Mechanism in ALK-Positive Lung Cancer

Author Manuscript Published OnlineFirst on February 21, 2020; DOI: 10.1158/1078-0432.CCR-19-3906 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

1 MET Alterations are a Recurring and Actionable Resistance 2 Mechanism in ALK-Positive Lung Cancer

3 Ibiayi Dagogo-Jack1,2*, Satoshi Yoda1,2*, Jochen K. Lennerz3, Adam Langenbucher1, 4 Jessica J. Lin1,2, Marguerite Rooney1, Kylie Prutisto-Chang1, Audris Oh1, Nathaniel A. 5 Adams1, Beow Y. Yeap1, Emily Chin1, Andrew Do1, Hetal D. Marble3, Sara E. Stevens1, 6 Subba R. Digumarthy4, Ashish Saxena5, Rebecca J. Nagy6, Cyril H. Benes1,2, 7 Christopher G. Azzoli1,2, Michael Lawrence1, Justin F. Gainor1,2, Alice T. Shaw1,2‡**, and 8 Aaron N. Hata1,2‡**

9 1Massachusetts General Hospital Cancer Center and Department of Medicine, 10 Massachusetts General Hospital, 2Harvard Medical School, 3Department of Pathology, 11 Center for Integrated Diagnostics, Massachusetts General Hospital, 4Department of 12 Radiology, Massachusetts General Hospital, 5Department of Medicine, Weill Cornell 13 Medicine, 6Guardant Health, Inc

14 ‡ Corresponding authors 15 *These authors contributed equally 16 **These authors contributed equally 17 18 Running title: MET Alterations in ALK-Positive Lung Cancer 19 Keywords: ALK, Lung Cancer, Resistance, MET Amplification 20 Figures/Tables: 6/0 21 Word Count: ~ 4500 22 Abstract Word Count: 252 words 23 Statement of Translational Relevance: 152 words 24 References: 26 25 Supplement: 5 Figures, 3 Tables 26 27 Corresponding Authors: 28 Alice T. Shaw, M.D., Ph.D. 29 Massachusetts General Hospital 30 Department of Medicine 31 32 Fruit Street 32 Boston, MA, USA, 02114 33 email: [email protected] 34 35 Aaron N. Hata, M.D., Ph.D. 36 Massachusetts General Hospital 37 Department of Medicine 38 149 13th Street, Charlestown, MA, 02129. 39 email: [email protected]

Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on February 21, 2020; DOI: 10.1158/1078-0432.CCR-19-3906 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

40 ABSTRACT (252/250) 41 Background: Most ALK-positive lung cancers will develop ALK-independent resistance 42 after treatment with next-generation ALK inhibitors. MET amplification has been 43 described in patients progressing on ALK inhibitors, but frequency of this event has not 44 been comprehensively assessed.

45 Methods: We performed fluorescence in-situ hybridization and/or next-generation 46 sequencing on 207 post-treatment tissue (n=101) or plasma (n=106) specimens from 47 patients with ALK-positive lung cancer to detect MET genetic alterations. We evaluated 48 ALK inhibitor sensitivity in cell lines with MET alterations and assessed antitumor activity 49 of ALK/MET blockade in ALK-positive cell lines and two patients with MET-driven 50 resistance.

51 Results: MET amplification was detected in 15% of tumor biopsies from patients 52 relapsing on next-generation ALK inhibitors, including 12% and 22% of biopsies from 53 patients progressing on second-generation inhibitors or lorlatinib, respectively. Patients 54 treated with a second-generation ALK inhibitor in the first-line setting were more likely to 55 develop MET amplification than those who had received next-generation ALK inhibitors 56 after crizotinib (p=0.019). Two tumor specimens harbored an identical ST7-MET 57 rearrangement, one of which had concurrent MET amplification. Expressing ST7-MET in 58 the sensitive H3122 ALK-positive cell line induced resistance to ALK inhibitors that was 59 reversed with dual ALK/MET inhibition. MET inhibition re-sensitized a patient-derived cell 60 line harboring both ST7-MET and MET amplification to ALK inhibitors. Two patients with 61 ALK-positive lung cancer and acquired MET alterations achieved rapid responses to 62 ALK/MET combination therapy.

63 Conclusions: Treatment with next-generation ALK inhibitors, particularly in the first-line 64 setting, may select for MET-driven resistance. Patients with acquired MET alterations 65 may derive clinical benefit from therapies that target both ALK and MET.

67 STATEMENT OF TRANSLATIONAL RELEVANCE

68 With the development of highly potent and selective next-generation ALK inhibitors, 69 resistance is increasingly mediated by ALK-independent mechanisms. Here through 70 molecular profiling of >200 resistant tissue and plasma specimens, including the largest 71 dataset of post-lorlatinib specimens to date, we identified MET amplification in 15% of 72 tumor biopsies from patients relapsing on next-generation ALK inhibitors and detected 73 rare, novel ST7-MET rearrangements in two cases. Upregulation of MET signaling 74 through MET amplification and/or fusion of MET with ST7 decreased sensitivity of ALK- 75 positive cells to ALK inhibitors. In cell lines, combined blockade of ALK and MET 76 overcame resistance driven by MET bypass signaling. Consistent with these findings, 77 two patients with ALK-positive lung cancer and acquired MET alterations achieved rapid 78 clinical and radiographic responses to ALK/MET combination therapy. Our study 79 demonstrates that MET bypass signaling is a recurring and actionable mechanism of 80 resistance to ALK inhibitors, providing rationale for pursuing ALK/MET combination 81 therapy in the clinic.

