met alterations are a recurring and actionable resistance mechanism in alk … · 2 40 . abstract...

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

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

1Massachusetts General Hospital Cancer Center and Department of Medicine, 9

Massachusetts General Hospital, 2Harvard Medical School, 3Department of Pathology, 10

Center for Integrated Diagnostics, Massachusetts General Hospital, 4Department of 11

Radiology, Massachusetts General Hospital, 5Department of Medicine, Weill Cornell 12

Medicine, 6Guardant Health, Inc 13

‡ Corresponding authors 14 *These authors contributed equally 15 **These authors contributed equally 16 17 Running title: MET Alterations in ALK-Positive Lung Cancer 18

Keywords: ALK, Lung Cancer, Resistance, MET Amplification 19

Figures/Tables: 6/0 20

Word Count: ~ 4500 21

Abstract Word Count: 252 words 22

Statement of Translational Relevance: 152 words 23

References: 26 24

Supplement: 5 Figures, 3 Tables 25

26

Corresponding Authors: 27

Alice T. Shaw, M.D., Ph.D. 28 Massachusetts General Hospital 29 Department of Medicine 30 32 Fruit Street 31 Boston, MA, USA, 02114 32 email: [email protected] 33 34 Aaron N. Hata, M.D., Ph.D. 35 Massachusetts General Hospital 36 Department of Medicine 37 149 13th Street, Charlestown, MA, 02129. 38 email: [email protected] 39

Research. on April 26, 2020. © 2020 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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

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ABSTRACT (252/250) 40

Background: Most ALK-positive lung cancers will develop ALK-independent resistance 41

after treatment with next-generation ALK inhibitors. MET amplification has been 42

described in patients progressing on ALK inhibitors, but frequency of this event has not 43

been comprehensively assessed. 44

Methods: We performed fluorescence in-situ hybridization and/or next-generation 45

sequencing on 207 post-treatment tissue (n=101) or plasma (n=106) specimens from 46

patients with ALK-positive lung cancer to detect MET genetic alterations. We evaluated 47

ALK inhibitor sensitivity in cell lines with MET alterations and assessed antitumor activity 48

of ALK/MET blockade in ALK-positive cell lines and two patients with MET-driven 49

resistance. 50

Results: MET amplification was detected in 15% of tumor biopsies from patients 51

relapsing on next-generation ALK inhibitors, including 12% and 22% of biopsies from 52

patients progressing on second-generation inhibitors or lorlatinib, respectively. Patients 53

treated with a second-generation ALK inhibitor in the first-line setting were more likely to 54

develop MET amplification than those who had received next-generation ALK inhibitors 55

after crizotinib (p=0.019). Two tumor specimens harbored an identical ST7-MET 56

rearrangement, one of which had concurrent MET amplification. Expressing ST7-MET in 57

the sensitive H3122 ALK-positive cell line induced resistance to ALK inhibitors that was 58

reversed with dual ALK/MET inhibition. MET inhibition re-sensitized a patient-derived cell 59

line harboring both ST7-MET and MET amplification to ALK inhibitors. Two patients with 60

ALK-positive lung cancer and acquired MET alterations achieved rapid responses to 61

ALK/MET combination therapy. 62

Conclusions: Treatment with next-generation ALK inhibitors, particularly in the first-line 63

setting, may select for MET-driven resistance. Patients with acquired MET alterations 64

may derive clinical benefit from therapies that target both ALK and MET. 65

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STATEMENT OF TRANSLATIONAL RELEVANCE 67

With the development of highly potent and selective next-generation ALK inhibitors, 68

resistance is increasingly mediated by ALK-independent mechanisms. Here through 69

molecular profiling of >200 resistant tissue and plasma specimens, including the largest 70

dataset of post-lorlatinib specimens to date, we identified MET amplification in 15% of 71

tumor biopsies from patients relapsing on next-generation ALK inhibitors and detected 72

rare, novel ST7-MET rearrangements in two cases. Upregulation of MET signaling 73

through MET amplification and/or fusion of MET with ST7 decreased sensitivity of ALK-74

positive cells to ALK inhibitors. In cell lines, combined blockade of ALK and MET 75

overcame resistance driven by MET bypass signaling. Consistent with these findings, 76

two patients with ALK-positive lung cancer and acquired MET alterations achieved rapid 77

clinical and radiographic responses to ALK/MET combination therapy. Our study 78

demonstrates that MET bypass signaling is a recurring and actionable mechanism of 79

resistance to ALK inhibitors, providing rationale for pursuing ALK/MET combination 80

therapy in the clinic. 81

82

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CONFLICTS OF INTEREST/DISCLOSURES: 84 85 IDJ has received honoraria from Foundation Medicine, consulting fees from Boehringer 86

Ingelheim, research support from Array, Genentech, Novartis, Pfizer, and Guardant 87

Health, and travel support from Array and Pfizer. JJL has received honoraria or served 88

as a compensated consultant for Chugai, Boehringer Ingelheim, and Pfizer; institutional 89

research support from Loxo Oncology and Novartis; and CME honorarium from OncLive. 90

RJN is an employee of Guardant Health. SRD provides independent image analysis 91

through the institution for clinical research trials sponsored by Merck, Pfizer, Bristol 92

Myers Squibb, Novartis, Roche, Polaris, Cascadian, Abbvie, Gradalis, Bayer, Zai 93

Laboratories; and has received honoraria from Siemens Medical Solutions. AS has 94

served on advisory boards for AstraZeneca and Takeda and as a consultant for 95

Genentech, AstraZeneca, and Medtronic. JFG has served as a compensated consultant 96

or received honoraria from Bristol-Myers Squibb, Genentech, Ariad/Takeda, Loxo, 97

Pfizer, Incyte, Novartis, Merck, Agios, Amgen, Jounce, Regeneron, Oncorus, Helsinn, 98

Jounce, Array, and Clovis Oncology, has an immediate family member who is an 99

employee of Ironwood Pharmaceuticals, has received research funding from Novartis, 100

Genentech/Roche, and Ariad/Takeda, and institutional research support from Tesaro, 101

