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
2
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
66
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
3
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
83
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
4
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
113
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
5
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
124
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
6
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
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
7
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
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
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
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
9
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
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
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
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
11
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
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
12
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
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
13
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
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
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
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
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
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
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
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
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
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
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
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
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
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
20
References
1. Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature. 2007;448(7153):561-566.
2. Camidge DR, Kim HR, Ahn MJ, et al. Brigatinib versus Crizotinib in ALK-Positive Non-Small-Cell Lung Cancer. N Engl J Med. 2018;379(21):2027-2039.
3. Peters S, Camidge DR, Shaw AT, et al. Alectinib versus Crizotinib in Untreated ALK-Positive Non-Small-Cell Lung Cancer. N Engl J Med. 2017;377(9):829-838.
4. Solomon BJ, Mok T, Kim DW, et al. First-line crizotinib versus chemotherapy in ALK-positive lung cancer. N Engl J Med. 2014;371(23):2167-2177.
5. Soria JC, Tan DS, Chiari R, et al. First-line ceritinib versus platinum-based chemotherapy in advanced ALK-rearranged non-small-cell lung cancer (ASCEND-4): a randomised, open-label, phase 3 study. Lancet. 2017;389(10072):917-929.
6. Shaw AT, Felip E, Bauer TM, et al. Lorlatinib in non-small-cell lung cancer with ALK or ROS1 rearrangement: an international, multicentre, open-label, single-arm first-in-man phase 1 trial. Lancet Oncol. 2017;18(12):1590-1599.
7. Gainor JF, Dardaei L, Yoda S, et al. Molecular Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in ALK-Rearranged Lung Cancer. Cancer Discov. 2016;6(10):1118-1133.
8. Shaw AT, Solomon BJ, Besse B, et al. ALK Resistance Mutations and Efficacy of Lorlatinib in Advanced Anaplastic Lymphoma Kinase-Positive Non-Small-Cell Lung Cancer. J Clin Oncol. 2019;37(16):1370-1379.
9. Yoda S, Lin JJ, Lawrence MS, et al. Sequential ALK Inhibitors Can Select for Lorlatinib-Resistant Compound. Cancer Discov. 2018;8(6):714-729.
10. Recondo G, Mezquita L, Facchinetti F, et al. Diverse Resistance Mechanisms to the Third-Generation ALK Inhibitor Lorlatinib in ALK-Rearranged Lung Cancer. Clin Cancer Res. 2019.
11. Hrustanovic G, Olivas V, Pazarentzos E, et al. RAS-MAPK dependence underlies a rational polytherapy strategy in EML4-ALK-positive lung cancer. Nat Med. 2015;21(9):1038-1047.
12. Isozaki H, Ichihara E, Takigawa N, et al. Non-Small Cell Lung Cancer Cells Acquire Resistance to the ALK Inhibitor Alectinib by Activating Alternative Receptor Tyrosine Kinases. Cancer Res. 2016;76(6):1506-1516.
13. Crystal AS, Shaw AT, Sequist LV, et al. Patient-derived models of acquired resistance can identify effective drug combinations for cancer. Science. 2014;346(6216):1480-1486.
14. Piotrowska Z, Isozaki H, Lennerz JK, et al. Landscape of Acquired Resistance to Osimertinib in. Cancer Discov. 2018;8(12):1529-1539.
15. Yu HA, Arcila ME, Rekhtman N, et al. Analysis of tumor specimens at the time of acquired resistance to EGFR-TKI therapy in 155 patients with EGFR-mutant lung cancers. Clin Cancer Res. 2013;19(8):2240-2247.
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.
17. Gouji T, Takashi S, Mitsuhiro T, Yukito I. Crizotinib can overcome acquired resistance to CH5424802: is amplification of the MET gene a key factor? J Thorac Oncol. 2014;9(3):e27-28.
18. Sakakibara-Konishi J, Kitai H, Ikezawa Y, et al. Response to Crizotinib Re-administration After Progression on Lorlatinib in a Patient With ALK-rearranged Non-small-cell Lung Cancer. Clin Lung Cancer. 2019;20(5):e555-e559.