84 CONFLICTS OF INTEREST/DISCLOSURES: 85 86 IDJ has received honoraria from Foundation Medicine, consulting fees from Boehringer 87 Ingelheim, research support from Array, Genentech, Novartis, Pfizer, and Guardant 88 Health, and travel support from Array and Pfizer. JJL has received honoraria or served 89 as a compensated consultant for Chugai, Boehringer Ingelheim, and Pfizer; institutional 90 research support from Loxo Oncology and Novartis; and CME honorarium from OncLive. 91 RJN is an employee of Guardant Health. SRD provides independent image analysis 92 through the institution for clinical research trials sponsored by Merck, Pfizer, Bristol 93 Myers Squibb, Novartis, Roche, Polaris, Cascadian, Abbvie, Gradalis, Bayer, Zai 94 Laboratories; and has received honoraria from Siemens Medical Solutions. AS has 95 served on advisory boards for AstraZeneca and Takeda and as a consultant for 96 Genentech, AstraZeneca, and Medtronic. JFG has served as a compensated consultant 97 or received honoraria from Bristol-Myers Squibb, Genentech, Ariad/Takeda, Loxo, 98 Pfizer, Incyte, Novartis, Merck, Agios, Amgen, Jounce, Regeneron, Oncorus, Helsinn, 99 Jounce, Array, and Clovis Oncology, has an immediate family member who is an 100 employee of Ironwood Pharmaceuticals, has received research funding from Novartis, 101 Genentech/Roche, and Ariad/Takeda, and institutional research support from Tesaro, 102 Moderna, Blueprint, BMS, Jounce, Array, Adaptimmune, Novartis, Genentech/Roche, 103 Alexo and Merck. ATS has served as a compensated consultant or received honoraria 104 from Achilles, Archer, ARIAD, Bayer, Blueprint Medicines, Chugai, Daiichi Sankyo, EMD 105 Serono, Foundation Medicine, Genentech/Roche, Guardant, Ignyta, KSQ Therapeutics, 106 LOXO, Natera, Novartis, Pfizer, Servier, Syros, Taiho Pharmaceutical, Takeda, and TP 107 Therapeutics, has received research (institutional) funding from Daiichi Sankyo, Ignyta, 108 Novartis, Pfizer, Roche/Genentech, and TP Therapeutics; has received travel support 109 from Pfizer and Genentech/Roche; and is an employee of Novartis. ANH has received 110 research grants/funding from Pfizer, Novartis, Amgen, Eli Lilly, Relay Therapeutics, and 111 Roche/Genentech. The remaining authors have no disclosures to report. 112 113

114 115 FUNDING: 116 This work was support by a Conquer Cancer/Amgen Young Investigator Award (I.D.J), 117 an institutional research grant from American Cancer Society (I.D.J.), a National Cancer 118 Institute Career Development Award (K12CA087723-16 to I.D.J.), a Lung Cancer 119 Research Foundation Annual Grant Program award (S.Y.), a Conquer 120 Cancer/AstraZeneca Young Investigator Award (J.J.L.), grants from the National Cancer 121 Institute (R01CA164273 to A.T.S. and R01CA225655 to J.K.L.), by Be a Piece of the 122 Solution, and by Targeting a Cure for Lung Cancer Research Fund at MGH. 123 124

125 INTRODUCTION 126 ALK-rearranged (i.e., ALK-positive) non-small cell lung cancers (NSCLC) depend on 127 constitutive ALK signaling for growth and survival.1 This oncogene dependency underlies 128 the marked sensitivity of these tumors to tyrosine kinase inhibitors (TKIs) targeting 129 ALK.2,3 Historically, the ALK/ROS1/MET TKI crizotinib was the standard first-line therapy 130 for advanced ALK-positive NSCLC.4 However, crizotinib has recently been supplanted 131 by more potent and selective second-generation ALK TKIs (e.g., ceritinib, alectinib, 132 brigatinib).2,3,5 In addition, the third-generation ALK TKI lorlatinib was recently approved 133 for patients previously treated with a second-generation TKI.6 134 135 Despite initial sensitivity to ALK TKIs, ALK-positive tumors invariably develop resistance. 136 In approximately one-half of cases, resistance to second-generation ALK TKIs is due to 137 secondary mutations in the ALK kinase domain.7 These tumors remain ALK-dependent 138 and responsive to treatment with lorlatinib.8 The remaining cases have presumably 139 developed ALK-independent resistance mechanisms, most often due to activation of 140 alternative or bypass signaling pathways.7,9 Recent studies of lorlatinib resistance 141 suggest an even more complicated array of resistance mechanisms, including diverse 142 compound ALK mutations in one-third of patients and ALK-independent resistance 143 mechanisms in the remaining two-thirds of patients.9,10 144 145 To date, ALK-independent resistance mechanisms remain poorly characterized. In 146 preclinical studies, multiple bypass mechanisms have been described, including 147 activation of MET, EGFR, SRC, and IGF-1R.11-13. Of these, MET is a particularly 148 attractive target given the availability of potent MET TKIs. In addition, MET activation has 149 been previously studied as a bypass pathway in EGFR-mutant NSCLC where MET 150 amplification has been reported in 5-20% of resistant cases.14,15 In phase I/II studies, co- 151 targeting EGFR and MET can re-induce responses in resistant EGFR-mutant tumors 152 with MET amplification.16 In contrast to EGFR-mutant NSCLC, relatively little is known 153 about the contribution of aberrant MET activation to resistance in ALK-positive NSCLC. 154 Several case reports suggest that MET amplification can mediate resistance to ALK 155 TKIs and that the ALK/ROS1/MET TKI crizotinib may be able to overcome MET-driven 156 resistance.17,18 However, larger studies are needed to validate MET as a clinically 157 relevant driver of resistance in ALK-positive NSCLC. 158

159 Here we analyze tumor and/or blood specimens from 136 patients with ALK-positive 160 NSCLC relapsing on ALK TKIs. We identify genetic alterations of MET in approximately 161 15% of resistant cases. In cell lines and in patients, MET activation drives resistance to 162 ALK TKIs but confers sensitivity to dual ALK/MET blockade. These studies support the 163 development of novel combinations targeting ALK and MET in ALK-positive NSCLC. 164 165 METHODS 166 Data Collection 167 Resistant specimens were obtained from 136 patients with metastatic ALK-positive 168 NSCLC who underwent molecular profiling between 2014 and 2019 (Figure 1A). 169 Medical records from these patients were retrospectively reviewed to extract data on 170 demographics, treatment histories and molecular profiling results. Data were updated as 171 of August 31, 2019. This study was approved by the Massachusetts General Hospital 172 Institutional Review Board. Patient studies were conducted according to the Declaration 173 of Helsinki, the Belmont Report, and the U.S. Common Rule. 174 175 Molecular Testing 176 MET copy number was quantified in tissue using FISH or the FoundationOne assay as 177 previously described.19 For cases assessed by FISH, amplification was defined as a 178 ratio of MET to centromere 7 (MET/CEP7) of ≥ 2.2.20 Cases with MET/CEP7 ≥ 4 were 179 classified as high-level amplification.20 For tumors genotyped with the FoundationOne 180 assay, MET amplification was defined as ≥ 6 gene copies.19 With the Guardant360 181 assay, MET amplification was defined as absolute plasma copy number ≥ 2.1.21 As MET 182 copy number analysis based on MGH SNaPshot NGS has not been formally validated, 183 this assay was not used to detect MET amplification. MET rearrangements and 184 mutations and ALK mutations were detected in tissue using either FoundationOne or the 185 MGH SNaPshot/Solid Fusion Assays.19,22 Guardant360 was used to identify MET and 186 ALK mutations in plasma, but is not designed to detect MET rearrangements.21 All 187 patients included in this study provided written informed consent for molecular analysis. 188 189 Cell Lines 190 Using methods previously described,13 we generated cell lines from pleural fluid 191 collected from MGH915 prior to lorlatinib (MGH915-3) and from a xenograft of pleural 192 fluid at relapse on lorlatinib (MGH915-4). The cell lines were sequenced to confirm the