Moderna, Blueprint, BMS, Jounce, Array, Adaptimmune, Novartis, Genentech/Roche, 102

Alexo and Merck. ATS has served as a compensated consultant or received honoraria 103

from Achilles, Archer, ARIAD, Bayer, Blueprint Medicines, Chugai, Daiichi Sankyo, EMD 104

Serono, Foundation Medicine, Genentech/Roche, Guardant, Ignyta, KSQ Therapeutics, 105

LOXO, Natera, Novartis, Pfizer, Servier, Syros, Taiho Pharmaceutical, Takeda, and TP 106

Therapeutics, has received research (institutional) funding from Daiichi Sankyo, Ignyta, 107

Novartis, Pfizer, Roche/Genentech, and TP Therapeutics; has received travel support 108

from Pfizer and Genentech/Roche; and is an employee of Novartis. ANH has received 109

research grants/funding from Pfizer, Novartis, Amgen, Eli Lilly, Relay Therapeutics, and 110

Roche/Genentech. The remaining authors have no disclosures to report. 111

112

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114

FUNDING: 115 This work was support by a Conquer Cancer/Amgen Young Investigator Award (I.D.J), 116

an institutional research grant from American Cancer Society (I.D.J.), a National Cancer 117

Institute Career Development Award (K12CA087723-16 to I.D.J.), a Lung Cancer 118

Research Foundation Annual Grant Program award (S.Y.), a Conquer 119

Cancer/AstraZeneca Young Investigator Award (J.J.L.), grants from the National Cancer 120

Institute (R01CA164273 to A.T.S. and R01CA225655 to J.K.L.), by Be a Piece of the 121

Solution, and by Targeting a Cure for Lung Cancer Research Fund at MGH. 122

123

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INTRODUCTION 125

ALK-rearranged (i.e., ALK-positive) non-small cell lung cancers (NSCLC) depend on 126

constitutive ALK signaling for growth and survival.1 This oncogene dependency underlies 127

the marked sensitivity of these tumors to tyrosine kinase inhibitors (TKIs) targeting 128

ALK.2,3 Historically, the ALK/ROS1/MET TKI crizotinib was the standard first-line therapy 129

for advanced ALK-positive NSCLC.4 However, crizotinib has recently been supplanted 130

by more potent and selective second-generation ALK TKIs (e.g., ceritinib, alectinib, 131

brigatinib).2,3,5 In addition, the third-generation ALK TKI lorlatinib was recently approved 132

for patients previously treated with a second-generation TKI.6 133

134

Despite initial sensitivity to ALK TKIs, ALK-positive tumors invariably develop resistance. 135

In approximately one-half of cases, resistance to second-generation ALK TKIs is due to 136

secondary mutations in the ALK kinase domain.7 These tumors remain ALK-dependent 137

and responsive to treatment with lorlatinib.8 The remaining cases have presumably 138

developed ALK-independent resistance mechanisms, most often due to activation of 139

alternative or bypass signaling pathways.7,9 Recent studies of lorlatinib resistance 140

suggest an even more complicated array of resistance mechanisms, including diverse 141

compound ALK mutations in one-third of patients and ALK-independent resistance 142

mechanisms in the remaining two-thirds of patients.9,10 143

144

To date, ALK-independent resistance mechanisms remain poorly characterized. In 145

preclinical studies, multiple bypass mechanisms have been described, including 146

activation of MET, EGFR, SRC, and IGF-1R.11-13. Of these, MET is a particularly 147

attractive target given the availability of potent MET TKIs. In addition, MET activation has 148

been previously studied as a bypass pathway in EGFR-mutant NSCLC where MET 149

amplification has been reported in 5-20% of resistant cases.14,15 In phase I/II studies, co-150

targeting EGFR and MET can re-induce responses in resistant EGFR-mutant tumors 151

with MET amplification.16 In contrast to EGFR-mutant NSCLC, relatively little is known 152

about the contribution of aberrant MET activation to resistance in ALK-positive NSCLC. 153

Several case reports suggest that MET amplification can mediate resistance to ALK 154

TKIs and that the ALK/ROS1/MET TKI crizotinib may be able to overcome MET-driven 155

resistance.17,18 However, larger studies are needed to validate MET as a clinically 156

relevant driver of resistance in ALK-positive NSCLC. 157

158




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Here we analyze tumor and/or blood specimens from 136 patients with ALK-positive 159

NSCLC relapsing on ALK TKIs. We identify genetic alterations of MET in approximately 160

15% of resistant cases. In cell lines and in patients, MET activation drives resistance to 161

ALK TKIs but confers sensitivity to dual ALK/MET blockade. These studies support the 162

development of novel combinations targeting ALK and MET in ALK-positive NSCLC. 163

164

METHODS 165

Data Collection 166

Resistant specimens were obtained from 136 patients with metastatic ALK-positive 167

NSCLC who underwent molecular profiling between 2014 and 2019 (Figure 1A). 168

Medical records from these patients were retrospectively reviewed to extract data on 169

demographics, treatment histories and molecular profiling results. Data were updated as 170

of August 31, 2019. This study was approved by the Massachusetts General Hospital 171

Institutional Review Board. Patient studies were conducted according to the Declaration 172

of Helsinki, the Belmont Report, and the U.S. Common Rule. 173

174

Molecular Testing 175

MET copy number was quantified in tissue using FISH or the FoundationOne assay as 176

previously described.19 For cases assessed by FISH, amplification was defined as a 177

ratio of MET to centromere 7 (MET/CEP7) of ≥ 2.2.20 Cases with MET/CEP7 ≥ 4 were 178

classified as high-level amplification.20 For tumors genotyped with the FoundationOne 179

assay, MET amplification was defined as ≥ 6 gene copies.19 With the Guardant360 180

assay, MET amplification was defined as absolute plasma copy number ≥ 2.1.21 As MET 181

copy number analysis based on MGH SNaPshot NGS has not been formally validated, 182

this assay was not used to detect MET amplification. MET rearrangements and 183

mutations and ALK mutations were detected in tissue using either FoundationOne or the 184

MGH SNaPshot/Solid Fusion Assays.19,22 Guardant360 was used to identify MET and 185

