell lines and xenografts and biopsies obtained from a variety of sites. Itis possible that patient-derived models may not fully recapitulate thespectrum of subclones in the tumor and that spatial heterogeneitycontributed to differences in sequential biopsies. Fifth, most of thepatients in this study had previously received crizotinib which couldhave prevented expansion of resistant subclones harboring METalterations, thereby decreasing the chance ofMET activation emergingas a bypass pathway with subsequent next-generation ALK TKIs. As

more selective second-generation ALK TKIs have recently replacedcrizotinib in the front-line setting, the frequency of MET bypasssignaling driving resistance may be higher than estimated in thisstudy. Finally, although we identified rare ST7-MET rearrangementsin a subset of cases, we could not fully validate the functional role of therearrangement or confirm that the rearrangement was an independentmediator of resistance due to limitations of our experimental system.Additional studies—ideally using patient-derived models that do notharbor concurrent MET amplification—are needed to robustly char-acterize these rearrangements.

In summary, by performing the most comprehensive analysis ofMET alterations in ALK-positive NSCLC to date, we demonstrate thatMET is an important bypass mechanism in tumors exposed tonext-generation ALK TKIs. The detection of MET amplification inone-third of patients who received next-generation ALK TKIs in thefront-line setting provides justification for exploring ALK/MET com-binations as an initial treatment strategy for advanced ALK-positiveNSCLC.

Disclosure of Potential Conflicts of InterestI. Dagogo-Jack is a paid consultant for Boehringer Ingelheim and Foundation

Medicine, reports receiving other commercial research support from Genentech andPfizer, and reports receiving other remuneration (travel support) from Pfizer andArray. J.J. Lin is a paid consultant for Nuvalent, C4 Therapeutics, and Pfizer, reportsreceiving commercial research grants from Novartis, Turning Point Therapeutics,Neon Therapeutics, and Hengrui, and reports receiving other remunerationfrom OncLive. S.R. Digumarthy reports receiving speakers bureau honoraria fromSiEMENS. A. Saxena is a paid consultant for Genentech, AstraZeneca, andMedtronic, and reports receiving speakers bureau honoraria from AstraZeneca andTakeda. R.J. Nagy is an employee of and holds ownership interest (including patents)in Guardant Health, Inc. C.H. Benes reports receiving commercial researchgrants from Novartis and Eli-Lilly. J.F. Gainor is a paid consultant for Bristol-Myers Squibb, Merck, Genentech, Blueprint, LOXO, Lilly, Regeneron, Pfizer,Ironwood Pharmaceuticals, Moderna, and Jounce, and reports receiving othercommercial research support from Novartis. A.T. Shaw is an employee of Novartisand a paid consultant for Pfizer, Genentech/Roche, Ariad/Takeda, Syros, BlueprintMedicine, KSQ Therapeutics, TP Therapeutics, Chugai, Daiichi-Sankyo, LOXO/Bayer, Achilles, Archer, Foundation Medicine, and Guardant. A.N. Hatareports receiving commercial research grants from Pfizer, Novartis, Amgen,Roche/Genentech, Eli Lilly, and Relay Therapeutics. No potential conflicts ofinterest were disclosed by the other authors.

Authors’ ContributionsConception and design: I. Dagogo-Jack, J.F. Gainor, A.T. Shaw, A.N. HataDevelopment of methodology: I. Dagogo-Jack, S. Yoda, J.K. Lennerz, K. Prutisto-ChangAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): I. Dagogo-Jack, S. Yoda, J.K. Lennerz, J.J. Lin, M.M. Rooney,K. Prutisto-Chang, A. Oh, N.A. Adams, A. Do, S.E. Stevens, A. Saxena, R.J. Nagy,C.H. Benes, C.G. Azzoli, J.F. Gainor, A.T. ShawAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): I. Dagogo-Jack, S. Yoda, J.K. Lennerz, A. Langenbucher,K. Prutisto-Chang, B.Y. Yeap, H.D. Marble, R.J. Nagy, C.H. Benes, M.S. Lawrence,J.F. Gainor, A.T. ShawWriting, review, and/or revision of the manuscript: I. Dagogo-Jack, S. Yoda,J.K. Lennerz, J.J. Lin, M.M. Rooney, B.Y. Yeap, A. Do, S.R. Digumarthy,A. Saxena, R.J. Nagy, C.G. Azzoli, J.F. Gainor, A.T. Shaw, A.N. HataAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): I. Dagogo-Jack,M.M. Rooney, N.A. Adams, E. Chin, A. Do,H.D. Marble, A.T. ShawStudy supervision: A.T. Shaw, A.N. HataOther (image interpretation): S.R. Digumarthy

AcknowledgmentsThis work was supported by a Conquer Cancer/Amgen Young Investigator Award

(to I. Dagogo-Jack), an institutional research grant fromAmerican Cancer Society (to

Dagogo-Jack et al.





I. Dagogo-Jack), a NCI Career Development Award (K12CA087723-16, to I. Dagogo-Jack), a LungCancer Research FoundationAnnualGrant Program award (to S. Yoda),a Conquer Cancer/AstraZeneca Young Investigator Award (to J.J. Lin), grants fromthe NCI (R01CA164273, to A.T. Shaw; R01CA225655, to J.K. Lennerz), by Be a Pieceof the Solution, and by Targeting a Cure for Lung Cancer Research Fund at MGH(Boston, MA).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 5, 2019; revised January 22, 2020; accepted February 17, 2020;published first February 21, 2020.

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2020;26:2535-2545. Published OnlineFirst February 21, 2020.Clin Cancer Res Ibiayi Dagogo-Jack, Satoshi Yoda, Jochen K. Lennerz, et al. Mechanism in ALK-Positive Lung CancerMET Alterations Are a Recurring and Actionable Resistance

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