193 presence of ALK rearrangement, MET rearrangement, and MET amplification. The ST7- 194 MET fusion transcript was confirmed by sequencing a 700 bp region spanning the fusion 195 breakpoint in cDNA generated from RNA extracted from MGH915-4. The corresponding 196 ST7-MET fusion sequence was constructed by fusing ST7 exon 1 (NM_021908.3) to 197 exons 2-21 of MET (NM_000245.4) which includes the full-length MET coding sequence. 198 This resulting fusion sequence incorporates the ST7 ATG start codon corresponding to 199 position 41 exon 1 followed by 151 bp of ST7 exon 1 and 14 bp of MET exon 2 5’ to the 200 wild-type MET ATG start codon. Wild-type MET sequence was obtained from an ORF 201 cDNA clone (GenScript OHu28578, NM_001127500.2). The sequence was cloned into 202 pEN TTmcs (Addgene #25755), recombined with pSLIK-Neo (Addgene #25735) by 203 using Gateway LR Clonase Enzyme (Invitrogen), and lentiviral particles were generated 204 in HEK293FT cells by transfection with ViraPower packaging Mix (Invitrogen) following 205 the manufacturers' instructions. H3122 cells were transduced by lentiviral infection and 206 selected with G418 (Gibco) for 5 days. The expression of ST7-MET was induced by 207 doxycycline followed by addition of lorlatinib to select the ST7-MET-expressing resistant 208 population. After selection, cells were maintained with continuous culture in 1 μg/mL of 209 doxycycline and 300 nM of lorlatinib. For drug sensitivity assays using DOX- controls, 210 doxycycline was removed 3 days prior to the experiment to allow for ST7-MET 211 expression levels to return to baseline. 212 213 Drug Sensitivity Assays, Cell Proliferation Assays, and Western Blot Analysis 214 2,000 cells were plated in triplicate into 96-well plates. Viability was determined by 215 CellTiter-Glo (Promega) three days after drug treatment in drug sensitivity assays. 216 Luminescence was measured with a SpectraMax M5 Multi-Mode Microplate Reader 217 (Molecular Devices). GraphPad Prism (GraphPad Software) was used to analyze data. 218 For Western blotting, a total of 0.25–2x106 cells was treated in 6-well plates for 6 hours. 219 Equal amounts of total cell lysate were processed for immunoblotting.

220 Whole Genome Sequencing 221 Whole genome sequencing was performed as described in the Supplementary Methods. 222 223 Statistical Analysis

224 Fisher’s exact test was used to compare MET amplification frequency between 225 treatment groups. All p-values were based on a two-sided hypothesis and computed 226 using Stata 12.1. 227 228 RESULTS 229 230 Study Population 231 To survey the landscape of molecular alterations associated with resistance to ALK 232 TKIs, we analyzed 207 tissue (n=101) and plasma (n=106) specimens from 136 patients 233 with ALK-positive NSCLC relapsing on ALK TKIs (Figure 1A). Four assays were used 234 for the analysis (see Methods), including two tissue-based next-generation sequencing 235 (NGS) assays (FoundationOne and MGH SNaPshot/Solid Fusion Assay),19,22 a plasma 236 NGS assay (Guardant360),21 and fluorescence in-situ hybridization (FISH) for MET 237 amplification (Supplementary Table 1). 238 239 The clinicopathologic characteristics of the study patients were consistent with an ALK- 240 positive NSCLC population (Supplementary Table 2). Of the 207 specimens, twelve 241 (6%) were from patients relapsing on crizotinib who had not been exposed to other ALK 242 TKIs. The majority of specimens (n=136/207, 66%) were obtained at progression on a 243 second-generation ALK TKI. The remaining fifty-nine (29%) specimens were collected 244 from patients relapsing on lorlatinib. Overall, ALK kinase domain mutations were 245 detected in 34 (47%) of 73 tissue biopsies genotyped by NGS, including 25/46 (54%) 246 specimens obtained after second-generation TKIs and 9/25 (36%) post-lorlatinib 247 biopsies (Supplementary Figure 1). These results suggest that a significant fraction of 248 resistant cases lack secondary ALK mutations and likely harbor ALK-independent 249 mechanisms of resistance. 250 251 Genetic Alterations of MET 252 MET Amplification in Tissue Biopsies 253 To evaluate the role of MET as a resistance mechanism, we first assessed MET copy 254 number in 86 tumor biopsies using FISH or the FoundationOne assay (Figure 1A, 255 Supplementary Table 1). Eleven (13%) biopsies harbored MET amplification (Figure 256 1B), including four with low-level MET amplification (MET/CEP7 2.4-3.9) and six with 257 high-level MET amplification based on FISH (MET/CEP7 5.2 to >25) or NGS (16-19