ALK mutations in plasma, but is not designed to detect MET rearrangements.21 All 186

patients included in this study provided written informed consent for molecular analysis. 187

188

Cell Lines 189

Using methods previously described,13 we generated cell lines from pleural fluid 190

collected from MGH915 prior to lorlatinib (MGH915-3) and from a xenograft of pleural 191

fluid at relapse on lorlatinib (MGH915-4). The cell lines were sequenced to confirm the 192




8

presence of ALK rearrangement, MET rearrangement, and MET amplification. The ST7-193

MET fusion transcript was confirmed by sequencing a 700 bp region spanning the fusion 194

breakpoint in cDNA generated from RNA extracted from MGH915-4. The corresponding 195

ST7-MET fusion sequence was constructed by fusing ST7 exon 1 (NM_021908.3) to 196

exons 2-21 of MET (NM_000245.4) which includes the full-length MET coding sequence. 197

This resulting fusion sequence incorporates the ST7 ATG start codon corresponding to 198

position 41 exon 1 followed by 151 bp of ST7 exon 1 and 14 bp of MET exon 2 5’ to the 199

wild-type MET ATG start codon. Wild-type MET sequence was obtained from an ORF 200

cDNA clone (GenScript OHu28578, NM_001127500.2). The sequence was cloned into 201

pEN TTmcs (Addgene #25755), recombined with pSLIK-Neo (Addgene #25735) by 202

using Gateway LR Clonase Enzyme (Invitrogen), and lentiviral particles were generated 203

in HEK293FT cells by transfection with ViraPower packaging Mix (Invitrogen) following 204

the manufacturers' instructions. H3122 cells were transduced by lentiviral infection and 205

selected with G418 (Gibco) for 5 days. The expression of ST7-MET was induced by 206

doxycycline followed by addition of lorlatinib to select the ST7-MET-expressing resistant 207

population. After selection, cells were maintained with continuous culture in 1 μg/mL of 208

doxycycline and 300 nM of lorlatinib. For drug sensitivity assays using DOX- controls, 209

doxycycline was removed 3 days prior to the experiment to allow for ST7-MET 210

expression levels to return to baseline. 211

212

Drug Sensitivity Assays, Cell Proliferation Assays, and Western Blot Analysis 213

2,000 cells were plated in triplicate into 96-well plates. Viability was determined by 214

CellTiter-Glo (Promega) three days after drug treatment in drug sensitivity assays. 215

Luminescence was measured with a SpectraMax M5 Multi-Mode Microplate Reader 216

(Molecular Devices). GraphPad Prism (GraphPad Software) was used to analyze data. 217

For Western blotting, a total of 0.25–2x106 cells was treated in 6-well plates for 6 hours. 218

Equal amounts of total cell lysate were processed for immunoblotting. 219

Whole Genome Sequencing 220

Whole genome sequencing was performed as described in the Supplementary Methods. 221

222

Statistical Analysis 223




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Fisher’s exact test was used to compare MET amplification frequency between 224

treatment groups. All p-values were based on a two-sided hypothesis and computed 225

using Stata 12.1. 226

227

RESULTS 228

229

Study Population 230

To survey the landscape of molecular alterations associated with resistance to ALK 231

TKIs, we analyzed 207 tissue (n=101) and plasma (n=106) specimens from 136 patients 232

with ALK-positive NSCLC relapsing on ALK TKIs (Figure 1A). Four assays were used 233

for the analysis (see Methods), including two tissue-based next-generation sequencing 234

(NGS) assays (FoundationOne and MGH SNaPshot/Solid Fusion Assay),19,22 a plasma 235

NGS assay (Guardant360),21 and fluorescence in-situ hybridization (FISH) for MET 236

amplification (Supplementary Table 1). 237

238

The clinicopathologic characteristics of the study patients were consistent with an ALK-239

positive NSCLC population (Supplementary Table 2). Of the 207 specimens, twelve 240

(6%) were from patients relapsing on crizotinib who had not been exposed to other ALK 241

TKIs. The majority of specimens (n=136/207, 66%) were obtained at progression on a 242

second-generation ALK TKI. The remaining fifty-nine (29%) specimens were collected 243

from patients relapsing on lorlatinib. Overall, ALK kinase domain mutations were 244

detected in 34 (47%) of 73 tissue biopsies genotyped by NGS, including 25/46 (54%) 245

specimens obtained after second-generation TKIs and 9/25 (36%) post-lorlatinib 246

biopsies (Supplementary Figure 1). These results suggest that a significant fraction of 247

resistant cases lack secondary ALK mutations and likely harbor ALK-independent 248

mechanisms of resistance. 249

250

Genetic Alterations of MET 251

MET Amplification in Tissue Biopsies 252

To evaluate the role of MET as a resistance mechanism, we first assessed MET copy 253

number in 86 tumor biopsies using FISH or the FoundationOne assay (Figure 1A, 254

Supplementary Table 1). Eleven (13%) biopsies harbored MET amplification (Figure 255

1B), including four with low-level MET amplification (MET/CEP7 2.4-3.9) and six with 256

high-level MET amplification based on FISH (MET/CEP7 5.2 to >25) or NGS (16-19 257




10

MET copies). One sample had focal MET amplification by NGS, and FISH was too 258

variable to estimate copy number. In 3 of 11 cases, there was available tissue to assess 259

MET copy number in a biopsy from an earlier timepoint, none of which had MET 260

amplification, supporting the notion that MET amplification was newly acquired. No co-261

alterations in other genes potentially associated with resistance or bypass signaling were 262

identified in the 11 biopsies. In addition, MET amplification was mutually exclusive with 263

ALK resistance mutations, with the exception of one case, MGH9226 (Figure 1B, 264

Supplementary Figure 1). This patient developed ALK I1171N (without MET 265

amplification) in plasma after relapsing on first-line alectinib, and then responded to 266

second-line brigatinib for 5.5 months. After relapsing on brigatinib, tumor biopsy 267

demonstrated persistence of ALK I1171N and acquisition of MET amplification, 268

suggesting that MET was driving resistance. 269

270

All 11 cases with MET amplification were patients relapsing on second- or third-271

generation ALK TKIs (Figure 1B). MET amplification was identified in six (12%) of 52 272

biopsies taken after a second-generation ALK TKI and in five (22%) of 23 post-lorlatinib 273

biopsies. MET amplification was not detected in patients relapsing on crizotinib. In 274

addition, six (55%) of the 11 positive cases had never received crizotinib (Figure 1B,C). 275