19. Frampton GM, Fichtenholtz A, Otto GA, et al. Development and validation of a clinical cancer genomic profiling test based on massively parallel DNA sequencing. Nat Biotechnol. 2013;31(11):1023-1031.
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
21
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. .
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
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
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
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.
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
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
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
A
B
C
D
0 0.1 1 10 100 10000
25
50
75
100
Drug Concentration (nM)
Cell
Via
bili
ty
(% o
f vehic
le c
ontr
ol)
Crizotinib
Ceritinib
Alectinib
Brigatinib
Lorlatinib
MGH915-4
MGH915-4
0 0.1 1 10 100 10000
25
50
75
100
Lorlatinib (nM)
Cell
Via
bili
ty
(% o
f vehic
le c
ontr
ol)
Lorlatinib
Lorlatinib + 100nM crizotinib
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
25
50
75
100
Lorlatinib (nM)
Cell
Via
bili
ty
(% o
f vehic
le c
ontr
ol)
Lorlatinib
Lorlatinib + 100nM crizotinib
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
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
B
C
A H3122 ST7-MET
H3122 ST7-MET
0 0.1 1 10 100 10000
25
50
75
100
Drug Concentration (nM)
Cell
Via
bili
ty
(% o
f vehic
le c
ontr
ol)
Capmatinib + 100nM lorlatinib (DOX+)
Capmatinib (DOX+)
0 0.1 1 10 100 10000
25
50
75
100
Drug Concentration (nM)
Cell
Via
bili
ty
(% o
f vehic
le c
ontr
ol)
Crizotinib (DOX+)
Crizotinib + 100nM lorlatinib (DOX+)
0 0.1 1 10 100 10000
25
50
75
100
Drug Concentration (nM)
Cell
Via
bili
ty
(% o
f vehic
le c
ontr
ol)
Lorlatinib (DOX+)
Lorlatinib + 100nM capmatinib (DOX+)
Lorlatinib + 100nM savolitinib (DOX+)
Lorlatinib + 100nM crizotinib (DOX+)
0 0.1 1 10 100 10000
25
50
75
100
Drug Concentration (nM)
Cell
Via
bili
ty
(% o
f vehic
le c
ontr
ol)
Savolitinib (DOX+)
Savolitinib + 100nM lorlatinib (DOX+)
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
0 0.1 1 10 100 10000
25
50
75
100
Drug Concentration (nM)
Cell
Via
bili
ty
(% o
f vehic
le c
ontr
ol) Lorlatinib (DOX+)
Lorlatinib (DOX–)
0 0.1 1 10 100 10000
25
50
75
100
Drug Concentration (nM)
Cell
Via
bili
ty
(% o
f vehic
le c
ontr
ol) Crizotinib (DOX+)
Crizotinib (DOX–)
0 0.1 1 10 100 10000
25
50
75
100
Drug Concentration (nM)
Cell
Via
bili
ty
(% o
f vehic
le c
ontr
ol) Ceritinib (DOX+)
Ceritinib (DOX–)
0 0.1 1 10 100 10000
25
50
75
100
Drug Concentration (nM)
Cell
Via
bili
ty
(% o
f vehic
le c
ontr
ol) Alectinib (DOX+)
Alectinib (DOX–)
0 0.1 1 10 100 10000
25
50
75
100
Drug Concentration (nM)
Cell
Via
bili
ty
(% o
f vehic
le c
ontr
ol) Brigatinib (DOX+)
Brigatinib (DOX–)
Figure 3
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
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
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
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
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
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
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
Updated version
10.1158/1078-0432.CCR-19-3906doi:
Access the most recent version of this article at:
Material
Supplementary
http://clincancerres.aacrjournals.org/content/suppl/2020/02/21/1078-0432.CCR-19-3906.DC1
Access the most recent supplemental material at:
Manuscript
Authoredited. Author manuscripts have been peer reviewed and accepted for publication but have not yet been
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)
.http://clincancerres.aacrjournals.org/content/early/2020/02/21/1078-0432.CCR-19-3906To request permission to re-use all or part of this article, use this link
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