258 MET copies). One sample had focal MET amplification by NGS, and FISH was too 259 variable to estimate copy number. In 3 of 11 cases, there was available tissue to assess 260 MET copy number in a biopsy from an earlier timepoint, none of which had MET 261 amplification, supporting the notion that MET amplification was newly acquired. No co- 262 alterations in other genes potentially associated with resistance or bypass signaling were 263 identified in the 11 biopsies. In addition, MET amplification was mutually exclusive with 264 ALK resistance mutations, with the exception of one case, MGH9226 (Figure 1B, 265 Supplementary Figure 1). This patient developed ALK I1171N (without MET 266 amplification) in plasma after relapsing on first-line alectinib, and then responded to 267 second-line brigatinib for 5.5 months. After relapsing on brigatinib, tumor biopsy 268 demonstrated persistence of ALK I1171N and acquisition of MET amplification, 269 suggesting that MET was driving resistance. 270 271 All 11 cases with MET amplification were patients relapsing on second- or third- 272 generation ALK TKIs (Figure 1B). MET amplification was identified in six (12%) of 52 273 biopsies taken after a second-generation ALK TKI and in five (22%) of 23 post-lorlatinib 274 biopsies. MET amplification was not detected in patients relapsing on crizotinib. In 275 addition, six (55%) of the 11 positive cases had never received crizotinib (Figure 1B,C). 276 To determine whether the development of MET amplification may be impacted by prior 277 crizotinib exposure, we evaluated the frequency of MET amplification according to prior 278 TKI therapy. Tumors from patients previously treated with crizotinib followed by next- 279 generation TKI(s) were significantly less likely to harbor MET amplification than those 280 from patients treated only with next-generation TKI(s) (9% vs 33%, p=0.019, Figure 1C). 281 Thus, prior exposure to crizotinib, an ALK inhibitor with documented MET activity, may 282 have suppressed emergence of MET-amplified clones in these patients. 283 284 MET Copy Number Gain by Plasma Genotyping 285 We then analyzed MET copy number in 106 plasma specimens from patients relapsing 286 on next-generation ALK TKIs using the Guardant360 assay (Figure 1A). MET copy 287 number gain was detected in eight (7.5%) plasma specimens (Supplementary Figure 288 2), one of which was due to polysomy of chromosome 7. The seven cases with focal 289 amplification of MET included two (3%) of 77 plasma specimens from patients relapsing 290 on a second-generation ALK TKI and five (17%) of 29 post-lorlatinib specimens (Figure 291 1B). Among 23 patients with contemporaneous plasma and tissue specimens, MET

292 amplification was detected in both plasma and tissue in four cases (Supplementary 293 Table 3). Eighteen pairs demonstrated absence of MET amplification. One patient, 294 MGH960, had low-level MET amplification (2.2 copies) in plasma which was not 295 detected in a contemporaneous biopsy. Thus, when tissue was used as the reference, 296 plasma genotyping demonstrated 100% sensitivity, 95% specificity, and 80% positive 297 predictive value for detecting MET amplification. The remaining two patients with high- 298 level MET amplification (4.9-6.1 copies) in plasma did not have a paired tumor biopsy. 299 300 Four of the seven specimens with focal MET amplification harbored both an ALK 301 mutation and MET amplification in plasma (Supplementary Figure 2), including 302 MGH9226, as described above. Apart from MGH9226’s ALK I1171N mutation, the 303 remaining three plasma specimens contained the gatekeeper ALK L1196M mutation. 304 ALK L1196M is known to cause resistance to crizotinib but is sensitive to next- 305 generation ALK TKIs.7 Indeed, all three patients had received crizotinib prior to the next- 306 generation ALK TKI(s) and presumably developed this secondary mutation after 307 crizotinib exposure. The known activity of next-generation ALK TKIs against ALK 308 L1196M suggests that MET amplification rather than the ALK mutation was likely 309 mediating drug resistance. In addition to MET amplification (6.1 copies), MGH9224’s 310 plasma demonstrated KRAS amplification (2.6 copies) and a PIK3CA E545K mutation 311 (allelic frequency 0.04%) which may have also contributed to resistance. The remaining 312 plasma specimens did not contain other putative drivers of resistance. 313 314 Other Genetic Alterations of MET 315 We next analyzed resistant specimens for other genetic alterations that could lead to 316 aberrant MET activation. To identify MET mutations, we reviewed molecular profiling 317 results from 179 tissue and plasma specimens genotyped by MGH SNaPshot/Solid 318 Fusion NGS (n= 43), FoundationOne (n=30), or Guardant360 (n=106) (Supplementary 319 Figures 1,2). One plasma specimen from a patient relapsing after sequential alectinib 320 and brigatinib harbored MET exon 14 skipping without MET amplification (Figure 1B). 321 Two additional plasma specimens contained MET mutations (D1101H and G500C, 322 Supplementary Figure 2) of unknown significance. MET mutations were not detected in 323 any of the tissue specimens. 324

325 In two (3%) of 73 tissue specimens, we detected a rearrangement fusing exons 2-21 of 326 MET with exon 1 of ST7 (suppressor of tumorigenicity 7), both of which reside in close 327 proximity on chromosome 7q (Figure 2A). Both cases were detected using an RNA- 328 based NGS assay (MGH Solid Fusion Assay).22 In the first case, the ST7-MET fusion 329 was seen in MGH9284’s pericardial fluid at progression on first-line alectinib without 330 concurrent MET amplification. The patient had primary progression on second-line 331 lorlatinib at which time molecular profiling of pleural fluid did not demonstrate the 332 rearrangement but revealed low-level MET amplification (MET/CEP7 2.4, Figure 1B). In 333 the second case, concomitant ST7-MET and high-level MET amplification were detected 334 in MGH915’s pleural fluid after failure of lorlatinib (Figure 1B), neither of which were 335 present prior to lorlatinib. Interestingly, contemporaneous biopsy of an axillary node 336 showed only high-level MET amplification and no detectable ST7-MET. 337 338 Functional Validation of ST7-MET as a Targetable Mechanism of Resistance 339 To evaluate the functional role of MET in driving resistance to ALK TKIs, we established 340 cell lines from MGH915’s pre-lorlatinib and post-lorlatinib malignant pleural effusion 341 (MGH915-3 and MGH915-4, respectively). Consistent with the pleural fluid analysis, the 342 MGH915-4 cell line harbored both MET amplification (MET/chromosome 7 ratio 4.2) and 343 the ST7-MET rearrangement, but MGH915-3 did not (Supplementary Figure 3A, B). 344 High MET expression was confirmed in MGH915-4 cell line at the mRNA and protein 345 level (Supplementary Figure 3B, C). In cell viability studies, the MGH915-4 cell line 346 was resistant to second- and third-generation ALK TKIs, none of which have MET 347 activity, but was sensitive to crizotinib (Figure 2B). Treatment with MET-specific TKIs 348 capmatinib or savolitinib, none of which have ALK activity, partially suppressed 349 proliferation (Supplementary Figure 3D). However, the combination of the ALK- 350 selective TKI lorlatinib with capmatinib, savolitinib, or crizotinib potently suppressed cell 351 proliferation (Figure 2C, Supplementary Figure 3D). Consistent with the cell growth 352 assays, lorlatinib monotherapy potently inhibited ALK phosphorylation in immunoblotting 353 studies, but failed to inhibit downstream ERK or PI3K/AKT signaling (Figure 2D). Only 354 dual inhibition of ALK and MET by crizotinib or by the combination of lorlatinib plus a 355 MET TKI effectively suppressed both ALK and downstream signaling pathways (Figure 356 2D, Supplementary Figure 3E). 357