To determine whether the development of MET amplification may be impacted by prior 276

crizotinib exposure, we evaluated the frequency of MET amplification according to prior 277

TKI therapy. Tumors from patients previously treated with crizotinib followed by next-278

generation TKI(s) were significantly less likely to harbor MET amplification than those 279

from patients treated only with next-generation TKI(s) (9% vs 33%, p=0.019, Figure 1C). 280

Thus, prior exposure to crizotinib, an ALK inhibitor with documented MET activity, may 281

have suppressed emergence of MET-amplified clones in these patients. 282

283

MET Copy Number Gain by Plasma Genotyping 284

We then analyzed MET copy number in 106 plasma specimens from patients relapsing 285

on next-generation ALK TKIs using the Guardant360 assay (Figure 1A). MET copy 286

number gain was detected in eight (7.5%) plasma specimens (Supplementary Figure 287

2), one of which was due to polysomy of chromosome 7. The seven cases with focal 288

amplification of MET included two (3%) of 77 plasma specimens from patients relapsing 289

on a second-generation ALK TKI and five (17%) of 29 post-lorlatinib specimens (Figure 290

1B). Among 23 patients with contemporaneous plasma and tissue specimens, MET 291




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amplification was detected in both plasma and tissue in four cases (Supplementary 292

Table 3). Eighteen pairs demonstrated absence of MET amplification. One patient, 293

MGH960, had low-level MET amplification (2.2 copies) in plasma which was not 294

detected in a contemporaneous biopsy. Thus, when tissue was used as the reference, 295

plasma genotyping demonstrated 100% sensitivity, 95% specificity, and 80% positive 296

predictive value for detecting MET amplification. The remaining two patients with high-297

level MET amplification (4.9-6.1 copies) in plasma did not have a paired tumor biopsy. 298

299

Four of the seven specimens with focal MET amplification harbored both an ALK 300

mutation and MET amplification in plasma (Supplementary Figure 2), including 301

MGH9226, as described above. Apart from MGH9226’s ALK I1171N mutation, the 302

remaining three plasma specimens contained the gatekeeper ALK L1196M mutation. 303

ALK L1196M is known to cause resistance to crizotinib but is sensitive to next-304

generation ALK TKIs.7 Indeed, all three patients had received crizotinib prior to the next-305

generation ALK TKI(s) and presumably developed this secondary mutation after 306

crizotinib exposure. The known activity of next-generation ALK TKIs against ALK 307

L1196M suggests that MET amplification rather than the ALK mutation was likely 308

mediating drug resistance. In addition to MET amplification (6.1 copies), MGH9224’s 309

plasma demonstrated KRAS amplification (2.6 copies) and a PIK3CA E545K mutation 310

(allelic frequency 0.04%) which may have also contributed to resistance. The remaining 311

plasma specimens did not contain other putative drivers of resistance. 312

313

Other Genetic Alterations of MET 314

We next analyzed resistant specimens for other genetic alterations that could lead to 315

aberrant MET activation. To identify MET mutations, we reviewed molecular profiling 316

results from 179 tissue and plasma specimens genotyped by MGH SNaPshot/Solid 317

Fusion NGS (n= 43), FoundationOne (n=30), or Guardant360 (n=106) (Supplementary 318

Figures 1,2). One plasma specimen from a patient relapsing after sequential alectinib 319

and brigatinib harbored MET exon 14 skipping without MET amplification (Figure 1B). 320

Two additional plasma specimens contained MET mutations (D1101H and G500C, 321

Supplementary Figure 2) of unknown significance. MET mutations were not detected in 322

any of the tissue specimens. 323

324




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In two (3%) of 73 tissue specimens, we detected a rearrangement fusing exons 2-21 of 325

MET with exon 1 of ST7 (suppressor of tumorigenicity 7), both of which reside in close 326

proximity on chromosome 7q (Figure 2A). Both cases were detected using an RNA-327

based NGS assay (MGH Solid Fusion Assay).22 In the first case, the ST7-MET fusion 328

was seen in MGH9284’s pericardial fluid at progression on first-line alectinib without 329

concurrent MET amplification. The patient had primary progression on second-line 330

lorlatinib at which time molecular profiling of pleural fluid did not demonstrate the 331

rearrangement but revealed low-level MET amplification (MET/CEP7 2.4, Figure 1B). In 332

the second case, concomitant ST7-MET and high-level MET amplification were detected 333

in MGH915’s pleural fluid after failure of lorlatinib (Figure 1B), neither of which were 334

present prior to lorlatinib. Interestingly, contemporaneous biopsy of an axillary node 335

showed only high-level MET amplification and no detectable ST7-MET. 336

337

Functional Validation of ST7-MET as a Targetable Mechanism of Resistance 338

To evaluate the functional role of MET in driving resistance to ALK TKIs, we established 339

cell lines from MGH915’s pre-lorlatinib and post-lorlatinib malignant pleural effusion 340

(MGH915-3 and MGH915-4, respectively). Consistent with the pleural fluid analysis, the 341

MGH915-4 cell line harbored both MET amplification (MET/chromosome 7 ratio 4.2) and 342

the ST7-MET rearrangement, but MGH915-3 did not (Supplementary Figure 3A, B). 343

High MET expression was confirmed in MGH915-4 cell line at the mRNA and protein 344

level (Supplementary Figure 3B, C). In cell viability studies, the MGH915-4 cell line 345

was resistant to second- and third-generation ALK TKIs, none of which have MET 346

activity, but was sensitive to crizotinib (Figure 2B). Treatment with MET-specific TKIs 347

capmatinib or savolitinib, none of which have ALK activity, partially suppressed 348

proliferation (Supplementary Figure 3D). However, the combination of the ALK-349

selective TKI lorlatinib with capmatinib, savolitinib, or crizotinib potently suppressed cell 350

proliferation (Figure 2C, Supplementary Figure 3D). Consistent with the cell growth 351

assays, lorlatinib monotherapy potently inhibited ALK phosphorylation in immunoblotting 352

studies, but failed to inhibit downstream ERK or PI3K/AKT signaling (Figure 2D). Only 353

dual inhibition of ALK and MET by crizotinib or by the combination of lorlatinib plus a 354

