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

Abstract

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

Experimental design: We performed FISH and/or next-generation sequencing on 207 posttreatment tissue (n = 101) or plasma (n = 106) specimens from patients with ALK-positive lung cancer to detect MET genetic alterations. We evaluated ALK inhibitor sensitivity in cell lines with MET alterations and assessed antitumor activity of ALK/MET blockade in ALK-positive cell lines and 2 patients with MET-driven resistance.

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

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

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; 2^nd 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. 2^nd-gen. ALKi: second-generation ALK inhibitor.

Figure 2.. Acquired Resistance Due to ST7-MET and MET Amplification.

(A) Sanger sequencing of the RT-PCR product in MGH915’s lorlatinib-resistant pleural fluid sample shows fusion of ST7 exon 1 to MET exon 2. (B) and (C) Cell viability after 3 days of monotherapy or combination therapy, as indicated. Viability was determined using CellTiter-Glo. Data are mean ± s.e.m. of three biological replicates. (D) Immunoblotting to assess phosphorylation of ALK, MET, and downstream targets in cells treated with lorlatinib and the MET TKIs shown. The arrowhead at 120 kDa indicates the band for phospho-ALK. Cells were treated with each drug at 300 nM for 6 hours.

Figure 3.. Expression of ST7-MET Confers Resistance to ALK TKIs and Can Be Overcome by Dual ALK and MET Inhibition.

(A) and (B) Cell viability of H3122 cells with or without doxycycline-induced ST7-MET expression (DOX+ and DOX-, respectively) was measured after 3 days of monotherapy or combination therapy, as indicated. Viability was determined using CellTiter-Glo. Data are mean ± s.e.m. of three biological replicates. (C) Immunoblotting to assess phosphorylation of ALK, MET, and downstream targets in cells treated with lorlatinib and the MET TKIs shown. The arrowheads at 175 kDa and 145 kDa indicate the band for pro-MET and MET beta-chain respectively. Cells were treated with each drug at 300 nM for 6 hours. DOX: doxycycline.

Figure 4.. Clinical Response to Crizotinib in a Patient with ALK-Positive Lung Cancer and Acquired MET amplification:

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

Figure 5.. Clinical Activity of Dual ALK/MET TKIs in MET-Driven Resistance

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

Figure 6.. Convergent Evolution Upon MET Activation Drives Resistance to Lorlatinib.

(A) Metastatic disease sites analyzed by whole genome sequencing. (B) Copy number profiles of MET locus demonstrate amplification encompassing MET and first exon of ST7 in post-lorlatinib samples. Circos plots depicting chromosomal structure are shown on right. (C) Proposed sequence of genomic alterations leading to generation of ST7-MET fusion and amplification. The EML4-ALK rearrangement (*) and MET locus (**) are indicated. (D) Phylogenetic relationship of serial tumors samples based on shared and private mutations and MET alterations: MGH915–1 (pre-treatment tumor biopsy), MGH915–3 (cell line derived from post-alectinib pleural effusion), MGH914–4 (post-lorlatinib pleural effusion and patient-derived xenograft), MGH915–9 (post-lorlatinib/crizotinib axillary lymph node biopsy). Number of somatic mutations and selected mutations private to each branch point are indicated.