358 As MGH915-4 harbors the ST7-MET rearrangement, we next evaluated whether ST7- 359 MET rearrangement could drive resistance to ALK inhibition. The sensitive ALK-positive 360 cell line H3122 was transduced with lentivirus expressing a TET-inducible ST7-MET 361 construct corresponding to the ST7-MET fusion identified in MGH915-4 (Figure 2A, 362 Supplementary Figure 3C). Whereas the H3122 cells without expression of ST7-MET 363 were highly sensitive to lorlatinib and other ALK TKIs, doxycycline-induced expression of 364 ST7-MET in H3122 cells caused resistance to all ALK TKIs except crizotinib (Figure 365 3A). Treatment with MET TKIs restored sensitivity to lorlatinib (Figure 3B). Similarly, 366 treatment with a selective MET TKI alone did not suppress proliferation of H3122 cells 367 expressing ST7-MET, but the addition of lorlatinib to the MET TKIs resensitized the cells 368 to treatment. (Figure 3B). The combination of lorlatinib plus crizotinib had greater 369 antiproliferative effect than crizotinib alone, likely due to more potent inhibition of ALK 370 (Figure 3B). 371 372 We next examined the effect of ST7-MET expression on ALK and MET signaling 373 pathways. In the absence of doxycycline induction of ST7-MET, we observed very little 374 MET phosphorylation, and suppression of ALK phosphorylation with ALK TKIs was 375 sufficient to inhibit downstream MAPK and PI3K signaling (Figure 3C). Upon 376 doxycycline-induced expression of ST7-MET, we observed increased phospho-MET, 377 which could be suppressed with MET TKIs. Consistent with the effects on cell viability, 378 the combination of MET plus ALK inhibitor, but not either agent alone, was sufficient to 379 inhibit downstream signaling (Figure 3C). Of note, we were unable to distinguish 380 between ST7-MET and endogenous MET by western blot due to their similar sizes and 381 lack of suitable antibodies capable of detecting ST7 exon 1. However, upon doxycycline 382 induction of ST7-MET, we detected an increase in two bands corresponding to immature 383 pro-MET (175 kDa) and the mature MET beta-chain (145 kDa), with an increase in 384 phosphorylation of mature MET only after doxycycline induction (Figure 3C). To 385 compare the functional effect of ST7-MET relative to amplification of wild-type MET, we 386 also generated H3122 cells overexpressing wild-type MET. Similar to ST7-MET, 387 doxycycline-induced expression of MET caused resistance to all ALK inhibitors except 388 crizotinib, and the combination of ALK and MET inhibitors suppressed downstream 389 signaling and cell proliferation (Supplementary Figure 4). In our overexpression 390 system, the effect of ST7-MET and wild-type MET were comparable. While the nature of 391 our experimental system precludes the ability to discriminate between increased MET

392 activity resulting from a hyper-functional ST7-MET protein or increased MET expression 393 level, these studies mimic the MGH915-4 clinical scenario and suggest that resistance 394 resulting from increased expression of ST7-MET is potentially targetable. 395 396 Clinical Activity of Combined ALK/MET Inhibition in MET-Driven Resistance 397 Crizotinib Monotherapy 398 Based on our preclinical findings, we treated two resistant ALK-positive patients with 399 combination ALK/MET TKIs. The first patient, MGH939, received first-line alectinib and 400 relapsed after 9 months (Figure 4A). Biopsy of a progressing lung lesion demonstrated 401 no ALK mutations. However, high-level MET amplification with MET/CEP7 >25 was 402 identified (Figure 1B). Similarly, plasma testing showed no detectable ALK mutations, 403 but evidence of MET amplification (2.5 copies). The patient was treated with 404 carboplatin/pemetrexed for 5 cycles and then transitioned to crizotinib after disease 405 progression. Scans performed five weeks after initiating crizotinib demonstrated 406 significant response to therapy (Figure 4B). However, the patient relapsed 10 weeks 407 later. Biopsy of a chest wall mass confirmed persistent MET amplification. There was 408 insufficient tissue for NGS, but plasma testing revealed multiple ALK resistance 409 mutations that were not detected pre-crizotinib/post-alectinib, in addition to the known 410 MET amplification. These findings suggest that while crizotinib is active against both 411 ALK and MET, its activity may ultimately be limited by secondary ALK mutations. 412 413 Combination Lorlatinib plus Crizotinib 414 The second patient, MGH915, was treated with first-line ceritinib with response lasting 415 31 months. Subsequent therapies included alectinib-based combinations, chemotherapy 416 plus immunotherapy, chemotherapy with alectinib, and lorlatinib (Figure 5A). As noted 417 previously, at the time of relapse on lorlatinib, analysis of pleural fluid demonstrated 418 ST7-MET and high-level MET amplification (MET/CEP7 ratio 5.2, Figure 5B). A 419 concurrent biopsy of a growing axillary node also revealed MET amplification 420 (MET/CEP7 ratio 5.7) but did not demonstrate the MET rearrangement. Based on these 421 findings, the patient received the combination of lorlatinib and crizotinib. Due to potential 422 overlapping toxicities, the lorlatinib dose was reduced to 50 mg when crizotinib 250 mg 423 BID was added. After two weeks on the combination, lorlatinib was escalated to 75 mg 424 daily. The patient tolerated combined therapy well with side effects of grade 1 nausea 425 and grade 1 lipid elevation. He experienced rapid symptomatic improvement, and first