MET TKI effectively suppressed both ALK and downstream signaling pathways (Figure 355

2D, Supplementary Figure 3E). 356

357




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As MGH915-4 harbors the ST7-MET rearrangement, we next evaluated whether ST7-358

MET rearrangement could drive resistance to ALK inhibition. The sensitive ALK-positive 359

cell line H3122 was transduced with lentivirus expressing a TET-inducible ST7-MET 360

construct corresponding to the ST7-MET fusion identified in MGH915-4 (Figure 2A, 361

Supplementary Figure 3C). Whereas the H3122 cells without expression of ST7-MET 362

were highly sensitive to lorlatinib and other ALK TKIs, doxycycline-induced expression of 363

ST7-MET in H3122 cells caused resistance to all ALK TKIs except crizotinib (Figure 364

3A). Treatment with MET TKIs restored sensitivity to lorlatinib (Figure 3B). Similarly, 365

treatment with a selective MET TKI alone did not suppress proliferation of H3122 cells 366

expressing ST7-MET, but the addition of lorlatinib to the MET TKIs resensitized the cells 367

to treatment. (Figure 3B). The combination of lorlatinib plus crizotinib had greater 368

antiproliferative effect than crizotinib alone, likely due to more potent inhibition of ALK 369

(Figure 3B). 370

371

We next examined the effect of ST7-MET expression on ALK and MET signaling 372

pathways. In the absence of doxycycline induction of ST7-MET, we observed very little 373

MET phosphorylation, and suppression of ALK phosphorylation with ALK TKIs was 374

sufficient to inhibit downstream MAPK and PI3K signaling (Figure 3C). Upon 375

doxycycline-induced expression of ST7-MET, we observed increased phospho-MET, 376

which could be suppressed with MET TKIs. Consistent with the effects on cell viability, 377

the combination of MET plus ALK inhibitor, but not either agent alone, was sufficient to 378

inhibit downstream signaling (Figure 3C). Of note, we were unable to distinguish 379

between ST7-MET and endogenous MET by western blot due to their similar sizes and 380

lack of suitable antibodies capable of detecting ST7 exon 1. However, upon doxycycline 381

induction of ST7-MET, we detected an increase in two bands corresponding to immature 382

pro-MET (175 kDa) and the mature MET beta-chain (145 kDa), with an increase in 383

phosphorylation of mature MET only after doxycycline induction (Figure 3C). To 384

compare the functional effect of ST7-MET relative to amplification of wild-type MET, we 385

also generated H3122 cells overexpressing wild-type MET. Similar to ST7-MET, 386

doxycycline-induced expression of MET caused resistance to all ALK inhibitors except 387

crizotinib, and the combination of ALK and MET inhibitors suppressed downstream 388

signaling and cell proliferation (Supplementary Figure 4). In our overexpression 389

system, the effect of ST7-MET and wild-type MET were comparable. While the nature of 390

our experimental system precludes the ability to discriminate between increased MET 391




14

activity resulting from a hyper-functional ST7-MET protein or increased MET expression 392

level, these studies mimic the MGH915-4 clinical scenario and suggest that resistance 393

resulting from increased expression of ST7-MET is potentially targetable. 394

395

Clinical Activity of Combined ALK/MET Inhibition in MET-Driven Resistance 396

Crizotinib Monotherapy 397

Based on our preclinical findings, we treated two resistant ALK-positive patients with 398

combination ALK/MET TKIs. The first patient, MGH939, received first-line alectinib and 399

relapsed after 9 months (Figure 4A). Biopsy of a progressing lung lesion demonstrated 400

no ALK mutations. However, high-level MET amplification with MET/CEP7 >25 was 401

identified (Figure 1B). Similarly, plasma testing showed no detectable ALK mutations, 402

but evidence of MET amplification (2.5 copies). The patient was treated with 403

carboplatin/pemetrexed for 5 cycles and then transitioned to crizotinib after disease 404

progression. Scans performed five weeks after initiating crizotinib demonstrated 405

significant response to therapy (Figure 4B). However, the patient relapsed 10 weeks 406

later. Biopsy of a chest wall mass confirmed persistent MET amplification. There was 407

insufficient tissue for NGS, but plasma testing revealed multiple ALK resistance 408

mutations that were not detected pre-crizotinib/post-alectinib, in addition to the known 409

MET amplification. These findings suggest that while crizotinib is active against both 410

ALK and MET, its activity may ultimately be limited by secondary ALK mutations. 411

412

Combination Lorlatinib plus Crizotinib 413

The second patient, MGH915, was treated with first-line ceritinib with response lasting 414

31 months. Subsequent therapies included alectinib-based combinations, chemotherapy 415

plus immunotherapy, chemotherapy with alectinib, and lorlatinib (Figure 5A). As noted 416

previously, at the time of relapse on lorlatinib, analysis of pleural fluid demonstrated 417

ST7-MET and high-level MET amplification (MET/CEP7 ratio 5.2, Figure 5B). A 418

concurrent biopsy of a growing axillary node also revealed MET amplification 419

(MET/CEP7 ratio 5.7) but did not demonstrate the MET rearrangement. Based on these 420

findings, the patient received the combination of lorlatinib and crizotinib. Due to potential 421

overlapping toxicities, the lorlatinib dose was reduced to 50 mg when crizotinib 250 mg 422

BID was added. After two weeks on the combination, lorlatinib was escalated to 75 mg 423

daily. The patient tolerated combined therapy well with side effects of grade 1 nausea 424

and grade 1 lipid elevation. He experienced rapid symptomatic improvement, and first 425




15

restaging scans confirmed improvement in disease (Figure 5C). However, after 3 426

months, the patient developed progression of lung and axillary metastases (Figure 5C). 427

Biopsy of an axillary node demonstrated persistent high-level MET amplification with 428

MET/CEP7 ratio >25 (Figure 5B). 429

430

Convergent Evolution of MET Alterations During Treatment with ALK Inhibitors 431

To investigate the evolutionary origin of MET alterations, we performed whole genome 432

sequencing (WGS) on MGH915’s serial tumor specimens and patient-derived models 433

(Figure 6A, Supplementary Figure 5A,B). We first focused on the MET locus and 434

observed that the post-lorlatinib specimens (MGH915-4) harbored a focal amplification 435

(copy number 14-16) extending from the intergenic region preceding the MET gene to 436

intron 1 of ST7 (Figure 6B). This region was contained within a larger ~3 Mb 437

amplification (Supplementary Figure 5C), consistent with an initial tandem duplication 438

of the MET-ST7 locus, followed by amplification of the resulting ST7-MET and wild-type 439

MET loci (~7 copies each) (Figure 6C). The duplication placed exon 1 of ST7 22kb 440

upstream of the MET transcriptional start site, likely generating a chimeric read-through 441

fusion whereby mRNA processing causes the first exon of ST7 to be spliced to MET 442

exon 2. Neither the tandem duplication nor MET amplification were present in the pre-443

lorlatinib (post-alectinib) pleural effusion (MGH915-3). In the post-lorlatinib/crizotinib 444

combination sample (MGH915-9), we observed an independent MET amplification event 445

that did not involve the amplified 3 Mb region housing the ST7-MET tandem duplication 446

(Figure 6B, Supplementary Figure 5C). When all non-MET mutation clusters and MET 447

alterations were assessed, there was significant overlap between mutations in the post-448

alectinib pleural effusion (MGH915-3) and the post-lorlatinib/crizotinib axillary node 449

(MGH915-9), but MET amplification only occurred in MGH915-9 (Figure 6D). The post-450

lorlatinib pleural effusion (MGH915-4), however, represented a distinct branch of the 451

phylogenetic tree which included >3400 private mutations in addition to MET 452

amplification and ST7-MET. Thus, reconstruction of phylogenetic relationships supports 453

convergent evolution of independent genomic events leading to MET-driven acquired 454

resistance to ALK TKIs. 455

456

DISCUSSION 457

We analyzed >200 tissue and plasma specimens to identify genetic alterations leading 458

to MET bypass signaling in resistant ALK-positive NSCLC. We detected MET 459




16

amplification and/or a MET fusion in 15% of tumors after failure of a next-generation ALK 460

TKI. Importantly, resistant cell line models and patients harboring MET amplification or 461

rearrangement could be re-sensitized to treatment with dual ALK/MET inhibition. 462

463

MET gene amplification was initially discovered as a bypass mechanism in EGFR-464

mutant NSCLC in 2007 and has subsequently been identified in up to 20% of resistant 465

specimens.14,23 In this study, we demonstrate that the prevalence of MET alterations in 466

patients relapsing on ALK TKIs may be comparable to that seen in EGFR-mutant 467

NSCLC. Interestingly, the incidence of MET amplification in EGFR-mutant NSCLC is 468

higher after exposure to the third-generation EGFR TKI osimertinib compared with less 469

potent EGFR TKIs.14,15 Similarly, in our series we observed that the frequency of MET 470

amplification was highest in tumors resistant to the third generation, broad-spectrum 471

ALK TKI lorlatinib (Figure 1C). These findings suggest that an association may exist 472

between TKI potency and likelihood of developing ALK-independent resistance 473

mechanisms such as MET amplification. In addition to TKI potency, TKI selectivity may 474

also impact the evolution of resistance. In support of this hypothesis, we observed MET 475

amplification in one-third of cases that received front-line second-generation ALK TKIs 476

(which have no activity against MET) compared to less than 10% of patients initially 477

treated with crizotinib. 478

479

MET signaling can be activated through a variety of mechanisms, including copy number 480

gain, mutation, fusions, and ligand upregulation.24 In this study, we found that copy 481

number gain was most frequently used to activate MET bypass signaling. The range of 482

MET copies in tissue was broad, spanning MET/CEP7 ratios of 2.4 to greater than 25 483

(Figure 1B). In cases where MET amplification was detected in paired tissue and 484

plasma specimens, the number of MET copies in plasma was consistently lower than in 485

tissue. Tumor DNA represents a small fraction of total cell-free DNA and some disease 486

sites are more likely to shed tumor DNA than others. Thus, calculation of MET copy 487

number in plasma is challenging. The relationship between MET copy number and 488

dependency on MET bypass signaling in resistant ALK-positive NSCLC remains to be 489

determined. While NSCLCs with de novo high-level MET amplification appear to be 490

more sensitive to crizotinib than NSCLCs with low-level MET amplification, MET/CEP7 491

ratio of 4-5 is an imperfect threshold for predicting sensitivity to crizotinib and some 492

tumors with low-level amplification respond to crizotinib.20 In the context of acquired 493




17

resistance, the relationship between MET copies and sensitivity to MET TKIs may be 494

more complex. Specifically, in resistant ALK-positive NSCLCs incompletely inhibited by 495

ALK TKIs, it is conceivable that even low-level MET amplification may be adequate to 496

restore proliferative signals. Furthermore, intratumoral molecular heterogeneity resulting 497

from exposure to multiple lines of therapy may impact response to MET TKIs even 498

among ALK-positive tumors with high-level MET amplification. 499

500

In addition to MET amplification, we detected identical ST7-MET rearrangements in two 501

patients relapsing on next-generation ALK TKIs. Rare ST7-MET rearrangements with 502

different breakpoints have been described in gliomas and ovarian cancer, but until now 503

the pathogenicity of these fusions was unknown.25,26 MGH915’s patient-derived cell line 504

harbored both ST7-MET and MET amplification, preventing us from assessing the 505

independent contribution of each MET alteration to ALK TKI resistance. We demonstrate 506

that introducing ST7-MET into a sensitive ALK cell line is sufficient to confer resistance 507

to ALK targeted therapy. However, as engineering the cells to express the ST7-MET 508

rearrangement also increases the total expression ST7-MET, we are unable to 509

conclusively determine whether the fusion alone (in the absence of overexpression) is 510

sufficient to drive resistance. Interestingly, ST7-MET was only identified in one of 511