426 restaging scans confirmed improvement in disease (Figure 5C). However, after 3 427 months, the patient developed progression of lung and axillary metastases (Figure 5C). 428 Biopsy of an axillary node demonstrated persistent high-level MET amplification with 429 MET/CEP7 ratio >25 (Figure 5B). 430 431 Convergent Evolution of MET Alterations During Treatment with ALK Inhibitors 432 To investigate the evolutionary origin of MET alterations, we performed whole genome 433 sequencing (WGS) on MGH915’s serial tumor specimens and patient-derived models 434 (Figure 6A, Supplementary Figure 5A,B). We first focused on the MET locus and 435 observed that the post-lorlatinib specimens (MGH915-4) harbored a focal amplification 436 (copy number 14-16) extending from the intergenic region preceding the MET gene to 437 intron 1 of ST7 (Figure 6B). This region was contained within a larger ~3 Mb 438 amplification (Supplementary Figure 5C), consistent with an initial tandem duplication 439 of the MET-ST7 locus, followed by amplification of the resulting ST7-MET and wild-type 440 MET loci (~7 copies each) (Figure 6C). The duplication placed exon 1 of ST7 22kb 441 upstream of the MET transcriptional start site, likely generating a chimeric read-through 442 fusion whereby mRNA processing causes the first exon of ST7 to be spliced to MET 443 exon 2. Neither the tandem duplication nor MET amplification were present in the pre- 444 lorlatinib (post-alectinib) pleural effusion (MGH915-3). In the post-lorlatinib/crizotinib 445 combination sample (MGH915-9), we observed an independent MET amplification event 446 that did not involve the amplified 3 Mb region housing the ST7-MET tandem duplication 447 (Figure 6B, Supplementary Figure 5C). When all non-MET mutation clusters and MET 448 alterations were assessed, there was significant overlap between mutations in the post- 449 alectinib pleural effusion (MGH915-3) and the post-lorlatinib/crizotinib axillary node 450 (MGH915-9), but MET amplification only occurred in MGH915-9 (Figure 6D). The post- 451 lorlatinib pleural effusion (MGH915-4), however, represented a distinct branch of the 452 phylogenetic tree which included >3400 private mutations in addition to MET 453 amplification and ST7-MET. Thus, reconstruction of phylogenetic relationships supports 454 convergent evolution of independent genomic events leading to MET-driven acquired 455 resistance to ALK TKIs. 456 457 DISCUSSION 458 We analyzed >200 tissue and plasma specimens to identify genetic alterations leading 459 to MET bypass signaling in resistant ALK-positive NSCLC. We detected MET

460 amplification and/or a MET fusion in 15% of tumors after failure of a next-generation ALK 461 TKI. Importantly, resistant cell line models and patients harboring MET amplification or 462 rearrangement could be re-sensitized to treatment with dual ALK/MET inhibition. 463 464 MET gene amplification was initially discovered as a bypass mechanism in EGFR- 465 mutant NSCLC in 2007 and has subsequently been identified in up to 20% of resistant 466 specimens.14,23 In this study, we demonstrate that the prevalence of MET alterations in 467 patients relapsing on ALK TKIs may be comparable to that seen in EGFR-mutant 468 NSCLC. Interestingly, the incidence of MET amplification in EGFR-mutant NSCLC is 469 higher after exposure to the third-generation EGFR TKI osimertinib compared with less 470 potent EGFR TKIs.14,15 Similarly, in our series we observed that the frequency of MET 471 amplification was highest in tumors resistant to the third generation, broad-spectrum 472 ALK TKI lorlatinib (Figure 1C). These findings suggest that an association may exist 473 between TKI potency and likelihood of developing ALK-independent resistance 474 mechanisms such as MET amplification. In addition to TKI potency, TKI selectivity may 475 also impact the evolution of resistance. In support of this hypothesis, we observed MET 476 amplification in one-third of cases that received front-line second-generation ALK TKIs 477 (which have no activity against MET) compared to less than 10% of patients initially 478 treated with crizotinib. 479 480 MET signaling can be activated through a variety of mechanisms, including copy number 481 gain, mutation, fusions, and ligand upregulation.24 In this study, we found that copy 482 number gain was most frequently used to activate MET bypass signaling. The range of 483 MET copies in tissue was broad, spanning MET/CEP7 ratios of 2.4 to greater than 25 484 (Figure 1B). In cases where MET amplification was detected in paired tissue and 485 plasma specimens, the number of MET copies in plasma was consistently lower than in 486 tissue. Tumor DNA represents a small fraction of total cell-free DNA and some disease 487 sites are more likely to shed tumor DNA than others. Thus, calculation of MET copy 488 number in plasma is challenging. The relationship between MET copy number and 489 dependency on MET bypass signaling in resistant ALK-positive NSCLC remains to be 490 determined. While NSCLCs with de novo high-level MET amplification appear to be 491 more sensitive to crizotinib than NSCLCs with low-level MET amplification, MET/CEP7 492 ratio of 4-5 is an imperfect threshold for predicting sensitivity to crizotinib and some 493 tumors with low-level amplification respond to crizotinib.20 In the context of acquired

494 resistance, the relationship between MET copies and sensitivity to MET TKIs may be 495 more complex. Specifically, in resistant ALK-positive NSCLCs incompletely inhibited by 496 ALK TKIs, it is conceivable that even low-level MET amplification may be adequate to 497 restore proliferative signals. Furthermore, intratumoral molecular heterogeneity resulting 498 from exposure to multiple lines of therapy may impact response to MET TKIs even 499 among ALK-positive tumors with high-level MET amplification. 500 501 In addition to MET amplification, we detected identical ST7-MET rearrangements in two 502 patients relapsing on next-generation ALK TKIs. Rare ST7-MET rearrangements with 503 different breakpoints have been described in gliomas and ovarian cancer, but until now 504 the pathogenicity of these fusions was unknown.25,26 MGH915’s patient-derived cell line 505 harbored both ST7-MET and MET amplification, preventing us from assessing the 506 independent contribution of each MET alteration to ALK TKI resistance. We demonstrate 507 that introducing ST7-MET into a sensitive ALK cell line is sufficient to confer resistance 508 to ALK targeted therapy. However, as engineering the cells to express the ST7-MET 509 rearrangement also increases the total expression ST7-MET, we are unable to 510 conclusively determine whether the fusion alone (in the absence of overexpression) is 511 sufficient to drive resistance. Interestingly, ST7-MET was only identified in one of 512 MGH915’s two contemporaneous biopsies, while high-level MET amplification was 513 present in both of the resistant disease sites. In MGH9284’s case, ST7-MET was 514 identified in pericardial fluid while low-level MET amplification without the rearrangement 515 was detected in pleural fluid two months later. These findings suggest that a single 516 tumor can acquire distinct resistance alterations that converge on MET activation and 517 also highlight the potential for intratumoral heterogeneity of MET genetic alterations. 518 519 In two patients with MET-driven resistance, inhibiting both ALK and MET led to clinical 520 responses. However, despite initial responses, both patients relapsed after 521 approximately 3 months. At relapse on crizotinib monotherapy, MGH939’s plasma 522 analysis demonstrated multiple crizotinib-resistant (but next-generation ALK TKI- 523 sensitive) ALK mutations, providing rationale for exploring combinations pairing more 524 potent next generation ALK TKIs with a MET TKI. Indeed, MGH915 did receive 525 combination therapy with lorlatinib and crizotinib, but still had a short-lived response. 526 Repeat biopsy after relapse on the combination revealed persistent MET amplification 527 without additional genetic alterations associated with resistance to ALK and/or MET TKIs