MGH915’s two contemporaneous biopsies, while high-level MET amplification was 512

present in both of the resistant disease sites. In MGH9284’s case, ST7-MET was 513

identified in pericardial fluid while low-level MET amplification without the rearrangement 514

was detected in pleural fluid two months later. These findings suggest that a single 515

tumor can acquire distinct resistance alterations that converge on MET activation and 516

also highlight the potential for intratumoral heterogeneity of MET genetic alterations. 517

518

In two patients with MET-driven resistance, inhibiting both ALK and MET led to clinical 519

responses. However, despite initial responses, both patients relapsed after 520

approximately 3 months. At relapse on crizotinib monotherapy, MGH939’s plasma 521

analysis demonstrated multiple crizotinib-resistant (but next-generation ALK TKI-522

sensitive) ALK mutations, providing rationale for exploring combinations pairing more 523

potent next generation ALK TKIs with a MET TKI. Indeed, MGH915 did receive 524

combination therapy with lorlatinib and crizotinib, but still had a short-lived response. 525

Repeat biopsy after relapse on the combination revealed persistent MET amplification 526

without additional genetic alterations associated with resistance to ALK and/or MET TKIs 527




18

(Figure 3B, 4A). Interestingly, the MET/CEP7 ratio increased from 5.7 to >25 at relapse 528

on the lorlatinib/crizotinib combination. As there can be cell-to-cell variability in MET 529

copy number which may be difficult to capture by FISH, it is possible that there was 530

expansion of a more highly-MET amplified subpopulation under the selective pressure of 531

crizotinib. In this scenario, it is conceivable that the MET copy number increase was not 532

the primary driver of relapse in MGH915’s case. Resistant cells may have developed 533

other alterations (including non-genetic aberrations) that decreased sensitivity to dual 534

ALK/MET combination treatment. To inform the clinical development of ALK/MET TKI 535

combinations, additional studies will be required to characterize molecular determinants 536

of resistance to ALK/MET combination therapies, to determine whether sensitivity to 537

combination therapy is impacted by line of therapy, and to assess anti-tumor activity of 538

other more potent MET TKIs like capmatinib and savolitinib (Supplementary Figure 539

3D). Indeed, there is clinical rationale for exploring ALK/MET combinations besides 540

lorlatinib/crizotinib. For example, while cross-trial comparisons suggest that most 541

adverse events, including nausea/vomiting and edema, occur at comparable rates with 542

the three MET TKIs evaluated in cell lines (crizotinib, capmatinib, savolitinib),4,27,28 the 543

blood-brain barrier penetration of capmatinib is superior to crizotinib. Capmatinib may 544

therefore be a more optimal partner for lorlatinib in patients with brain metastases. 545

546

This study has several important limitations. First, due to limited tissue availability, we 547

could not perform analyses for all MET alterations in all specimens. In addition, we used 548

several assays to detect MET alterations, each with its own limitations. Second, patients 549

in our study did not consistently undergo repeat biopsy each time they relapsed on an 550

ALK TKI, precluding definitive timing of the acquisition of the MET alteration in the 551

majority of cases. Third, we limited our analysis to genetic alterations of MET in resistant 552

tumors and did not examine non-genetic mechanisms of MET activation, including 553

upregulation of MET’s ligand hepatocyte growth factor. Fourth, in the WGS analysis, 554

clonal relationships were inferred from patient-derived cell lines and xenografts and 555

biopsies obtained from a variety of sites. It is possible that patient-derived models may 556

not fully recapitulate the spectrum of subclones in the tumor and that spatial 557

heterogeneity contributed to differences in sequential biopsies. Fifth, most of the patients 558

in this study had previously received crizotinib which could have prevented expansion of 559

resistant subclones harboring MET alterations, thereby decreasing the chance of MET 560

activation emerging as a bypass pathway with subsequent next-generation ALK TKIs. As 561




19

more selective second-generation ALK TKIs have recently replaced crizotinib in the 562

front-line setting, the frequency of MET bypass signaling driving resistance may be 563

higher than estimated in this study. Finally, although we identified rare ST7-MET 564

rearrangements in a subset of cases, we could not fully validate the functional role of the 565

rearrangement or confirm that the rearrangement was as an independent mediator of 566

resistance due to limitations of our experimental system. Additional studies—ideally 567

using patient-derived models that do not harbor concurrent MET amplification—are 568

needed to robustly characterize these rearrangements. 569

570

In summary, by performing the most comprehensive analysis of MET alterations in ALK-571

positive NSCLC to date, we demonstrate that MET is an important bypass mechanism in 572

tumors exposed to next-generation ALK TKIs. The detection of MET amplification in one-573

third of patients who received next-generation ALK TKIs in the front-line setting provides 574

justification for exploring ALK/MET combinations as an initial treatment strategy for 575

advanced ALK-positive NSCLC. 576

577




20

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16. Wu YL, Zhang L, Kim DW, et al. Phase Ib/II Study of Capmatinib (INC280) Plus Gefitinib After Failure of Epidermal Growth Factor Receptor (EGFR) Inhibitor Therapy in Patients With EGFR-Mutated, MET Factor-Dysregulated Non-Small-Cell Lung Cancer. J Clin Oncol. 2018;36(31):3101-3109.

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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.

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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. .




22

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




23

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.




Figure 1

A

C

207 specimens from 136 patients with ALK+ NSCLC

Plasma samples

(106)

Tumor biopsies

(101)

FISH

(n=56*)

28

11 17

Post-crizotinib

Post-lorlatinib

Post-2nd gen TKI

77

29

28 78 73

Guardant360

(n=106)

FoundationOne

(n=30)

24

6

SNaPshot

(n=43)

22 19

2

B

0 25 50 75 100

% MET amplification

2/38

3/19

4/14

2/4

Prior

Crizotinib

No prior

Crizotinib

0/11 Crizotinib

2nd gen. ALKi

Lorlatinib

Treatment history:

P=0.019

Prio

r Tre

atm

ent

MET F

usion/

Mut

ation

MET/C

EP7

ratio

(FIS

H)

MET C

N (t

issu

e NGS)

MET C

N (p

lasm

a)

Tis

su

e-P

ositiv

e

MGH049 Yes Ceritinib No -- 3 -- --

MGH902 Yes Ceritinib No No 3.9 -- -- Crizotinib

MGH9134 Yes Bri gatinib Yes No 2.5 -- -- Ceritinib

MGH962 Yes Alectinib Yes No 7.3 -- 2.6 Brigatinib

MGH9106 Yes Ceritinib Alectinib Yes No >25 -- -- Alectinib

MGH075 Ceritinib --- No Focal* -- -- Lorlatinib

MGH9085 Ceritinib Alectinib No -- 16 --

MGH9226 Alectinib Bri gatinib No -- 19 2.8

MGH939 Alectinib --- __ >25 -- 2.5

MGH9284 (Pericardial) ST7-MET 1.1 -- --

MGH9284 (Pleural) Yes No 2.4 -- --

MGH915 (Pleural) Ceritinib Alectinib Yes ST7-MET 5.2 -- 2.4

MGH915 (Axilla) Ceritinib Alectinib Yes No 5.7 -- 2.4

MGH960 Yes Alectinib Yes No** 1 -- 2.2

MGH9158 Yes Alectinib Yes No** -- -- 4.9

MGH9224 Yes Alectinib Yes No** -- -- 6.1

MGH9133 Alectinib Bri gatinib Yes Ex14 skip 1 -- <2.1T

issu

e-P

ositiv

eP

lasm

a o

nly




A

B

C

D

0 0.1 1 10 100 10000

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Cell

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Alectinib

Brigatinib

Lorlatinib

MGH915-4

MGH915-4

0 0.1 1 10 100 10000

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Lorlatinib + 100nM capmatinib

Lorlatinib + 100nM savolitinib

MGH915-4

Lorlatinib

120 kDa

– + + – – – – –

– – – – – + + –

– – – – + – – +

– – + + – – + +

Capmatinib

Savolitinib

Crizotinib

β-Actin

S6

p-S6 (S240/244)

AKT

ERK1/2

p-ERK1/2 (T202/Y204)

p-AKT (S473)

MET

p-MET (Y1234/1235)

p-ALK (Y1282/1283)

ALK

0 0.1 1 10 100 10000

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Lorlatinib (nM)

Cell

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Lorlatinib + 100nM capmatinib

Lorlatinib + 100nM savolitinib

Kinase TM

ST7 Exon 1 MET Exons 2-21

ST7-MET fusion transcript (cDNA)

G C A T A A A A A A A T A T T T T A A A T A C C C C C C C G G G G

Figure 2




B

C

A H3122 ST7-MET

H3122 ST7-MET

0 0.1 1 10 100 10000

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Capmatinib (DOX+)

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Lorlatinib + 100nM savolitinib (DOX+)

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0 0.1 1 10 100 10000

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175 kDa

145 kDa

p-ALK Y1282/1283

ALK

p-MET Y1234/1235

MET

p-AKT S473

AKT

p-ERK1/2 T202/Y204

ERK1/2

p-S6 S240/244

S6

β-Actin

DOX–

Lorlatinib

Capmatinib

Savolitinib

Crizotinib

– + – – – + + +

– – + – – + – –

– – – + – – + –

– – – – + – – +

DOX+

– + – – – + + +

– – + – – + – –

– – – + – – + –

– – – – + – – +

H3122 ST7-MET

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(% o

f vehic

le c

ontr

ol) Brigatinib (DOX+)

Brigatinib (DOX–)

Figure 3




Months: 0 3 6 9 12 15 18 21

Plasma:

EML4-ALK

No ALK mutations

MET AMP

A

B Baseline Response to crizotinib

Alectinib

Lung biopsy:

Adenocarcinoma

NGS: EML4-ALK v3,

No ALK mutations,

MET FISH (+)

Carboplatin/pemetrexed

Crizotinib

R Pleural biopsy:

Adenocarcinoma

NGS: EML4-ALK v3,

ALK FISH (+)

MET FISH (-)

Chest wall biopsy:

Adenocarcinoma

NGS: EML4-ALK v3,

ALK mutations: qns

MET FISH (+)

Plasma:

EML4-ALK

ALK F1174L,

L1196M, S1206F

MET AMP

Plasma:

EML4-ALK

No ALK mutations

MET AMP

Figure 4




Progression on

alectinib + bevacizumab

Progression on

lorlatinib + crizotinib

Progression on

lorlatinib (pleural fluid)

Progression on

lorlatinib (axillary LN)

MET/CEP7 1.0 MET/CEP7 5.2 MET/CEP7 5.7 MET/CEP7 >25

A

B

Progression on lorlatinib Progression on lorlatinib + crizotinib Response to lorlatinib + crizotinib

R axillary LN biopsy:

Adenocarcinoma

NGS: EML4-ALK v1,

no ALK mutations

MET FISH >25:1

Years: 0 2 3 4 5 6

Lung biopsy:

Adenocarcinoma

ALK FISH (+)

MET FISH n.d.

Ceritinib

Lorlatinib

Alectinib/Bevacizumab

1

Alectinib/Pemetrexed Carboplatin/Pemetrexed/Nivolumab

Alectinib/Cobimetinib

Lorlatinib/Crizotinib

Lung biopsy:

Adenocarcinoma

NGS: EML4-ALK v1,

no ALK mutations

MET FISH n.d.

Pleural Effusion:

Adenocarcinoma

NGS: EML4-ALK v1,

no ALK mutations,

ST7-MET

MET FISH 5.2

Lung biopsy:

Adenocarcinoma

NGS: EML4-ALK v1,

no ALK mutations,

MET FISH 1.0 (-)

R axillary LN biopsy:

Adenocarcinoma

NGS: EML-ALK v1,

no ALK mutations

MET FISH 5.7

C

Pleural effusion

(no clinical

genotyping)

Figure 5




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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