528 (Figure 3B, 4A). Interestingly, the MET/CEP7 ratio increased from 5.7 to >25 at relapse 529 on the lorlatinib/crizotinib combination. As there can be cell-to-cell variability in MET 530 copy number which may be difficult to capture by FISH, it is possible that there was 531 expansion of a more highly-MET amplified subpopulation under the selective pressure of 532 crizotinib. In this scenario, it is conceivable that the MET copy number increase was not 533 the primary driver of relapse in MGH915’s case. Resistant cells may have developed 534 other alterations (including non-genetic aberrations) that decreased sensitivity to dual 535 ALK/MET combination treatment. To inform the clinical development of ALK/MET TKI 536 combinations, additional studies will be required to characterize molecular determinants 537 of resistance to ALK/MET combination therapies, to determine whether sensitivity to 538 combination therapy is impacted by line of therapy, and to assess anti-tumor activity of 539 other more potent MET TKIs like capmatinib and savolitinib (Supplementary Figure 540 3D). Indeed, there is clinical rationale for exploring ALK/MET combinations besides 541 lorlatinib/crizotinib. For example, while cross-trial comparisons suggest that most 542 adverse events, including nausea/vomiting and edema, occur at comparable rates with 543 the three MET TKIs evaluated in cell lines (crizotinib, capmatinib, savolitinib),4,27,28 the 544 blood-brain barrier penetration of capmatinib is superior to crizotinib. Capmatinib may 545 therefore be a more optimal partner for lorlatinib in patients with brain metastases. 546 547 This study has several important limitations. First, due to limited tissue availability, we 548 could not perform analyses for all MET alterations in all specimens. In addition, we used 549 several assays to detect MET alterations, each with its own limitations. Second, patients 550 in our study did not consistently undergo repeat biopsy each time they relapsed on an 551 ALK TKI, precluding definitive timing of the acquisition of the MET alteration in the 552 majority of cases. Third, we limited our analysis to genetic alterations of MET in resistant 553 tumors and did not examine non-genetic mechanisms of MET activation, including 554 upregulation of MET’s ligand hepatocyte growth factor. Fourth, in the WGS analysis, 555 clonal relationships were inferred from patient-derived cell lines and xenografts and 556 biopsies obtained from a variety of sites. It is possible that patient-derived models may 557 not fully recapitulate the spectrum of subclones in the tumor and that spatial 558 heterogeneity contributed to differences in sequential biopsies. Fifth, most of the patients 559 in this study had previously received crizotinib which could have prevented expansion of 560 resistant subclones harboring MET alterations, thereby decreasing the chance of MET 561 activation emerging as a bypass pathway with subsequent next-generation ALK TKIs. As

562 more selective second-generation ALK TKIs have recently replaced crizotinib in the 563 front-line setting, the frequency of MET bypass signaling driving resistance may be 564 higher than estimated in this study. Finally, although we identified rare ST7-MET 565 rearrangements in a subset of cases, we could not fully validate the functional role of the 566 rearrangement or confirm that the rearrangement was as an independent mediator of 567 resistance due to limitations of our experimental system. Additional studies—ideally 568 using patient-derived models that do not harbor concurrent MET amplification—are 569 needed to robustly characterize these rearrangements. 570 571 In summary, by performing the most comprehensive analysis of MET alterations in ALK- 572 positive NSCLC to date, we demonstrate that MET is an important bypass mechanism in 573 tumors exposed to next-generation ALK TKIs. The detection of MET amplification in one- 574 third of patients who received next-generation ALK TKIs in the front-line setting provides 575 justification for exploring ALK/MET combinations as an initial treatment strategy for 576 advanced ALK-positive NSCLC. 577

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20. Camidge D, GA O, Clark J, et al. Crizotinib in patients with MET-amplified non-small cell lung cancer: Updated safety and efficacy findings from a phase 1 trial. In. Vol 36. Journal of Clinical Oncology2018:9062. 21. Odegaard JI, Vincent JJ, Mortimer S, et al. Validation of a Plasma-Based Comprehensive Cancer Genotyping Assay Utilizing Orthogonal Tissue- and Plasma-Based Methodologies. Clin Cancer Res. 2018;24(15):3539-3549. 22. Zheng Z, Liebers M, Zhelyazkova B, et al. Anchored multiplex PCR for targeted next-generation sequencing. Nat Med. 2014;20(12):1479-1484. 23. Engelman JA, Zejnullahu K, Mitsudomi T, et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science. 2007;316(5827):1039-1043. 24. Drilon A, Cappuzzo F, Ou SI, Camidge DR. Targeting MET in Lung Cancer: Will Expectations Finally Be MET? J Thorac Oncol. 2017;12(1):15-26. 25. Earp MA, Raghavan R, Li Q, et al. Characterization of fusion genes in common and rare epithelial ovarian cancer histologic subtypes. Oncotarget. 2017;8(29):46891-46899. 26. Ferguson SD, Zhou S, Huse JT, et al. Targetable Gene Fusions Associate With the IDH Wild-Type Astrocytic Lineage in Adult Gliomas. J Neuropathol Exp Neurol. 2018;77(6):437-442. 27. Gan HK, Millward M, Hua Y, et al. First-in-Human Phase I Study of the Selective MET Inhibitor, Savolitinib, in Patients with Advanced Solid Tumors: Safety, Pharmacokinetics, and Antitumor Activity. Clin Cancer Res. 2019;25(16):4924-4932. 28. Wolf J, Seto T, Han J-Y, et al. Capmatinib (INC280) in METΔex14-mutated advanced non-small cell lung cancer (NSCLC): Efficacy data from the phase II GEOMETRY mono-1 study. Journal of Clinical Oncology 37, no. 15_suppl(May 20, 2019) 9004-9004. .

Figure Legends Figure 1. Summary of MET Alterations in Resistant ALK-Positive NSCLC. (A) Schematic depicts number of specimens analyzed using four different assays. Pie charts are color-coded to reflect whether specimens were tissue (blue) or plasma (purple). The ALK inhibitor received immediately prior to biopsy is shown. The SNaPshot cohort includes specimens genotyped using both SNaPshot and Solid Fusion Assays. Asterisk (*) includes 28 cases analyzed by both FISH and SNaPshot/Solid Fusion Assay. One patient (MGH915) had molecular profiling of two disease sites at relapse on lorlatinib but is only counted once in the FISH cohort given identical findings at both sites. TKI: tyrosine kinase inhibitor; 2nd gen: second-generation (B) Table lists MET alterations identified in resistant specimens. *MGH075 had focal MET amplification that was too variable to estimate copy number or MET/CEP7. **Plasma testing evaluated MET mutations but did not assess for MET rearrangement. MET/CEP7: ratio of MET to centromere 7 probe; CN: copy number; FISH: fluorescence in-situ hybridization; NGS: next-generation sequencing, ex14 skip: exon 14 skipping. (C) Bar graphs illustrate frequency of MET amplification according to prior ALK inhibitors received. The p-value corresponds to the comparison of MET amplification frequency in crizotinib-naïve vs crizotinib-pretreated patients who received next-generation ALK inhibitors. 2nd-gen. ALKi: second-generation ALK inhibitor.

Figure 2. Acquired Resistance Due to ST7-MET and MET Amplification. (A) Sanger sequencing of the RT-PCR product in MGH915’s lorlatinib-resistant pleural fluid sample shows 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 determined using CellTiter-Glo. Data are mean ± s.e.m. of three biological replicates. (D) Immunoblotting to assess phosphorylation of ALK, MET, and downstream targets in 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 drug at 300 nM for 6 hours.

Figure 3. 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 without doxycycline-induced ST7-MET expression (DOX+ and DOX-, respectively) was measured after 3 days of monotherapy or combination therapy, as indicated. Viability was determined using CellTiter-Glo. Data are mean ± s.e.m. of three biological replicates. (C) Immunoblotting to assess phosphorylation of ALK, MET, and

downstream targets in cells treated with lorlatinib and the MET TKIs shown. The arrowheads at 175 kDa and 145 kDa indicate the band for pro-MET and MET beta-chain respectively. Cells were treated with each drug at 300 nM for 6 hours. DOX: doxycycline.

Figure 4. Clinical Response to Crizotinib in a Patient with ALK-Positive Lung Cancer and Acquired MET amplification: (A) Timeline illustrates treatments received and molecular profiling results from serial tissue and plasma specimens analyzed during MGH939’s disease course. NGS: next-generation sequencing; v3: EML4-ALK variant 3, or fusion of exon 6 of EML4 to exon 20 of ALK; FISH: fluorescence in-situ hybridization, qns: quantity not sufficient; AMP: amplification. (B) Positron emission computed tomography scans depict metabolic response to treatment in the chest wall and liver during treatment with crizotinib.

Figure 5. Clinical Activity of Dual ALK/MET TKIs in MET-Driven Resistance (A) Timeline of treatments and biopsies for MGH915. NGS: next-generation sequencing; n.d.: not performed; v1: EML4-ALK variant 1, or fusion of exon 13 of EML4 to exon 20 of ALK; R: right; LN: lymph node; FISH: fluorescence in-situ hybridization (B) FISH images from serial biopsies demonstrate progressive increase in MET 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 pleural fluid and lung mass (red arrow, top panels) and decrease in right axillary node (bottom panels, red arrow). The patient later relapsed at these same sites due to resistance.

Figure 6. Convergent Evolution Upon MET Activation Drives Resistance to Lorlatinib. (A) Metastatic disease sites analyzed by whole genome sequencing. (B) Copy number profiles of MET locus demonstrate amplification encompassing MET and first exon of ST7 in post-lorlatinib samples. Circos plots depicting chromosomal structure are shown on right. (C) Proposed sequence of genomic alterations leading to generation of ST7-MET fusion and amplification. The EML4-ALK rearrangement (*) and MET locus (**) are indicated. (D) Phylogenetic relationship of serial tumors samples based on shared and private mutations and MET alterations: MGH915-1 (pre-treatment 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 each branch point are indicated.

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i ERK1/2

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e 50

C p-S6 S240/244 o

25 %

( S6 0 0 0.1 1 10 100 1000 β-Actin

A Carboplatin/pemetrexed Alectinib Crizotinib

Months: 0 3 6 9 12 15 18 21

R Pleural biopsy: Lung biopsy: Chest wall biopsy: Adenocarcinoma Adenocarcinoma Adenocarcinoma NGS: EML4-ALK v3, NGS: EML4-ALK v3, NGS: EML4-ALK v3, ALK FISH (+) No ALK mutations, ALK mutations: qns MET FISH (-) MET FISH (+) MET FISH (+)

Plasma: Plasma: Plasma: EML4-ALK EML4-ALK EML4-ALK No ALK mutations No ALK mutations ALK F1174L, MET AMP MET AMP L1196M, S1206F MET AMP B Baseline Response to crizotinib

Author Manuscript Published OnlineFirst on February 21, 2020; DOI: 10.1158/1078-0432.CCR-19-3906 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2020 American Association for Cancer Research. Figure 5 Alectinib/Cobimetinib A Carboplatin/Pemetrexed/Nivolumab Alectinib/Pemetrexed Lorlatinib Ceritinib Alectinib/Bevacizumab Lorlatinib/Crizotinib

Years: 0 1 2 3 4 5 6

Lung biopsy: Lung biopsy: Lung biopsy: Pleural Effusion: R axillary LN biopsy: Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma Adenocarcinoma ALK FISH (+) NGS: EML4-ALK v1, NGS: EML4-ALK v1, NGS: EML4-ALK v1, NGS: EML4-ALK v1, MET FISH n.d. no ALK mutations no ALK mutations, no ALK mutations, no ALK mutations MET FISH n.d. MET FISH 1.0 (-) ST7-MET MET FISH >25:1 MET FISH 5.2

Pleural effusion R axillary LN biopsy: (no clinical Adenocarcinoma genotyping) NGS: EML-ALK v1, no ALK mutations MET FISH 5.7

B Progression on Progression on Progression on Progression on alectinib + bevacizumab lorlatinib (pleural fluid) lorlatinib (axillary LN) lorlatinib + crizotinib

Author Manuscript Published OnlineFirst on February 21, 2020; DOI: 10.1158/1078-0432.CCR-19-3906 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

MET/CEP7 1.0 MET/CEP7 5.2 MET/CEP7 5.7 MET/CEP7 >25 C Progression on lorlatinib Response to lorlatinib + crizotinib Progression on lorlatinib + crizotinib

MET Alterations are a Recurring and Actionable Resistance Mechanism in ALK-Positive Lung Cancer

Ibiayi Dagogo-Jack, Satoshi Yoda, Jochen K. Lennerz, et al.

Clin Cancer Res Published OnlineFirst February 21, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-19-3906

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2020/02/21/1078-0432.CCR-19-3906.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

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